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Physiological and biochemical aspects of growth and yield stimulation of bean (Phaseolus vulgaris L.)… Rathore, Vikram S. 1968

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PHYSIOLOGICAL AND BIOCHEMICAL ASPECTS OF GROWTH AND YIELD STIMULATION OF BEAN (Phaseolus v u l g a r i s L.) PLANTS BY 2,4-D-MINERAL SPRAYS  by  VIKRAM S. RATHORE B . S c , Agra U n i v e r s i t y , Agra, I n d i a , 1958 M . S c , Agra U n i v e r s i t y , Agra, I n d i a , 1960  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of BOTANY  We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard.  THE UNIVERSITY OF BRITISH COLUMBIA June, 1968  In  presenting  for  an  that  advanced  the  I  thesis  for  Department  shall  further  agree  the  make  freely  representatives.  h its  of  of  this  thesis  may  for  permission.  Botany Columbia  June 2 7 t h , 1968.  be  of  for  granted  It  is  financial  of  British  available  permission  or  by  fulfilment  University  it  that  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, C a n a d a Date  partial  purposes  my w r i t t e n  Department  at  in  scholarly  publication  without  thesis  degree  Library  Study.  or  this  for  the  Columbia,  I  reference  and  extensive  by  the  requirements  copying  Head  understood  gain  shall  of  not  of  my  that  be  agree  this  ,  copying  allowed  ABSTRACT Recent r e p o r t s have emphasized t h a t f o l i a r  applications  of s t i m u l a t o r y c o n c e n t r a t i o n s of 2,4-dichlorophenoxyacetic a c i d (2,4-D) induce g r e a t e r growth and p r o d u c t i v i t y of s e v e r a l crop plants.  T h i s s t i m u l a t o r y a c t i o n of the hormone has been found  to be augmented by simultaneous a p p l i c a t i o n of such micron u t r i e n t s as i r o n , manganese, z i n c , copper, and boron(Wort, 1964).  However, no systematic attempt has so f a r been made to  i n v e s t i g a t e p h y s i o l o g i c a l and b i o c h e m i c a l changes induced i n a r e l a t i v e l y s h o r t p e r i o d f o l l o w i n g these treatments.  A corre-  l a t i o n of such changes w i t h f i n a l improvements i n growth and y i e l d may  p r o v i d e a b e t t e r understanding o f the mechanism o f  a c t i o n of the hormone.  In a d d i t i o n , the b i o c h e m i c a l nature  o f the augmentory r o l e p l a y e d by m i c r o n u t r i e n t s on 2,4-D i s y e t undefined.  I t was  action  t h e r e f o r e f e l t d e s i r a b l e to i n v e s t i g a t e  these a s p e c t s . Bean p l a n t s (Phaseolus v u l g a r i s L. Var. Top Crop) were grown i n p l a s t i c cans under c o n t r o l l e d environment room.  i n a growth  When the p l a n t s were 2 weeks o l d , the f o l l o w i n g  ments were a p p l i e d as f o l i a r sprays: micronutrient solution  (Fe, Mn,  (1)  2,4-D  (1 ppm),  Zn, Cu, B; 5x10""* M)  2,4-D-mineral s o l u t i o n , w i t h the same components as 1 ppm  2,4-D.  treat-  and  (2) (3)  (2) p l u s  Measurements of j u v e n i l e growth, chemical composition  ii  of the p l a n t s , activities  of  r e s p i r a t i o n and p h o t o s y n t h e t i c  rates  and  some o f t h e k e y enzymes o f c a r b o h y d r a t e  and  n i t r o g e n m e t a b o l i s m w e r e made 5 , 1 0 , a n d 15 d a y s a f t e r treatment,  to determine the c o u r s e and b a s i s o f  the  stimulation.  Y i e l d o f pods and seeds and t h e v i t a m i n C c o n t e n t o f pods also  were  determined. The r e s u l t s  revealed that:  (1)  maximum s t i m u l a t i o n  from the use of hormone-mineral s p r a y , 2,4-D resulted in progressive  (2)  treatments  resulted  involving  increase in plant height,  number,  and l e a f a r e a ; f r e s h and d r y w e i g h t s  leaves;  chlorophyll  of r o o t ,  leaf  stem and  c o n t e n t and t o t a l sugar i n l e a v e s ,  (3)  s u c r o s e and r e d u c i n g s u g a r s were l o w e r i n stems and r o o t s treated plants;  (4)  m o i s t u r e c o n t e n t was n o t a f f e c t e d  l y b y any t r e a t m e n t ; m i n a t i o n of  the f r e e ,  (5)  quanifeative chromatographic  nutrient plants  significantdeter-  e t h a n o l - s o l u b l e amino a c i d s r e v e a l e d a  r e d u c t i o n i n amino a c i d c o n t e n t particularly  of  in leaves,  in 2,4-D-treated plants,  and an i n c r e a s e i n p l a n t s t o w h i c h m i c r o -  spray had been a p p l i e d .  The a m i n o a c i d c o n t e n t  t r e a t e d w i t h 2 , 4 - D p l u s m i n e r a l s was  between those r e s u l t i n g from the o t h e r  of  intermediate  two t r e a t m e n t s ;  (6)  m e a s u r e m e n t made w i t h i n t a c t p l a n t s u s i n g a n i n f r a r e d CO2 a n a l y z e r r e v e a l e d i n c r e a s e s i n r a t e s o f r e s p i r a t i o n and p h o t o synthesis  of  the a e r i a l p o r t i o n s  10 a n d 15 d a y s a f t e r  treatment  ;  iii  (7) s i g n i f i c a n t i n c r e a s e s i n the a c t i v i t y o f phosphorylase, p h o s p h o g l y c e r y l k i n a s e , s u c c i n i c dehydrogenase,  catalase,  n i t r a t e r e d u c t a s e , and transaminase i n a l l organs were a l s o apparent a t 10 and 15 days a f t e r treatments i n v o l v i n g 2,4-D; (8) treatments i n v o l v i n g 2,4-D a l s o r e s u l t e d i n s i g n i f i c a n t increases  (P=0.05) i n number and f r e s h weight o f green pods,  i n seed number, and i n t o t a l seed weight.  Weight o f the  i n d i v i d u a l seed was not s i g n i f i c a n t l y a l t e r e d ;  (9) the green  pods from t r e a t e d p l a n t s had h i g h e r v i t a m i n C content a t h a r v e s t time, b u t the moisture content was not a l t e r e d by t r e a t ment.  The green pods of p l a n t s which had r e c e i v e d 2,4-D alone  or 2,4-D p l u s m i n e r a l s l o s t l e s s a s c o r b i c a c i d and moisture d u r i n g 4 days storage a t room  temperature.  The s t i m u l a t i o n o f enzyme a c t i v i t i e s i s e x p l a i n e d on the b a s i s o f h y p o t h e s i s proposed by Wort (1964) t h a t 2,4-D may p a r t i c i p a t e i n the f o r m a t i o n o f substrate-enzyme-regulator complex.  M i n e r a l s may a f f e c t the a c t i v i t y o f formation o f t h i s  complex. In p r o p o s i n g a p h y s i o l o g i c a l and b i o c h e m i c a l b a s i s f o r the s t i m u l a t i o n i n growth and y i e l d under 2,4-D a c t i o n , the f o l l o w i n g p o i n t s are emphasized:  (1) the s t i m u l a t e d r a t e o f p h o t o s y n t h e s i s  produced a l a r g e r amount o f photosynthate which c o u l d be u t i l i z e d i n the b i o s y n t h e s i s o f a l l c e l l c o n s t i t u e n t s o r serve as s u b s t r a t e  iv  f o r r e s p i r a t i o n ; (2) the s t i m u l a t e d r a t e of r e s p i r a t i o n and a c t i v i t y of such enzymes as phosphoglyceryl  kinase  and  s u c c i n i c dehydrogenase r e s u l t e d i n an i n c r e a s e d supply of able energy, as ATP and  and reduced n u c l e o t i d e s , f o r b i o s y n t h e s i s ,  i n l a r g e r amounts of keto a c i d s which p r o v i d e  s k e l e t o n s of amino a c i d s ; reductase  and  avail-  the carbon  (3) the g r e a t e r a c t i v i t y o f n i t r a t e  transaminase r e s u l t e d i n an augmented supply  of  amino a c i d s r e s p o n s i b l e f o r the enhanced s y n t h e s i s o f p r o t e i n e v i d e n t i n g r e a t e r growth and p r o d u c t i v i t y .  V  ACKNOWLEDGMENTS  In  deepest  guidance,  advice,  D.J.  Wort  throughout  also  wish  to  uation  of  the  D.P.  terest  entire  Ormrod,  for  his  work.  wish  very I  thanks  given to  T.  acknowledge  provided to  of  this  the  me b y  Professor  investigation.  constructive  go  to  Dr.  review  throughout  help  acknowledge  G.H.N.  Bisalputra  express  generous  also  his  to  and  I eval-  manuscript.  and Dr.  and a d v i c e I  wish  progress  him f o r  My g r a t e f u l Dr.  I  and encouragement  thank  the  gratitude,  for  this  my s i n c e r e and  his  their  continued  in-  study. thanks  suggestions help  Towers,  to  in  Mr.  the  in  checking  with  thanks  K.M.  Patel  experimental  the  typed  manuscript. The facilities Botany. are at  provided  Several  not one  author  to  time  or  my c o r d i a l  the  the  members extended  other.  students  In  Department of  the  their  addition,  the of  excellent  Biology  Department, help I  in  the  Department.  also  due  to  and  have To  and  whose  names  encouragement been  all  aided  these  I  by express  thanks. Thanks  Canada whose  are  financial  Finally, Mitchell  him by  Faculty  mentioned here,  many g r a d u a t e  of  acknowledges  for  neatly  support  a word o f typing  the  National  made  this  appreciation  the  manuscript.  is  Research  study due  Council  possible. to  Mrs.  Stella  vi  TABLE OF CONTENTS PAGE Abstract  •••  i  Acknowledgments Table  v  of Contents  List  of Figures  List  o f Tables  v i x xiii  INTRODUCTION  1  LITERATURE REVIEW  5  A.  E f f e c t o f 2,4-D, a n d 2,4-D-minerals and  B.  C.  on g r o w t h  yield  5  E f f e c t s on p l a n t  composition  content  and i t s l o s s  15  1.  Moisture  15  2.  Pigments  18  3.  Vitamins  20  E f f e c t s on m e t a b o l i s m  24  1.  2.  Carbohydrate  metabolism  24  Carbohydrate  content  24  Photosynthesis  29  Respiration  32  Nitrogen metabolism  35  Free  36  amino a c i d s  Enzymes  and p r o t e i n c o n t e n t  40  vii  TABLE OF CONTENTS  (Cont'd) PAGE  3.  Other m e t a b o l i c changes  MATERIALS AND METHODS  44 45  A.  Plant material  45  B.  Spray treatments  45  C.  Measurement times  46  D.  J u v e n i l e growth measurements  46  E.  Y i e l d measurements  47  F.  A n a l y t i c a l procedures  48  1.  Pigments  48  2.  Reducing  3.  Free amino a c i d s  49  4.  Ascorbic acid  51  G.  H.  sugars and sucrose  48  Photosynthesis and r e s p i r a t i o n r a t e s measurements  52  Determination of enzyme a c t i v i t i e s  53  1.  Phosphorylase  54  2.  Catalase  55  3.  Transaminase  56  4.  N i t r a t e reductase  57  5.  Phosphoglyceryl kinase  58  6.  S u c c i n i c dehydrogenase  60  viii  TABLE OF CONTENTS (Cont'd) PAGE 7.  Protein determination  RESULTS A.  B.  C.  D.  61 6  3  J u v e n i l e growth  63  1.  F r e s h weights  63  2.  Dry weights  66  3.  Shoot e l o n g a t i o n  68  4.  Leaf number and area  68  Chemical  composition  72  1.  M o i s t u r e content  72  2.  C h l o r o p h y l l and c a r o t e n o i d content  72  3.  Sugar content  75  4.  Free amino a c i d s  75  Photosynthesis and r e s p i r a t i o n r a t e s  82  1.  Photosynthesis  82  2.  Respiration  85  Enzyme a c t i v i t i e s  85  1.  Phosphorylase  86  2.  Phosphoglyceryl kinase  86  3.  S u c c i n i c dehydrogenase  90  4.  N i t r a t e reductase  90  5.  Transaminase  94  6.  Catalase  94  ix  TABLE OF CONTENTS  (Cont'd) PAGE  E.  F.  Yield  94  1.  Pod f r e s h weight  94  2.  Pod number  98  3.  Seed y i e l d  98  Q u a l i t y o f pods  98  1.  A s c o r b i c a c i d content  103  2.  A s c o r b i c a c i d l o s s d u r i n g storage  103  3.  Moisture  103  l o s s d u r i n g storage  DISCUSSION  104  CONCLUSIONS  130  BIBLIOGRAPHY  133  APPENDICES  153  X  LIST OF FIGURES FIGURE 1.  2. ]  3.  4.  5.  |  PAGE E f f e c t o f 2,4-D (D) , m i n e r a l s (M) , or 2,4-Dm i n e r a l (MD) sprays on f r e s h weights o f r o o t s , stem, and leaves o f bushbean (Phaseolus v u l g a r i s ) plants. (C= c o n t r o l ) . . . .  65  E f f e c t o f 2,4-D (D), m i n e r a l s (M), or 2,4-Dm i n e r a l (MD) sprays on dry weights o f r o o t s , stem, and leaves o f bushbean (Phaseolus v u l g a r i s ) p l a n t s . (C= c o n t r o l )  67  E f f e c t o f 2,4-D (D), m i n e r a l s (M), o r 2,4-Dm i n e r a l (MD) sprays on the h e i g h t o f bushbean (Phaseolus v u l g a r i s ) p l a n t s  70  E f f e c t o f 2,4-D (D), m i n e r a l s (M), or 2,4-Dm i n e r a l (MD) sprays on the number, area, and c h l o r o p h y l l content o f bushbean (Phaseolus v u l g a r i s ) leaves  71  E f f e c t o f 2,4-D (D), m i n e r a l s (M), or 2,4-Dm i n e r a l (MD) sprays on r a t e s o f r e s p i r a t i o n and p h o t o s y n t h e s i s o f a e r i a l p o r t i o n s o f i n t a c t bushbean (Phaseolus v u l g a r i s ) p l a n t s  84  E f f e c t o f 2,4-D (D) , m i n e r a l s (M) , or 2,4-Dm i n e r a l (MD) sprays on the a c t i v i t y o f phosphoryl a s e i n r o o t s , stem, and leaves o f bushbean (Phaseolus v u l g a r i s ) p l a n t s  88  E f f e c t o f 2,4-D (D), m i n e r a l s (M), or 2,4-Dm i n e r a l (MD) sprays on the a c t i v i t y o f phosphog l y c e r y l k i n a s e i n r o o t s , stem, and leaves o f bushbean (Phaseolus v u l g a r i s ) p l a n t s  89  E f f e c t o f 2,4-D (D), m i n e r a l s (M), or 2,4-Dm i n e r a l (MD) sprays on the a c t i v i t y o f s u c c i n i c dehydrogenase i n r o o t s , stem, and leaves o f bushbean (Phaseolus v u l g a r i s ) p l a n t s  92  i  6|.  ! 7. ;  8. I !  xi  LIST OF FIGURES (Cont'd) FIGURE 9.  10.  11.  12.  PAGE E f f e c t o f 2,4-D (D), m i n e r a l s (M), or 2,4-Dm i n e r a l (MD) sprays on the a c t i v i t y o f n i t r a t e reductase i n r o o t s , stem, and leaves o f bushbean (Phaseolus v u l g a r i s ) p l a n t s  93  E f f e c t o f 2,4-D (D), m i n e r a l s (M), or 2,4-Dm i n e r a l (MD) sprays on the a c t i v i t y o f g l u t a m i c o x a l a c e t i c transaminase i n r o o t s , stem, and leaves o f bushbean (Phaseolus v u l g a r i s ) p l a n t s . . .  95  E f f e c t o f 2,4-D (D), m i n e r a l s (M), or 2,4-Dm i n e r a l (MD) sprays on the a c t i v i t y o f c a t a l a s e i n r o o t s , stem, and leaves o f bushbean (Phaseolus v u l g a r i s ) p l a n t s  96  E f f e c t o f 2,4-D (D), m i n e r a l s (M), or 2,4-Dm i n e r a l (MD) sprays on the y i e l d o f bushbean (Phaseolus v u l g a r i s ) p l a n t s  101  13.  E f f e c t o f 2,4-D (D), m i n e r a l s (M), or 2,4-Dm i n e r a l (MD) sprays on a s c o r b i c a c i d content o f f r e s h bean (Phaseolus v u l g a r i s ) pods, and l o s s o f a s c o r b i c a c i d and moisture d u r i n g 4 days storage o f the pods 102  14.  R e l a t i v e changes i n the c o n c e n t r a t i o n o f asparagine as induced by 2,4-D (. . ) , m i n e r a l s (• — or 2,4-D-mineral (x . x) sprays i n bushbean (Phaseolus v u l g a r i s ) leaves  15.  > 112  B i o s y n t h e t i c scheme to show the p o i n t s where s t i m u l a t i o n by 2,4-D, or 2,4-D-minerals was observed i n the i n v e s t i g a t i o n  129  16.  Standard c h a r t f o r r e d u c i n g sugars  156  17.  Standard  158  18.  A schemetic r e p r e s e n t a t i o n o f two-dimensional chromatogram showing standard p o s i t i o n s o f spots o b t a i n e d w i t h known amino a c i d s  chart f o r ascorbic acid  161  xii  LIST OF FIGURES  (Cont'd)  FIGURE  PAGE  19.  Standard c h a r t f o r phosphorylase  164  20.  Standard c h a r t f o r s u c c i n i c  165  21.  Standard c h a r t f o r n i t r a t e reductase  166  22.  Standard c h a r t f o r transaminase  167  dehydrogenase  (GOT)  xiii  L I S T OF TABLES TABLE I II  III  IV  V  VI  VII  VIII  IX  PAGE The e f f e c t s  o f 2,4-D on enzyme a c t i v i t i e s  41-43  E f f e c t o f m i n e r a l s , 2,4-D, o r 2 , 4 - D - m i n e r a l s p r a y s on f r e s h w e i g h t s , a n d d r y w e i g h t s o f r o o t s , stem, a n d l e a v e s o f b u s h b e a n (Phaseolus v u l g a r i s ) p l a n t s  64  E f f e c t o f m i n e r a l s , 2,4-D, o r 2 , 4 - D - m i n e r a l s p r a y s on p l a n t h e i g h t , l e a f number, a n d l e a f area o f bushbean (Phaseolus v u l g a r i s ) p l a n t s  69  E f f e c t o f m i n e r a l s , 2,4-D, o r 2 , 4 - D - m i n e r a l s p r a y s on m o i s t u r e c o n t e n t o f r o o t s , stem, and l e a v e s o f b u s h b e a n ( P h a s e o l u s v u l g a r i s ) plants  73  E f f e c t o f m i n e r a l s , 2,4-D, o r 2 , 4 - D - m i n e r a l s p r a y s on c h l o r o p h y l l a n d c a r o t e n o i d c o n t e n t o f bushbean (Phaseolus v u l g a r i s ) l e a v e s  74  E f f e c t o f m i n e r a l s , 2,4-D, o r 2 , 4 - D - m i n e r a l s p r a y s on r e d u c i n g s u g a r s , a n d s u c r o s e c o n t e n t i n r o o t , stem, a n d l e a v e s o f b u s h b e a n (Phaseolus vulgaris) plants  76  E f f e c t o f m i n e r a l s , 2,4-D, o r 2 , 4 - D - m i n e r a l s p r a y s on t h e r e l a t i v e c o n c e n t r a t i o n s o f t h e e t h a n o l - s o l u b l e free-amino acids o f leaves o f bushbean (Phaseolus v u l g a r i s )  78  E f f e c t o f m i n e r a l s , 2,4-D, o r 2 , 4 - D - m i n e r a l s p r a y s on t h e r e l a t i v e c o n c e n t r a t i o n s o f t h e e t h a n o l s o l u b l e f r e e - a m i n o a c i d s o f stem o f b u s h b e a n (Phaseolus v u l g a r i s )  79  E f f e c t o f m i n e r a l s , 2,4-D, o r 2 , 4 - D - m i n e r a l s p r a y s on t h e r e l a t i v e c o n c e n t r a t i o n s o f e t h a n o l s o l u b l e free-amino a c i d s o f r o o t s o f bushbean (Phaseolus v u l g a r i s )  80  xiv  LIST OF TABLES (Cont'd) TABLE X  XI  XIII  XIII  XIV  XV  XVI  PAGE E f f e c t o f m i n e r a l s , 2,4-D, or 2,4-D-mineral sprays on p h o t o s y n t h e t i c and r e s p i r a t i o n r a t e s o f the a e r i a l p o r t i o n s o f i n t a c t bushbean (Phaseolus v u l g a r i s ) p l a n t s  83  E f f e c t o f m i n e r a l s , 2,4-D, or 2,4-D-mineral sprays on the a c t i v i t i e s o f phosphorylase, p h o s p h o g l y c e r y l k i n a s e , and s u c c i n i c dehydrogenase i n bushbean (Phaseolus v u l g a r i s ) p l a n t . . . .  87  E f f e c t o f m i n e r a l s , 2,4-D, or 2,4-D-mineral sprays on the a c t i v i t i e s o f n i t r a t e reductase, transaminase, and c a t a l a s e i n bushbean (Phaseolus v u l g a r i s ) p l a n t  91  Comparative e f f e c t s o f m i n e r a l , 2,4-D, or 2,4-Dm i n e r a l s on enzymes o f bushbean (Phaseolus vulgaris) plants  97  E f f e c t o f m i n e r a l s , 2,4-D, or 2,4-D-mineral sprays on y i e l d o f bushbean (Phaseolus vulgaris) plant  99  E f f e c t o f m i n e r a l s , 2,4-D, o r 2,4-D-mineral sprays on v i t a m i n C content, moisture content, and t h e i r l o s s d u r i n g 4 days storage i n bean (Phaseolus v u l g a r i s ) pods  100  Comparative e f f e c t s o f treatment o f bushbean (Phaseolus v u l g a r i s ) p l a n t s w i t h 2,4-D-alone and i n combination w i t h m i c r o n u t r i e n t s on f r e s h and dry weights o f r o o t s , stems, and leaves  107  INTRODUCTION  Since  their f i r s t discovery  almost three decades ago  (Haagen-Smit and Went, 1935; Zimmerman and Hitchcock,  1942)  s e v e r a l s y n t h e t i c auxins have been recognized,  b u t none has  r e c e i v e d as much a p p l i c a t i o n i n p l a n t r e s e a r c h  as  phenoxyacetic a c i d (2,4-D) . h e r b i c i d e i n higher  2,4-dichloro-  Although a c t i n g as a s e l e c t i v e  doses, the s t i m u l a t o r y r o l e o f 2,4-D i n  lower c o n c e n t r a t i o n s  i s f a i r l y well established.  There i s  indeed ample evidence t h a t when a p p l i e d a t the c o r r e c t ontogenetic  time and i n a p p r o p r i a t e  both v e g e t a t i v e species  doses, 2,4-D can s t i m u l a t e  as w e l l as r e p r o d u c t i v e  growth o f s e v e r a l crop  (Wedding e t a l . 1956; Wort, 1957, 1959, 1962, 1964a,  1964b, 1966; M i l l e r e t a l . 1962a, 1962b). which 2,4-D s t i m u l a t e s  The mechanism by  growth and y i e l d o f p l a n t s i s r e l a t i v e l y  unknown. For many years both minor and major elements as Fe, Zn, Cu, B, Mn, K, P, S, N, e t c . , have been a p p l i e d t o p l a n t s v i a t h e i r l e a v e s . The s u b j e c t o f f o l i a r been reviewed r e c e n t l y The  fertilization  o f p l a n t s has  (Wittwer and Teubner, 1959; Wittwer, 1964).  i n t e r a c t i o n between auxins and m e t a l l i c ions has  gained s i g n i f i c a n c e d u r i n g Takahashi, 1961).  the r e c e n t years  (Thimann and  Manganese has been shown to a c t u a l l y promote  the growth induced by i n d o l e a c e t i c a c i d  (IAA) i n c o l e o p t i l e  2. sections  (Bonner, 1949), and f e r r o u s s u l f a t e has been used to  i n c r e a s e the s e n s i t i v i t y of Avena c u r v a t u r e Yamaki, 1959).  T h i s powerful  test  (Shibaoka  i n t e r a c t i o n between auxins  m e t a l l i c ions has been emphasized i n r e c e n t reviews  and and  (Wort,  1962,  1964b; Thimann, 1963). A s e r i e s o f i n v e s t i g a t i o n s has been c a r r i e d out at the U n i v e r s i t y o f B r i t i s h Columbia concerned w i t h the a p p l i c a t i o n of sprays and dusts of 2,4-D  and 2,4-D w i t h m i c r o n u t r i e n t s  a number of economic p l a n t s p e c i e s 1954,  1957,  1959,  1961,  1962,  (Wort, 1950,  1951,  1964a, 1964b, 1966).  have shown a s t r o n g i n t e r a c t i o n between 2,4-D  1953,  These s t u d i e s  and m e t a l l i c ions  i n t h e i r e f f e c t s on p l a n t growth and metabolism.  I n c l u s i o n of  or more elements, p a r t i c u l a r l y such m i c r o n u t r i e n t s as Fe, Zn, Cu,  and B, i n s a l t or c h e l a t e form i n a p p r o p r i a t e l y  concentrations.  Mn,  has  e f f e c t s of h e r b i c i d a l  More s i g n i f i c a n t , however, i s the  one  low  c o n c e n t r a t i o n s , w i t h the dust or aqueous sprays o f 2,4-D, been found to l e s s e n the deformative  to  2,4-D  discovery  t h a t when these m i c r o n u t r i e n t s are used i n combination w i t h s t i m u l a t o r y 2,4-D  concentrations,  a s t i l l g r e a t e r s t i m u l a t i o n of  v e g e t a t i v e growth and y i e l d of s e v e r a l crops, such as bushbeans, sugarbeets, 1964b, 1966;  and potato may  be obtained  (Wort, 1953,  M i l l e r e t a l . 1962a, 1962b; Huffaker  The b i o c h e m i c a l  nature  1956,  1959,  e t a l . 1967).  of t h i s s t i m u l a t o r y  interaction  3 .  between auxins and m i c r o n u t r i e n t ions s t i l l remains In a d d i t i o n , there have been no attempts  so f a r to study the  changes i n v e g e t a t i v e growth, metabolic s t a t u s and composition of p l a n t s immediately applications.  Miller's  chemical  f o l l o w i n g 2,4-D-nutrient  (1962a) o b s e r v a t i o n s on the  growth of bean p l a n t s f o l l o w i n g sprays w i t h 2,4-D and 2,4-D  unexplored.  juvenile  alone,  w i t h f e r r o u s s u l f a t e and c h e l a t e d i r o n , were taken 10 to  14 days a f t e r the treatment.  I t must be emphasized, however, t h a t  o b s e r v a t i o n s taken a f t e r a c o n s i d e r a b l e l a p s e o f time may c o n s i d e r e d as r e f l e c t i v e of the changes brought primary a c t i o n of the treatment,  about by  not be the  e s p e c i a l l y i n view of the  dynamic nature of p l a n t growth. In view of the c o n s i d e r a t i o n s o u t l i n e d above, i t was f e l t v e r y d e s i r a b l e to i n v e s t i g a t e changes t h a t occur i n a r e l a t i v e l y s h o r t span o f time f o l l o w i n g 2,4-D-nutrient  treatments.  In the course of the p r e s e n t i n v e s t i g a t i o n , t h e r e f o r e , the f o l l o w i n g aspects were i n v e s t i g a t e d over a p e r i o d of 15  days,  at 5-day i n t e r v a l s , subsequent to the a p p l i c a t i o n of aqueous sprays c o n t a i n i n g 2,4-D  alone, m i n e r a l s alone, or 2,4-D  plus  m i n e r a l s , to 2-week-6ld bushbean p l a n t s (Phaseolus v u l g a r i s L. var. Top 1.  Crop): J u v e n i l e growth:  F r e s h and dry weights  of r o o t s ,  stem and l e a v e s ; p l a n t h e i g h t ; l e a f number and l e a f area.  2.  Chemical  composition: Moisture and  content;  sucrose  reducing  content;  sugars  chlorophyll  a and b and c a r o t e n o i d content; free and 3.  amino a c i d s i n l e a v e s ,  and  stem,  roots.  M e t a b o l i c changes: Rates  of respiration  synthesis;  activities  and  photo-  o f some k e y  enzymes o f c a r b o h y d r a t e  and n i t r o g e n  metabolism  (phosphorylase,  phos-  phogyceryl  kinase, succinic  de-  hydrogenase, c a t a l a s e , n i t r a t e r e -  4.  Yield  responses:  ductase  and transaminase), i n  leaves,  stem, and r o o t s .  Number a n d w e i g h t ascorbic pods;  loss  moisture age;  metabolism of  with  t h e pods has been  stor-  number and w e i g h t p e r  changes  o f 100  seeds.  i n growth and  improvement i n t h e y i e l d  attempted.  o f the green  o f a s c o r b i c a c i d and  and the weight  of the observed  the f i n a l  content  b y pods d u r i n g 4 days  seed  plant, A correlation  acid  o f pods p e r p l a n t ;  and q u a l i t y  5. LITERATURE REVIEW The l i t e r a t u r e c i t e d i n t h i s review i s r e l a t e d to the e f f e c t s o f 2,4-D and 2,4-D-minerals on growth, y i e l d , chemical composition, p h o t o s y n t h e s i s , r e s p i r a t i o n and enzyme a c t i v i t i e s of p l a n t s .  Emphasis has been put on the l i t e r a t u r e  pertaining  to 2,4-D and 2,4-D-mineral e f f e c t s , but wherever necessary f o r the sake o f comparison, have been i n c l u d e d .  r e p o r t s o f other s y n t h e t i c auxin e f f e c t s  S e v e r a l books and reviews concerned w i t h  these aspects o f s y n t h e t i c auxin e f f e c t s have been p u b l i s h e d . S y n t h e t i c auxin l i t e r a t u r e through 1950 S c h e l l e t a l . (1955).  has been reviewed by  Books d e a l i n g w i t h c h e m i s t r y and mode o f  auxin a c t i o n have been w r i t t e n or e d i t e d by Went and Thimann (1937), Skoog (1951), Thimann (1952), Thimann and Leopold Wain and Wightman (1956), Audus (1959), C r a f t s (1961), Ruhland  (1961), and Weyer (1967).  (1961),  Pilet  S e v e r a l review  i c l e s on n a t u r a l and s y n t h e t i c auxins have appeared Review o f P l a n t P h y s i o l o g y (Volume 6 to 19).  (1955),  art-  i n Annual  L i t e r a t u r e on the  e f f e c t s o f 2,4-D and 2,4-D-minerals on growth, composition, and metabolism  of v a r i o u s crop p l a n t s has been r e c e n t l y reviewed by  Wort (1961, 1962, A.  1964a, and 1964b).  E f f e c t s o f 2,4-D and 2,4-D-Minerals on growth and  yield.  The s t o r y o f p r a c t i c a l a p p l i c a t i o n o f 2,4-D as a growth s t i m u l a n t f o r crop p l a n t s dates back to more than two decades when Taylor  (1946) showed t h a t low doses o f 2,4-D i n n u t r i e n t  6.  solutions increased  the dry weights of soybean p l a n t s .  as the growth o f dicotyledonous cambial a c t i v i t y , of 0.1%  p l a n t s i s governed by  aqueous sprays o f 2,4-D  significantly  taken as comparable to i n c r e a s e d Weaver (1946) r e p o r t e d  growth by  increased  the  the  also  be  treatment.  t h a t a p p l i c a t i o n of aqueous s o l u t i o n s of  2,4-D, a t the r a t e of 0.001, 0.01, square yard,  the  Swanson's (1946) r e p o r t t h a t f o l i a r a p p l i c a t i o n  cambial a c t i v i t y i n young red kidney bean p l a n t s , may  per  Inasmuch  and  0.1  g a c t u a l chemical  to the leaves of young red kidney bean and  bean p l a n t s , r e s u l t e d i n a reduced p l a n t weight and  soy-  delayed-pod-  appearance. Wedding et a l . (1956) r e p o r t e d 20 ppm  of 2,4-D  t h a t a p p l i c a t i o n o f 10  to  e s t e r to lima beans two weeks a f t e r emergence,  r e s u l t e d i n a 35% i n c r e a s e i n the y i e l d of s h e l l e d green beans. There were more pods per p l a n t , and seed was  also increased.  The  the s i z e of the i n d i v i d u a l  a p p l i c a t i o n of 10 ppm  2,4-D  ester  four weeks a f t e r emergence, however, reduced the t o t a l y i e l d marketable pods to o n l y 26% of t h a t formed by u n t r e a t e d  plants.  In a l a t e r communication, Wedding (1962) i n d i c a t e d that the of s u b l e t h a l c o n c e n t r a t i o n s and b e l l peppers i n San t i a l gains  20%.  The  The  The  gave substan-  y i e l d of pole beans  time of a p p l i c a t i o n was  importance o f time and  use  on lima beans, pole beans,  Diego County, C a l i f o r n i a ,  i n y i e l d o f these crops.  i n c r e a s e d by critical.  of 2,4-D  of  other  found to be  f a c t o r s to  was  very  obtain  7.  stimulatory  response t o 2,4-D treatment has been a l s o emphasized  i n a r e c e n t review by Wort (1964) i n the f o l l o w i n g  lines:  "The f i n a l r e s u l t s a r e determined not o n l y by the g e n e r a l nature o f the h e r b i c i d e , b u t a l s o by i t s chemical form, e.g., a c i d , amide, e s t e r , e t c . ; by the c o n c e n t r a t i o n used, the pH, the c a r r i e r , the w e t t i n g agent, the method o f a p p l i c a t i o n , and the s i z e o f d r o p l e t or dust p a r t i c l e a p p l i e d . The species of p l a n t , the p a r t o f the p l a n t t o which the chemical i s a p p l i e d , the p l a n t ' s age, v i g o r , growth r a t e and past h i s t o r y a l l p l a y t h e i r p a r t i n determining the response. Add to a l l these f a c t o r s the weather c o n d i t i o n s , e.g., temperature, l i g h t i n t e n s i t y , humidity, a t the time o f a p p l i c a t i o n o f the chemical and d u r i n g the p e r i o d when the h e r b i c i d e i s a c t i v e w i t h i n the p l a n t , the nature o f the s o i l and the l e v e l and a v a i l a b i l i t y o f i t s n u t r i e n t s and moisture, and i t w i l l be apparent t h a t the c o n d i t i o n o f the t r e a t e d l i v i n g p l a n t a t a given i n s t a n t has indeed been determined by many v a r i a b l e s . " There a r e s e v e r a l a d d i t i o n a l r e p o r t s which bear out the usefulness  o f 2,4-D to s t i m u l a t e  p r o d u c t i v i t y o f crop p l a n t s .  growth and i n c r e a s e  Leonard  (1958) r e p o r t e d  that  aqueous sprays o f trimethylammonium s a l t s o f 2,4-D (150 ppm)  in-  c r e a s e d f r e s h weights o f bean p l a n t s h a r v e s t e d 15 days a f t e r the a p p l i c a t i o n o f 2,4-D. Wort (1959) has r e p o r t e d  r e s u l t s o f extensive  s t u d i e s on  the a p p l i c a t i o n o f 2,4-D and 2,4-D f o r t i f i e d w i t h m i n e r a l s on growth and y i e l d o f beans.  A t a l c dust c o n t a i n i n g  0.1% 2,4-D  was a p p l i e d t o top crop bean p l a n t s when 15-days-old a t the r a t e of 12 l b / a c r e .  When h a r v e s t e d 6 weeks a f t e r the treatment, i t  was found t h a t w h i l e the f r e s h weights o f stems and leaves had been i n c r e a s e d  o n l y s l i g h t l y , the f r e s h weight o f the pods and  8.  threshed  b e a n s went up b y  creases by  in yield  as much as  o f p o d s and  a p p l i c a t i o n o f NCL  0.1  cupric  fate,  zinc  sulfate, ferrous  2.75%  Cu,  sulfur,  and  3.96%  Mn,  M a s t e r p i e c e b u s h b e a n s and have been c o n f i r m e d  3.46%  or  growth  at  (11.6  production "the  tration  or  2,4-D  f o l i a g e of  A  the  common b e a n ,  (16  to  20%).  The  the  acid with  hormone, and  the  instances,  0.1%  2,4-D  increases  to 40%),  2,4-D  and  with in in  augmented  widened the  safely applied  to  a  the  that, the  concenplants  to  yield".  similar  'safening  effect'  of mineral  by  Miller  e t a l . (1962).  effect  of  foliar  a p p l i c a t i o n of various  1,  Clay  i n v e s t i g a t o r concluded  of microelements w i t h  a c t i o n of  (23  to  P_. v u l g a r i s .  13.9%), i n y i e l d  seeds  19.58% f r e e  (Wort, 1 9 6 6 ) .  days o l d w i t h  give  These r e s u l t s  a l o n e i n some  o f pods  sul-  s u l f u r to and  navy beans.  ported  (0.5,  obtained  a l l in attaclay)  to  r a n g e w h i c h c o u l d be  increase  Fe,  lb/acre, resulted in significant  incorporation  stimulatory  elemental  report  In-  preparation  12  of  control.  2,4-dibromophenoxyacetic  T r e a t m e n t o f b e a n p l a n t s when 14 minerals,  dust  3.19%  2,4-D  i n a more r e c e n t  2,4-D  to the  Zn,  Michilite  complex o f m i c r o n u t r i e n t s , were a p p l i e d  the  b e a n s were a l s o  s u l f a t e and  acid equivalent  dusts containing  over  s u l f a t e , b o r a x p e n t a h y d r a t e , manganese  B,  0.1%  threshed  (a c o m m e r c i a l  containing  1.10%  55%  5,  10,  and  100  ppm),  and  ions  has  been  re-  These i n v e s t i g a t o r s s t u d i e d  2,4-D  concentrations i n these  of  the  2,4-D  concentrations  9. f o r t i f i e d with FeS0  4  (  1  5  0  0 p  p  m  p  e  )  o  r  i  r  o  n  c  h  e  i  a  t  e  .  Observations on (1) shoot elongation,. (2) shoot dry matter, (3) r o o t weight and n o d u l a t i o n and (4) l e a f area, were taken 1-0 to 14 days a f t e r the treatments.  The r e s u l t s showed t h a t 0.5 and 1  ppm o f 2,4-D aqueous sprays s t i m u l a t e d j u v e n i l e p l a n t growth. At 5 ppm or above, however, 2,4-D alone e x e r t e d an i n h i b i t o r y e f f e c t on growth as r e f l e c t e d i n reduced l e a f area and dry matter p r o d u c t i o n .  A d d i t i o n o f 1500 ppm Fe as FeSO^, or f e r r i c  i r o n c h e l a t e d w i t h ethylenediamine d i - o - h y d r o x y p h e n y l a c e t i c a c i d (FeEDDHA) or EDDHA alone, e i t h e r b e f o r e , w i t h , or a f t e r 2,4-D treatment, e v e n t u a l l y l e d t o an i n c r e a s e d growth i n a l l t r e a t ments.  These i n v e s t i g a t o r s concluded t h a t , "where the con-  c e n t r a t i o n o f 2,4-D was 5 ppm o r h i g h e r , a l l o f the supplements e x h i b i t e d a 'safening e f f e c t  1  a g a i n s t normally i n j u r i o u s  c o n c e n t r a t i o n s o f 2,4-D, w h i l e p r e s e r v i n g the s t i m u l a t o r y characteristics.  FeSO^ was p a r t i c u l a r l y e f f e c t i v e when a p p l i e d  24 hours a f t e r the 2,4-D.  A t s t i m u l a t o r y l e v e l s o f 2,4-D, the  a d d i t i o n o f the chemical supplements t o f o l i a r sprays showed an additive beneficial effect"  ( M i l l e r e t a l . 1962a).  In l a t e r experiments, M i l l e r e t a l . (1962b) sought t o d i s cover i f (1) the growth s t i m u l a t i o n achieved a t an e a r l y stage would u l t i m a t e l y l e a d to an i n c r e a s e d y i e l d , and (2) the e f f e c t of 2,4-D on y i e l d would be i n f l u e n c e d by simultaneous of an i r o n supplement.  application  In greenhouse experiments, bean p l a n t s a t  10.  the age o f 14 days were sprayed w i t h 0.5, 1, 5, 10, and 100 ppm 2,4-D s o l u t i o n s and these c o n c e n t r a t i o n s o f 2,4-D f o r t i f i e d with FeS0  4  (300 ppm F e ) .  The r e s u l t s showed t h a t  (1)  the  number o f f l o w e r s was i n c r e a s e d by 100 ppm 2,4-D i n combination w i t h FeSO^, (2) the number o f beans per pod and seed s i z e were s i g n i f i c a n t l y i n c r e a s e d by most l e v e l s o f 2,4-D whether used alone or i n combination w i t h FeSO^, (3) f o l i a r treatments w i t h 0.5 and 1 ppm 2,4-D, both w i t h and without FeS04, and 100 ppm 2,4-D w i t h FeS04 gave s i g n i f i c a n t i n c r e a s e s i n dry matter production,  (4) seed y i e l d was i n c r e a s e d up to 34% by t r e a t -  ment w i t h 2,4-D p l u s FeSO^, and (5) even c o n c e n t r a t i o n s o f 2,4-D t h a t i n h i b i t e d growth when a p p l i e d alone, gave i n c r e a s e d y i e l d s i n combination w i t h FeSO^.  I t was concluded that these  r e s u l t s add a d d i t i o n a l evidence t h a t growth r e g u l a t o r s and supplements  a p p l i e d a t an e a r l y stage o f growth may f i n d  s i g n i f i c a n t p r a c t i c a l use i n improving the q u a l i t y and y i e l d . S i g n i f i c a n t i n c r e a s e s i n y i e l d o f S u t t e r Pink f i e l d beans have a l s o been r e p o r t e d by Huffaker e t a l . (1967) who a p p l i e d the i s o p r o p y l e s t e r o f 2,4-D i n v a r i o u s c o n c e n t r a t i o n s and 2,4-D w i t h i r o n supplements,  i n f i e l d t r i a l experiments  out a t T u l e l a k e and Davis i n C a l i f o r n i a .  carried  The r e s u l t s were  consonant w i t h those o f Wedding e t a l . (1956) i n t h a t the d i f f e r e n t environmental c o n d i t i o n s from year t o year a p p a r e n t l y changed the c o n c e n t r a t i o n s o f 2,4-D r e q u i r e d to i n c r e a s e y i e l d .  11.  Improvements i n t h e y i e l d crops  by  2,4-D  obtained. by  and  2,4-D  Podrazanskaja wheat b y alone. dust at  2,4-D-nutrient  Longchamp and  supplying  various  with minerals,  the  2,4-D  fortified  significance,  Sexsmith with  early.  field  plants-(Huffaker  lower y i e l d s  treatments.  an  20%  Miller,  a l s o been i n c r e a s e d by  field 2,4-D  1962).  the  reported  an  as b o r o n  increase  application.  of  2,4-D and  foliar  The  oats,  induced  in yield  that  i n the y i e l d  by lacked  o f b a r l e y when by  obtained  a p p l i c a t i o n of t o young p l a n t s  of  resulted in in  untreated  p r o d u c t i v i t y of  a p p l i c a t i o n o f low  (Yurkovitch,  barley  r e s u l t s v a r i e d from  than those  of 2,4,5-trichlorophenoxyacetic  such m i c r o n u t r i e n t s  trials  in California,  greater  of  fertilizer  increased y i e l d  The  c o n t a i n i n g 5%  and  and  e x p e r i m e n t s were c o n d u c t e d  Hannchen b a r l e y  and  The  o f wheat and  also reported  y i e l d s w h i c h were 16%  2,4-D  on  through increases  Similar f i e l d  (1953) who  durum wheat and  has  compared t o  yield  in yield  a p p l i e d t o wheat, o a t s ,  to s i g n i f i c a n t l y  nutrient-2,4-D  trations  i n c r e a s e o f 20%  data  in yield  dust,  hormone-nutrient dust  has  S i m i l a r l y , Mackov  l o c a t i o n s i n W e s t e r n Canada. increase  other  (1949) i n c r e a s e d g r a i n  to f e r t i l i z e r ,  a significant  treated  an  (1952) r e p o r t e d  several  t r e a t m e n t s have a l s o been  a t p l a n t i n g time.  a d d i n g 2,4-D  fortified  q u a l i t y of  Gautheret  (1950) o b t a i n e d  Ripley  and  tomatoes concen-  a c i d i n combination 1963).  of cotton  Arle  (1954)  obtained  by  with  12. Recently, Huffaker e t a l . (1967) a l s o r e p o r t e d r e s u l t s of  field  t r i a l s w i t h a p p l i c a t i o n o f 2,4-D and i r o n supplements  on y i e l d and p r o t e i n contents o f wheat and b a r l e y c a r r i e d out at  T u l e l a k e and Davis i n n o r t h e r n and c e n t r a l  respectively.  The i s o p r o p y l , i s o o c t y l e s t e r s and d i e t h y l a m i n e  s a l t s o f 2,4-D alone, and i n combination (Fe-ethylenediamine-di FeDTPA of  California,  w i t h F e S 0 , FeEDDHA 4  (O-hydroxyphenol) a c e t i c acid) and  ( F e - e t h y l e n e t r i a m i n e p e n t a a c e t i c acid) i n the c o n c e n t r a t i o n  500 mg per l i t r e w i t h r e s p e c t to i r o n were a p p l i e d to f i e l d  beans when about 2 weeks o l d , and t o b a r l e y and wheat p l a n t s a t the 5 to 7 l e a f stage.  A commercial dust f o r m u l a t i o n  (NCL),  c o n t a i n i n g the i s o p r o p y l e s t e r o f 2,4-D p l u s m i c r o n u t r i e n t s ( c u p r i c s u l f a t e , borax pentahydrate,  manganese s u l f a t e ,  zinc  s u l f a t e , f e r r o u s s u l f a t e and elemental s u l f u r ) , was a l s o i n these t r i a l s .  The r e s u l t s of t h i s q u i t e e x t e n s i v e  showed t h a t a p p l i c a t i o n of i s o p r o p y l e s t e r o f 2,4-D  used  experiment  consistently  i n c r e a s e d y i e l d o f wheat and b a r l e y a t both the l o c a t i o n s . Highest c e r e a l y i e l d s were o b t a i n e d when the i s o p r o p y l e s t e r of  2,4-D was used i n combination w i t h e i t h e r F e S 0  4  o r FeEDDHA.  The i n v e s t i g a t o r s concluded t h a t these r e s u l t s , together w i t h those w i t h beans, p r o v i d e support to the view t h a t i r o n a d d i t i v e s do e f f e c t the response o f p l a n t s i n the f i e l d i n the same manner as r e p o r t e d i n green house t r i a l s , earlier.  to 2,4-D  mentioned  The r e s u l t s a l s o p r o v i d e d support f o r the f i n d i n g s o f  13.  Wedding e t a l . (1956) and mulation the  of  2,4-D  Wort  u s e d , and  time o f treatment,  (1962, 1964a) t h a t  the  the  stage of growth o f  are  important  has  also obtained  for-  the  f a c t o r s i n the  plant  at  yield  response. Wort  (1959, 1966)  s u g a r b e e t s , m a i z e , and containing further  potatoes by  s u i t a b l y low  i n c l u s i o n of  augmented t h e  yield  taining  with  2,4-D  the  concentrations  increases  still  application foliage The  of  of the  the  and  Cu,  Zn,  further. Clay  The  f o l i a g e of  dust  containing  month o l d s e e d l i n g s  2,4-D, was  was  at the Cu,  B,  Mo  conalone  in  the  obtained 2,4-D  Mn,  12  Zn,  by to  the the  l b per and  stimulatory  Fe,  acre. and  action  hormone.  Limited  trials  conducted by  5%  2,4-D-mineral dust  in  an  increase Danpatatas  o f 3,  Wort  a p p l i e d a t 12 6,  and  10%  (1962) r e p o r t e d  commercial  formulation  Denmark b y  the  o f 2,4-D  (1959) w i t h  l b per  i n maize  acre,  commercial resulted  grains.  r e s u l t s of a p p l i c a t i o n of and  D a n i s h P r o d u c e r s ' and  in  sugarbeet  rate of  f o u n d t o augment t h e  The  and  dusts  of  dusts  o r 2,4-D  0.1%  B,  and iron.  largest increase  incorporation of micronutrients,  S, w i t h  2,4-D  s u g a r b e e t p l a n t s , 44%,  of a composite  o f one  of  in yield  sprays  a complex o f m i c r o n u t r i e n t s ,  saccharifera)seedlings.  root production  foliar  s u c h m i c r o e l e m e n t s as  some i n s t a n c e s , were a p p l i e d t o t h e (Beta  increases  micronutrient Exporters'  dusts  Potato  in Union.  14. Seven v a r i e t i e s of potato p l a n t s were t r e a t e d i n f i e l d s of 18 or more farmers.  Observations were made on t o t a l tuber y i e l d , number  of s e e d - s i z e potatoes, and the i n c i d e n c e of scab s c a b i e s ) , and r h i z o c t o n i a  (Corticum s o l a n i ) .  (Streptomyces  The r e s u l t s showed  t h a t i n 21 out of a t o t a l o f 22 p l o t s , treatment w i t h 2,4-Dm i n e r a l dust i n c r e a s e d y i e l d of tubers, and i n 18 o f 22 there was  cases  an i n c r e a s e i n the number of tubers of the s e e d - s i z e .  In most cases, tubers from t r e a t e d p l a n t s were found to be more r e s i s t a n t to the d i s e a s e s . Grainger  (1962) a p p l i e d commercial 2,4-D-mineral dust  (NCL)  i n S c o t l a n d on p l o t s of potatoes t h a t had been p r e t r e a t e d w i t h standard main crop f e r t i l i z e r a t the r a t e of 0, 5, 10, 20 30 B r i t i s h cwt per acre.  and  He summarized h i s f i n d i n g s as f o l l o w s :  "The NCL-100 gave i n c r e a s e d t o t a l y i e l d s on the u n f e r t i l i z e d crop, and t h a t o f 5 cwt per acre. u n t r e a t e d p l o t s at 10 cwt,  Y i e l d was  about equal to t h a t of  and s l i g h t l y l e s s a t 20 and 30  B l i g h t on the f o l i a g e of p l o t s t r e a t e d w i t h NCL-100 was  a  cwt. little  lower i n amount than t h a t on u n t r e a t e d p l o t s . " S i m i l a r l a r g e s c a l e t r i a l s on potatoes w i t h 2,4-D-mineral dust a p p l i c a t i o n have y i e l d e d s i g n i f i c a n t i n c r e a s e s i n Russett Burbank potatoes.  Six v a r i e t i e s of the commercial dust  for-  mulations were t r i e d , and i n each case t r e a t e d p l a n t s o u t y i e l d e d the u n t r e a t e d c o n t r o l s (J.R. Simplot Co.,  1960,  1961).  15. B.  E f f e c t s on p l a n t 1.  composition  M o i s t u r e content and  i t s loss:  Changes i n the moisture content are the v i s i b l e symptoms of hormone a c t i o n on the p l a n t . are r e f l e c t i v e of a l t e r e d p e r m e a b i l i t y or changed chemical composition.  These changes  of the c e l l  Both i n c r e a s e  The  decrease i n  have been r e -  response has been found to v a r y w i t h the con-  c e n t r a t i o n of the hormone, the s p e c i e s , the p l a n t .  membrane  and  moisture percentage of p l a n t s t r e a t e d w i t h 2,4-D ported.  first  and w i t h the organ of  In most cases, t r e a t e d p l a n t s possess an  c a p a c i t y o f water r e t e n t i o n .  Opinions d i f f e r as to  mechanism of the e f f e c t o f 2,4-D  increased the  on the water balance of  the  plant. Brown (1946) r e p o r t e d w i t h 2,4-D in  had  t h a t bean p l a n t s  a lower water content i n the l e a v e s , but  the stems. In a 5-day p e r i o d f o l l o w i n g 2,4-D  water t r a n s p i r e d by l o s t by u n t r e a t e d  the t r e a t e d p l a n t s was  plants.  34%  much as 2 5 to 40% w i t h i n An nasturtium and  Potapova (1959) oat p l a n t s w i t h  sprays reduced t r a n s p i r a t i o n as  7 days.  initial  increase  i n moisture content of  soybean p l a n t s t r e a t e d w i t h low  c e n t r a t i o n s , has been noted by  the  l e s s than t h a t  S i m i l a r l y , R a k i t i n and  aqueous 2,4-D  higher  application,  found t h a t treatment o f 10-day o l d sunflower and 0.075 to 0.15%  treated  2,4-D  F r e i b e r g and C l a r k  con-  (1952).  16.  Higher c o n c e n t r a t i o n s this i n i t i a l  of the chemical, however, f a i l e d to produce  r i s e i n water content of the p l a n t s .  vestigators also reported water a b s o r p t i o n by  t h a t the treatment reduced a c t i v e  by r o o t s as evidenced by  t r e a t e d corn p l a n t s , and  stumps.  This i n i t i a l  These i n -  the l a c k of  absence of b l e e d i n g  increase  guttation  from t h e i r  i n water content f o l l o w i n g hor-  mone treatment was  a l s o observed i n the i n v e s t i g a t i o n s of  M i t c h e l l and Marth  (1950).  chlorphenoxyacetic a c i d acid  was  There was and  and  2,4-,5-tri-  stems of bean p l a n t s  found to i n c r e a s e  treated plants during  ppm  (2,4,5-T), or 4-chlorophenoxyacetic  (4-CPA) to the leaves  to harvest,  A p p l i c a t i o n of 100  the f i r s t  the water content of  1.5  An opposite  prior  the  hours subsequent to  also a s i g n i f i c a n t reduction  f r u i t s of the t r e a t e d p l a n t s  just  harvest.  i n water l o s s from  during  e f f e c t was  leaves  storage. observed i n buck-  wheat p l a n t s t r e a t e d w i t h s u b l e t h a l sprays of 2,4-D  of  concentrations  following  (Wort, 1951).  treatment there was  In the f i r s t  12 hours  a d e c l i n e i n the moisture percentage of  t r e a t e d , as compared to the c o n t r o l p l a n t s . f o l l o w e d by  a steady i n c r e a s e  so t h a t by  treatment p l a n t s t r e a t e d w i t h 50 to 1000 to h o l d between 103  various  and  138%  This was  then  the e i g h t h day ppm  2,4-D  the  after  were found  moisture percentage of the  control  plants. There i s a dearth of l i t e r a t u r e on changes  i n water content o f p l a n t s t r e a t e d w i t h 2,4-D-minerals. o n l y a v a i l a b l e r e p o r t of t h i s nature i s by Wort  The  (1962).  Burbank Russet potato p l a n t s were t r e a t e d w i t h a commercial dust f o r m u l a t i o n c o n t a i n i n g 1.25%  2,4-D  a t the r a t e o f 6 l b per  acre when s t o l o n s were s t a r t i n g to s w e l l i n t o t u b e r s , and 2 to 3 weeks l a t e r .  The  again  tubers h a r v e s t e d from t r e a t e d p l a n t s  were found to l o s e o n l y one-half as much water as d i d tubers from the u n t r e a t e d c o n t r o l p l a n t s d u r i n g 3 months' storage a t 55° F (13° C). S e v e r a l suggestions have been advanced to e x p l a i n i n c r e a s e d r e t e n t i o n o f water by 2,4-D  treated plants  and organs.  (1952),  A c c o r d i n g to Bradbury and Ennis  t h i s reduced l o s s of water i s due to p a r t i a l c l o s u r e of stomata under the i n f l u e n c e of 2,4-D.  Experimental  support f o r such a  view came from o b s e r v a t i o n t h a t leaves of bean p l a n t s t r e a t e d w i t h 2,4-D of 2,4-D  show p a r t i a l l y c l o s e d stomata. (10 to 1000  process.  The  ppm)  Higher c o n c e n t r a t i o n s  even i n t e r f e r e d w i t h the  authors a t t r i b u t e d t h i s to a d i r e c t e f f e c t of  on the stomatal apparatus.  a c t i v i t y of phosphorylase application  decrease  and amylase of stomatal c e l l s  (Maciejewska-Potapczyk,  p o s s i b l e t h a t stomatal c l o s u r e may i n CO2  2,4-D  Wort (1964), on the other hand, has  made the s u g g e s t i o n t h a t i n view of the observed  2,4-D  reopening  1955)  i n the  by  i t seems q u i t e  be the r e s u l t of an i n c r e a s e  c o n c e n t r a t i o n of the l e a f c e l l s brought  about by  the  18. primary e f f e c t o f 2,4-D on p h o t o s y n t h e s i s . I t has a l s o been proposed t h a t the i n c r e a s e d absorption  and r e t e n t i o n o f water by the 2,4-D-treated  c o u l d be a consequence o f an i n c r e a s e d  plants  osmotic p r e s s u r e o f  c e l l s , r e s u l t i n g from h y d r o l y s i s o f nitrogenous compounds and carbohydrates, or from an accumulation o f m i n e r a l elements. Such an osmotic r e t e n t i o n o f water i s apparent i n the case o f banana f r u i t s dipped i n 2,4-D s o l u t i o n s  ( F r e i b e r g , 1955).  In  t h i s case, a s i g n i f i c a n t decrease i n water l o s s was accompanied by  a p a r a l l e l increase 2.  i n the sugar content o f the t r e a t e d  fruits.  Pigments Chlorophylls Application of i n d i v i d u a l droplets  i n g 2,4-D to Euonymus leaves  contain-  caused no change i n green c o l o r o f  the t i s s u e immediately under the d r o p l e t , b u t r e s u l t e d i n c h l o r o s i s o f the surrounding t i s s u e The  (Osborne and Hallaway, 1961).  e f f e c t o f 2,4-D on c h l o r o p h y l l content  i n two v a r i e t i e s o f maize and i n f l a x was i n v e s t i g a t e d by Mashtakov and Paromchik  (1966).  of 5 or 6 leaves by a p p l y i n g  Maize was t r e a t e d a t the stage  the sodium s a l t o f 2,4-D to the  s o i l a t the r a t e o f 9.5 Kgm per h e c t a r e ,  and f l a x was sprayed  w i t h 0.04% s o l u t i o n o f the sodium s a l t o f 2,4-D a t the r a t e o f 1.5 Kgm per hectare, The  when the p l a n t s were i n 6 to 7 l e a f - s t a g e .  r e s u l t s o f t h i s experiment showed t h a t although the t o t a l  19. c h l o r o p h y l l was  not  changed, the  p e t r o l e u m e t h e r was treatment  d e c r e a s e d the  whereas i n the  2,4-D  on  the  stability  by  the  of  exactly  activity  and  of  ions  work o f  Tullin  possibly  the of  synthesis  2-4-D  and  Mg  indicate  of  2-4-D.  the  the  t h a t Mg  was  Other  that  Bliss  potatoes  o b t a i n e d by  the  translocated  complex  2,4-D  interacts  i n f e r r e d from the  coleoptile  latter  This  has  i n s p i t e of  the  endo-  effect  been  the  was  When  same g r o w t h r e s p o n s e  and  Schaal  (1948) and  Triumph p o t a t o p l a n t s  Fults  supported these  The  effects  was  thought  presence  pigments  produced tubers with s k i n of results  be  found  photo-  injections into  remained green.  Fults reported  these  a 2,4-D-Mg complex.  together,  plants  complex,  reaction.  almost doubled the  formation of  were g i v e n  obtained, but to  may  (1962) i n w h i c h 2,4-D  to  Hill  applied  the  s t a b i l i t y was  e f f e c t s on  the  i n d u c e d c h l o r o t i c symptoms.  due  the  Furthermore,  to  productivity  sperm o f y o u n g wheat s e e d l i n g growth, but  In maize,  chlorophyll-p>rotein-lipid  parallel  in chlorophyll  with  chlorophyll  flax,  That e x t e r n a l l y w i t h Mg  species. the  treatment.  stability  were f o u n d t o be synthetic  i n both  r e s i s t a n t v a r i e t y of  to have i n c r e a s e d of  increased  amount e x t r a c t a b l e  and  an  treated with  unusually red  Payne  findings.  Ellis  color.  (1955) w i t h Red  (1949) 2,4-D Later,  McClure  20. S l i c e s o f Jerusalem  artichoke  (Helianthus  tuberous) incubated on f i l t e r papers impregnated w i t h 10 per l i t e r of 2,4-D No r e d c o l o r was crushed,  produced a r e d pigment i n the v a s c u l a r t i s s u e s . produced by 2,4-D  and i t s p r o d u c t i o n was  M arsenite.  sugarbeet  i f the t i s s u e were ground or  a l s o e f f e c t i v e l y stopped by  Treatment of stem s e c t i o n s of  and Xanthium strumarium with 2,4-D but i t was  mg  tuberous,  H.  lxlO ^ -  annus,  a l s o produced the pigment,  not formed i n s e c t i o n s from white potato p e t i o l e s and r o o t s , and tobacco  tubers,  stems (Swanson e t a l .  1956). 3.  Vitamins Most of the known vitamins are s y n t h e s i z e d i n  green p l a n t s , which are, by f a r , the primary  source o f  these  compounds. In a d d i t i o n , even a minute change i n the v i t a m i n content of the p l a n t may  be expected  to have profound  on the e n t i r e metabolism, and t h e r e f o r e on the of  the p l a n t , as the v i t a m i n s are now  v i t a l f u n c t i o n s i n the p l a n t c e l l  influence  growth and  known to perform  yield  the same  as they do i n the animal  cell.  Wittwer and Murneek (1946) a p p l i e d i n d o l e - 3 acetic acid in  (IAA), p-chlorophenoxyacetic  acid  (CPA)  low c o n c e n t r a t i o n s , i n the form of sprays and  and  dusts, to snap  beans a t weekly i n t e r v a l s d u r i n g the time o f f l o w e r i n g . obtained i n c r e a s e d y i e l d and a l s o found  They  t h a t the pods from the  t r e a t e d v i n e s were somewhat darker green and t h e i r v i t a m i n C content.  2,4-D  s l i g h t l y higher i n  The b e s t s t i m u l a t o r y c o n c e n t r a t i o n  found was  2 ppm.  On the other hand, Randhawa and Thompson  (1948) f a i l e d to d e t e c t any s i g n i f i c a n t d i f f e r e n c e s i n a s c o r b i c a c i d contents of the pods from p l a n t s t r e a t e d w i t h 4 chlorophenoxypropionic  a c i d and  CPA.  A p p l i c a t i o n of 1 drop s o l u t i o n of 2,4-D  ppm  (0.05 ml) o f  0.1%  to the base of the b l a d e of one of the  leaves of r e d kidney bean p l a n t s was  found to decrease  primary  the  content o f thiamine, r i b o f l a v i n and n i c o t i n i c a c i d i n the l e a v e s , t e s t e d 6 days a f t e r the treatment. v i t a m i n s had i n c r e a s e d i n the stems. t h i n i c a c i d was  A l l the three  The content of  a l s o g r e a t e r , but carotene was  panto-  l e s s i n both  stems and leaves of the t r e a t e d p l a n t s (Luecke e t a l . 1949). M i t c h e l l e t a l . (1949) determined o f p r e - h a r v e s t treatment of bean pods w i t h 2,4-D C content d u r i n g storage.  the  effect  on t h e i r v i t a m i n  At the time when o n e - t h i r d of the pods  were o f commercial s i z e , they were sprayed w i t h x y a c e t i c a c i d p l u s 1% Tween-20.  p-chloropheno-  Four days a f t e r the a p p l i c a t i o n  no s i g n i f i c a n t d i f f e r e n c e s c o u l d be d e t e c t e d between t r e a t e d and u n t r e a t e d f r u i t s . sprayed w i t h 400 ppm  In l a t e r experiments CPA.  o n l y the pods were  A l l the f r u i t s o f marketable  were h a r v e s t e d 4 days a f t e r the treatment  size  and spread out i n a  l a y e r one to 2 pods t h i c k , i n d i f f u s e l i g h t a t 74 to 75°F (24°C).  Six to 10 r e p l i c a t e s were analyzed a t i n t e r v a l s f o r  t h e i r a s c o r b i c a c i d content by the method o f L o e f f l e r  and  Ponting  (1942).  The r e s u l t s o f t h i s greenhouse t r i a l showed  t h a t 4 days a f t e r the treatment, u n t r e a t e d l o s t 37.5% o f the o r i g i n a l  ( p r i o r t o spray) a s c o r b i c a c i d , b u t  the t r e a t e d pods l o s t o n l y 11.7%. same type,  In a second t e s t o f the  the l o s s by the c o n t r o l and t r e a t e d pods was found  to be 42% and 18%, r e s p e c t i v e l y . field  c o n t r o l pods had  Extension  experiments showed an even g r e a t e r  o f the t r i a l to  protection of ascorbic  a c i d by the treatment, the pods l o s i n g o n l y 3.6% as a g a i n s t 31.8%  l o s t by the c o n t r o l pods i n 4 days. Wort  (1950) i n v e s t i g a t e d the i n f l u e n c e o f  2,4-D sprays on v i t a m i n  C ( a s c o r b i c acid) content o f buckwheat  (Fagopyrum esculentum), and o f bushbean  (Phaseolus v u l g a r i s ) .  Of the s e v e r a l doses o f 2,4-D spray a p p l i e d to one-month-old buckwheat p l a n t s , o n l y the s m a l l e s t dose, 10 ;ag per p l a n t , r e s u l t e d i n an i n c r e a s e d The  higher  a s c o r b i c a c i d content o f the l e a v e s .  concentrations  up to 1000 ug per p l a n t produced an  i n i t i a l r i s e o f v i t a m i n C i n the leaves, b u t the l e v e l showed a sharp d e c l i n e w i t h time. i n c r e a s e o f v i t a m i n C.  In stems, there was a continuous  In bean p l a n t s , a spray o f 500 ppm  2,4-D brought about a r e d u c t i o n  i n vitamin  C content o f both  leaves and stems over an 11-day t e s t p e r i o d . Key old  and Wold (1961) experimented w i t h 2.5 days  s e e d l i n g s o f soybean t r e a t e d w i t h 5 x l 0 ^ M potassium  of 2,4-D.  -  Within  salt  6 hours o f the treatment, a s c o r b i c a c i d had  i n c r e a s e d markedly i n the t r e a t e d s e e d l i n g s , taut i t had decreased i n the c o n t r o l p l a n t s . 2,4-D  These i n v e s t i g a t o r s concluded t h a t  had caused a s h i f t towards a more reduced s t a t e of  ascortaic a c i d , p r o t e i n , s o l u b l e SH compounds and p y r i d i n e nucleotides,  accompanied by a net i n c r e a s e i n the  concentration  o f the t o t a l a s c o r b i c a c i d and p y r i d i n e n u c l e o t i d e s .  The  r e l a t i o n s h i p between 2,4-D-induced growth and changes i n the content o f a s c o r b i c a c i d and dehydroascorbic a c i d s i n e x c i s e d soybean, corn, and Key  cucumber, and pea t i s s u e s was s t u d i e d by L i n  (1967).  stimulatory  The r e s u l t s i n d i c a t e d that the growth-  concentration  of auxin  d i d not b r i n g about any  s h i f t i n the a s c o r b i c to dehydroascorbic a c i d r a t i o .  The r a t i o  c o u l d be experimentally i n c r e a s e d without a f f e c t i n g the growth rate.  I n h i b i t i o n o f growth by supra-optimal auxin  concentrations  was a s s o c i a t e d w i t h a decrease i n the a s c o r b i c a c i d to dehydroascorbic a c i d r a t i o .  However, EDTA, which d i d not  a f f e c t growth r a t e , c o u l d a l s o lower the a s c o r b i c to dehydroascorbic a c i d r a t i o .  Therefore,  any c a u s a l r e l a t i o n s h i p  between a s c o r b i c a c i d system and auxin r e g u l a t i o n of c e l l e l o n g a t i o n was  thought to be u n l i k e l y . On the other  hand, P a t e l and Suthar  (1967)  p r e s e n t e d data showing t h a t auxin and g i b b e r e l l i n s f u n c t i o n i n the growth process by c a t a l y z i n g the b i o s y n t h e s i s of a s c o r b i c acid;  that a s c o r b i c a c i d not o n l y p a r t i c i p a t e s i n the a c t i v a t i o n  24.  of v a r i o u s enzyme systems, but a l s o s t i m u l a t e s the p r o d u c t i o n o f ATP by a c t i n g as an e l e c t r o n donor i n p h o t o s y n t h e t i c and o x i d a t i v e p h o s p h o r y l a t i o n ; and t h a t the above a c t i o n s of asc o r b i c a c i d c r e a t e a f a v o r a b l e b a l a n c e f o r s y n t h e s i s of n u c l e i c a c i d s , thus e n a b l i n g the process of c e l l d i v i s i o n and e n l a r g e ment to proceed a t a f a s t C.  E f f e c t s on 1.  rate.  metabolism Carbohydrate  metabolism  Carbohydrate  content:  L i t e r a t u r e p e r t a i n i n g to 2,4-D  effects  on the carbohydrate content of p l a n t s has been d i s c u s s e d by Blackman e t a l . (1951), Audus (1953), and Wort (1962, 1964). A survey of the e f f e c t s o f c h l o r i n a t e d phenoxyacetic  a c i d s on  s p e c i f i c carbohydrate f r a c t i o n s of bean p l a n t s was p u b l i s h e d by S e l l e t a l . (1957).  An examination of these reviews r e v e a l s  t h a t one of the most marked e f f e c t s o f 2,4-D  a p p l i c a t i o n to  such p l a n t s as bindweed  (Convolvulus a r v e n s i s ) , buckwheat  (Fagopyrum esculentum),  d a n d e l i o n (Taraxacum o f f i c i n a l e ) ,  bean  (Phaseolus v u l g a r i s ) and morning g l o r y  kidney  (Convolvulus sepium)  i s the r a p i d d e p l e t i o n of carbohydrate food r e s e r v e s , s t a r c h  and  sugars. In r e d kidney bean p l a n t s , a p p l i c a t i o n of 1000  ppm  of 2,4-D  ( S e l l e t a l . 1949;  Weller e t a l . 1950)  the f o l l o w i n g p a t t e r n 6 days a f t e r the  treatment:  showed  Stems: d e p l e t i o n of r e d u c i n g and non-reducing of  sugars; r e d u c t i o n  s t a r c h , crude f i b e r s and p o l y s a c c h a r i d e s .  Roots and Leaves:  d e p l e t i o n o f non-reducing in  sugars, no change  r e d u c i n g sugars, s t a r c h , p o l y s a c c h a r i d e s  and crude f i b e r c o n t e n t s . Wort (1951) i n v e s t i g a t e d the changes i n carbohydrate contents of 1 - f o o t - h i g h buckwheat ( F ^ esculentum) p l a n t s t r e a t e d w i t h 50, 100, sprays.  500 and 1000  ppm  2,4-D  as  foliar  Changes i n the sugar and s t a r c h contents o f the l e a v e s ,  stems and r o o t s were f o l l o w e d a t 0.5, a p p l i c a t i o n of 2,4-D.  1,2,4, and 8 days a f t e r  the  The r e s u l t s showed an i n c r e a s e i n the  sugar content of stems and l e a v e s o f the t r e a t e d p l a n t s w i t h i n 1 day o f the a p p l i c a t i o n of the chemical, but i t f e l l  as low as  48% o f the c o n t r o l p l a n t s by the e i g h t h day a f t e r the spray. In  the r o o t s , a continuous d e p l e t i o n of the carbohydrates  recorded. of  was  The s t a r c h - d e x t r i n f r a c t i o n i n stems f e l l below t h a t  the c o n t r o l l e v e l immediately  f o l l o w i n g the treatments, showed  a steep r i s e above the c o n t r o l and a g a i n d e c l i n e d r e a c h i n g a l e v e l of almost complete in  exhaustion by end of the e i g h t h day  those p l a n t s which had r e c e i v e d 1000  ppm  of the chemical.  A s i m i l a r t r e n d of carbohydrate d e p l e t i o n f o l l o w i n g 2,4-D  treat-  ment has been r e p o r t e d i n s e v e r a l other cases i n c l u d i n g morning glory  (Ipomoea lacunosa)  ( M i t c h e l l and Brown, 1945), bindweed  (Smith e t a l . 1947), d a n d e l i o n (Rasmussen, 1947), bean (Brown,  1946) and  rape ( B r a s s i c a napus) and broad bean ( V i c i a faba) (Hofmann  Schmeling,  1953). There are, n e v e r t h e l e s s ,  i s o l a t e d reports  of i n c r e a s e d carbohydrate content brought about by 2,4-D on p l a n t s .  action  Thus, on t h e i r s t u d i e s w i t h the a p p l i c a t i o n o f  0.002 to 0.04  mg  o f 2,4-D  to one  l e a f o f 1-month-old c o t t o n  p l a n t s , E r g l e and Dunlap (1949) r e p o r t e d i n c r e a s e d contents  of  sucrose, h e m i c e l l u l o s e s and c e l l u l o s e s i n the f o l i a g e from the t r e a t e d p l a n t s , as measured about 2 months l a t e r .  Reducing  sugars tended to keep an i n v e r s e r e l a t i o n s h i p w i t h the  2,4-D  doses w h i l e t o t a l sugars d i d not show any d i f f e r e n c e from t h a t i n the c o n t r o l p l a n t s .  Leaf s t a r c h was  reduced, but o n l y by  the h i g h e s t dosage of 2,4-D, to as low as 40% of the c o n t r o l value.  The  w i t h every  sum 2,4-D  of a l l these carbohydrates dose and the i n c r e a s e was  to the amount of 2,4-D  directly proportional  a p p l i e d to the s e e d l i n g .  i n the l e v e l of carbohydrates e t a l . (1950).  f r a c t i o n s increased  by 2,4-D  A similar  has been r e p o r t e d by Wolf  Soybean p l a n t s were grown i n n u t r i e n t s o l u t i o n s  of v a r i o u s n i t r o g e n l e v e l s , each b e i n g supplemented w i t h 20 2,4-D, except the c o n t r o l s . the 2,4-D  additions.  t o t a l and r e d u c i n g Hemicellulose  rise  P l a n t s were analyzed  In g e n e r a l , the s t a r c h was  ppm  14 days a f t e r lower  and  sugars h i g h e r i n the 2,4-D-supplied p l a n t s .  content was  i n c r e a s e d to almost 143% o f  the  c o n t r o l i n the p l a n t s r a i s e d i n medium-N s o l u t i o n supplemented  w i t h 2,4-D. A s e r i e s o f i n v e s t i g a t i o n s has been undertaken t o study the e f f e c t s o f 2,4-D and other s y n t h e t i c auxins on sucrose metabolism i n sugarcane.  Beauchamp (1950), who  o b t a i n e d an i n c r e a s e d sugar content o f canes by treatment 2,4-D and m a l e i c h y d r a z i d e on t h i s aspect.  with  (MH), has d i s c u s s e d the l i t e r a t u r e  H a l l and Khan (1955), and C h a k r a v a r t i e t a l .  (1955) a l s o r e p o r t e d i n c r e a s e d sucrose and decreased sugar contents o f the sap by treatment 2,4-D and other compounds.  o f the sugarcanes  with  Lugo-Lopez e t a l . (1953) c o u l d ,  however, f i n d no s i g n i f i c a n t d i f f e r e n c e i n the sugar when l e a v e s o f sugarcane  reducing  content  were g i v e n a s i n g l e a p p l i c a t i o n o f  v a r i o u s c o n c e n t r a t i o n s o f 2,4-D and MH. Alexander sugarcane  (1965) sprayed 10-week-old  p l a n t s w i t h IAA (0.175 p e r c e n t ) , 2,4-D (0.2%) and  0.15 percent MH s o l u t i o n s .  Sugar a n a l y s i s o f leaves 1,3,9, and  2 7 days f o l l o w i n g spray a p p l i c a t i o n showed maximum  sucrose,  f r u c t o s e and t o t a l r e d u c i n g sugar contents 9 days a f t e r the treatments.  Sucrose was p a r t i c u l a r l y a f f e c t e d by i A A and 2,4-D,  i n c r e a s i n g by 1.6 and 1.8 times, r e s p e c t i v e l y , over the c o n t r o l v a l u e s a t 9 days a f t e r the treatments. By the 2 7th day, t h i s enhancing  e f f e c t o f IAA had p r a c t i c a l l y disappeared, and the  sucrose content was found to be lower i n the 2,4-D t r e a t e d p l a n t s as compared to the c o n t r o l s .  28. An e a r l y a p p l i c a t i o n of s u b l e t h a l doses of 2,4-D  (or MH)  to the a e r i a l p o r t i o n s , caused a s i g n i f i c a n t  decrease  i n the r e d u c i n g sugars, b u t the l a t e a p p l i c a t i o n of  the r e g u l a t o r s r e s u l t e d i n an i n c r e a s e i n sugars i n potato tubers h a r v e s t e d 4 t o 5 months l a t e r  (Payne and F u l t s , 1955).  Payne  et a l . (1953) a l s o r e p o r t e d t h a t a p p l i c a t i o n o f sodium s a l t s o f 2,4-D  as aqueous sprays to Red McClure potato p l a n t s a t the  e a r l y bloom stage, r e s u l t e d i n potatoes of h i g h e r  specific  g r a v i t y , an i n d i c a t i o n of h i g h e r s t a r c h c o n t e n t s . Other r e c e n t f i n d i n g s i n d i c a t e i n c r e a s e d t o t a l carbohydrate  contents o f dwarf beans a f t e r 2,4-D  ment ( A l g h i s i and G h i l l i n i ,  1959)  and i n c r e a s e d s t a r c h , but  c r e a s e d sugars i n e x c i s e d t i p s of potato sprouts and disappearance shoots  treatde-  (Okazawa, 1958),  o f s t a r c h from the c o r t e x and p i t h of tomato  (Frohberger, 1951)  under the i n f l u e n c e o f 2,4-D  Wightman and Neish  treatment.  (1959) noted t h a t the  14 i n c o r p o r a t i o n of  C-sucrose  i n t o the c e l l u l o s e and h e m i c e l l u l o s e  f r a c t i o n s of wheat c o l e o p t i l e s and pea stem segments was enhanced by 2,4-D.  L i t e r a t u r e on the changes i n the  activities  of the enzyme systems of carbohydrate metabolism s h a l l be sented i n a l a t e r  section.  greatly  pre-  29.  Photosynthesis; One  of the f i r s t s t u d i e s o f 2,4-D  on p h o t o s y n t h e t i c e f f i c i e n c y of bushbean p l a n t s was out by F r e e l a n d (1949). p l a n t tops w i t h b e l l  was  carried  Using the simple device of c o v e r i n g  j a r s and p a s s i n g the  C02~absorption towers,  effects  he observed t h a t  o.utcoming a i r through assimilation rate  depressed to 70% o f the o r i g i n a l r a t e 3 days a f t e r  p l a n t s were sprayed w i t h 100 ppm spp. as w e l l , 30 and 100 ppm  2,4-D  2,4-D  solution.  the  In Anacharis  sprays were found to b r i n g  about r e d u c t i o n s i n the r a t e o f a s s i m i l a t i o n , the i n h i b i t i o n b e i n g g r e a t e r a t the h i g h e r c o n c e n t r a t i o n . and  Muzik  A few years l a t e r , L o u s t a l o t  (1953) r e p o r t e d t h e i r r e s u l t s on the p h o t o s y n t h e t i c  a c t i v i t y and developmental  morphology of v e l v e t bean  ( S t i z o l o g i u m d e r r i n g i a n u m ) s e e d l i n g s sprayed w i t h an aqueous s o l u t i o n of i s o p r o p y l e s t e r o f 2,4-D of 0.001  to 0.1%.  i n the c o n c e n t r a t i o n range  Photosynthesis was  measured 5 hours a f t e r  s p r a y i n g by drawing the a i r through a porometer cup a t t a c h e d to the lower s u r f a c e o f a mature l e a f and f o r c i n g the  oncoming a i r  through a C02~absorption tower a t a constant flow r a t e f o r 3 hours.  F i v e hours a f t e r the treatment,  p h o t o s y n t h e t i c r a t e was 2,4-D.  noted i n the p l a n t s t r e a t e d w i t h  P l a n t s t r e a t e d w i t h 0.01%  r a t e of CO2  a sharp r e d u c t i o n i n the  2,4-D  a s s i m i l a t i o n a t t h i s time.  a l s o showed a The  0.1%  lowered  lower c o n c e n t r a t i o n s  showed a delayed r e d u c t i o n i n the r a t e of p h o t o s y n t h e s i s .  30. Wedding e t a l . (1954) determined q u a n t i t a t i v e e f f e c t s of 2,4-D of orange l e a v e s .  on photosynthesis  D i s c s from leaves i n f i l t e r e d w i t h  c o n c e n t r a t i o n s o f 2,4-D  was  employed to e l i m i n a t e  v a r i a t i o n s i n c o n c e n t r a t i o n o f 2,4-D  The  various  A p a r a l l e l s e r i e s of experiments  u t i l i z i n g C h l o r e l l a pyrenoidosa  w i t h i n the l e a f , and  respiration  were used to minimize p o s s i b l e e f f e c t s  of a l t e r e d t r a n s l o c a t i o n .  openings.  and  the  which might have o c c u r r e d  the e f f e c t of the chemical  r a t e of photosynthesis  the  was  on the  stomatal  measured i n terms of  O2 e v o l u t i o n at 25°C i n ah . i l l u m i n a t e d Warburg respirometer  over  a p e r i o d of 1 hour, f o l l o w e d by 1 hour measurement of r e s p i r a t i o n i n the dark. was  The  sum  of the readings  taken as t r u e r a t e of p h o t o s y n t h e s i s .  photosynthesis  by 2,4-D  was  The  i n l i g h t and  dark  i n h i b i t i o n of  found to be r e l a t e d to the con-  c e n t r a t i o n of u n d i s s o c i a t e d 2,4-D  a c i d molecules i n the s o l u t i o n  used f o r treatment, r e g a r d l e s s of whether the d i f f e r e n c e i n u n d i s s o c i a t e d a c i d c o n c e n t r a t i o n was  o b t a i n e d by v a r y i n g the  of the b u f f e r s o l u t i o n s c o n t a i n i n g a constant or by v a r y i n g the 2,4-D  amount of  added to b u f f e r s of the same pH.  phases o f i n h i b i t i o n were shown: one,  pH  2,4-D Two  i n a low range of  u n d i s s o c i a t e d a c i d c o n c e n t r a t i o n , b e i n g p r o p o r t i o n a l to the l o g of t h a t c o n c e n t r a t i o n ; the other, c o v e r i n g a higher  concentration  range, b e i n g p r o p o r t i o n a l to the c o n c e n t r a t i o n of u n d i s s o c i a t e d 2,4-D.  The  authors b e l i e v e d t h a t the l a t t e r e f f e c t  represented  a d i r e c t or i n d i r e c t d e s t r u c t i o n o f the c h l o r o p h y l l by 2,4-D. R a k i t i n and Potapova  (1959) sprayed  a 0.15% s o l u t i o n o f 2,4-D on young sunflower, o a t , and chickweed plants.  One day a f t e r the a p p l i c a t i o n o f 2,4-D, the photo-  s y n t h e t i c r a t e s i n sunflower and o a t p l a n t s were found to have -decreased by 2 7 and 64 percent, r e s p e c t i v e l y .  One week l a t e r ,  the r a t e had almost r e c o v e r e d to the l e v e l o f the c o n t r o l i n oat, b u t not i n the sunflower  plants  plants.  W i l l i a m s and Dunn (1961) t h a t b l u e l i g h t was most e f f e c t i v e i n promoting  demonstrated  2,4-D i n t e r -  ference w i t h CO2 u t i l i z a t i o n , whereas r e d l i g h t was i n e f f e c t i v e . Since r e d l i g h t was found t o be most e f f e c t i v e i n r e d u c i n g c h l o r o p h y l l content i n combination w i t h 2,4-D, the observed growth r e p r e s s i o n  appeared  t o r e s u l t from lowered  chlorophyll  contents and consequently lowered p h o t o s y n t h e t i c e f f i c i e n c y . Huffaker e t a l . (1962) sprayed 2-weeko l d bushbean p l a n t s w i t h 0.5, 1, 10, and 100 ppm 2,4-D w i t h and without F e S 0 , i r o n c h e l a t e 4  (EDDHA) supplements,  (FeEDDHA) and c h e l a t i n g  to determine  agent  the e f f e c t s o f these  treatments  14 on the two dark genates  CO2 f i x a t i o n r e a c t i o n systems i n c e l l - f r e e homo-  from the t r e a t e d p l a n t s .  ( c a t a l y z e d by phosphoenol  pyruvic  5-phosphate r e a c t i o n system phoriboisomerase,  The PEP c a r b o x y l a s e  system  carboxylase) and the R i b u l o s e -  ( c a t a l y z e d i n sequence by phos-  phosphoribulose k i n a s e and r i b u l o s e  diphosphate  c a r b o x y l a s e enzymes) showed a c o n s i d e r a b l y i n c r e a s e d a c t i v i t y i n both the primary and the f i r s t mature t r i f o l i a t e leaves a t the growth s t i m u l a t o r y l e v e l s o f 2,4-D, both w i t h and without c h e l ated i r o n .  Assays made soon a f t e r the treatments w i t h both'  2,4-D and c h e l a t e d i r o n alone, or i n combination, showed i n creased a c t i v i t y o f the R-5-P r e a c t i o n system.  When a p p l i e d  together, a g r e a t e r s t i m u l a t i o n was observed than w i t h e i t h e r alone.  These s t i m u l a t o r y e f f e c t s on both enzyme systems tended  to fade a f t e r about 12 days, b u t c o u l d be maintained a t the h i g h e r l e v e l s p r o v i d e d EDDHA o r FeEDDHA was a p p l i e d together w i t h 2,4-D.  The i n v e s t i g a t o r s proposed t h a t t h i s i n c r e a s e i n CO2  f i x a t i o n c o u l d be important i n p r o v i d i n g b o t h sugars and o r g a n i c a c i d s and thereby r e s u l t i n an i n c r e a s e d m e t a b o l i c s t a t u s and growth o f the t r e a t e d p l a n t s . Respiration Reviews on 2,4-D e f f e c t s on p l a n t r e s p i r a t i o n c o v e r i n g l i t e r a t u r e up t o 1950 were w r i t t e n by Avery (1951) and Smith  (1951).  Recent  l i t e r a t u r e on r e s p i r a t o r y  aspects o f auxin a c t i o n has been d i s c u s s e d by Audus Cleland  (1959),  (1961), Wort (1964) and H i l t o n e t a l . (1963). S e v e r a l examples o f s t i m u l a t i o n o f p l a n t  r e s p i r a t i o n by 2,4-D have been c i t e d by Avery those i n r i p e n i n g pears (Southwick,  (1951),  including  (Hansen, 1946), apple and peach  1946), rhizomes and r o o t s o f bindweed  fruits  (Smith e t a l .  1947)  and i n t a c t p l a n t s o f beans and morning g l o r y  Rasmussen a l s o r e p o r t e d  a 92% i n c r e a s e  i n r e s p i r a t i o n o f Tara-  xacum o f f i c i n a l e r o o t s consequent t o s p r a y i n g 480  (Brown, 1946).  the p l a n t s w i t h  ppm 2,4-D s o l u t i o n . Smith (1948) a p p l i e d 0.1% 2,4-D as  2 drops on the base o f each o f the primary l e a f b l a d e s o f bean plants.  The i n t e r n o d a l t i s s u e was subsequently c u t i n t o t h i n  s l i c e s and t h e i r r e s p i r a t o r y a c t i v i t y was measured manometrically. The  s l i c e s from t r e a t e d p l a n t s showed a s i g n i f i c a n t l y h i g h e r  r e s p i r a t o r y a c t i v i t y than those from untreated the maximum r a t e i n 7 days.  Freeland's d e t e r m i n a t i o n  o f r e s p i r a t o r y a c t i v i t y o f bean leaves showed a 70% r e d u c t i o n and  an i n c r e a s e  stems,  reaching (1940)  sprayed w i t h 0.1% 2,4-D  i n the r e s p i r a t o r y r a t e on the f i r s t day  to 110% o f the c o n t r o l by the 3 r d day a f t e r the  treatment. Corns  (1950) i n v e s t i g a t e d the e f f e c t s  o f 50 and 1000 ppm 2,4-D and s o i l moisture on r e s p i r a t i o n , c a t a l a s e a c t i v i t y and p r o t e i n contents o f bean p l a n t s .  The r e -  s u l t s o f t h i s i n v e s t i g a t i o n emphasized t h a t s t i m u l a t i o n or i n h i b i t i o n o f r e s p i r a t i o n i s much dependent on 2,4-D  concentration,  s o i l moisture and the time elapsed between the treatment and the measurement. Wedding e t a l . (1954), studying the e f f e c t s o f 2,4-D, made the d i s c o v e r y  t h a t the same  concentration  34. o f 2,4-D  had  different  of photosynthesis were i n h i b i t o r y piration,  and  intermediate  and  quantitative effects respiration.  The  for photosynthesis  had  with  phosphorylative  both  o x i d a t i o n and  2,4-D  little  but  isolated  the  an  and  phosphate uptake  t i s s u e was  (Stevens  decreases  were d e t e c t e d  observed  e t a l . 1962).  (Khubatiya,  i n the  an  an  f r o m 2,4-D  growth o f t i s s u e  increase i n mitochondrial  effect  catabolism.  1957).  2,4-D  o f 2,4-D  acetate  treatment  noted  i n pea  (Key  root 1960)  phosphorylation  treated plants.  Key and  mito-  isolated  latter investigators  induced  by  2,4-D  involves  activity.  in shifting  Humphreys and  (Fang  et a l .  e l e c t r o n micrographs of mitochondria  t h a t the  in  associated swelling of tissue  The  in  inhibited  uptake o f  in oxidative  Recently, the  plants  (Switzer,  after  res-  soybean  a d d i t i o n o f 2,4-D  u p t a k e was  from t r e a t e d soybean h y p o c o t y l s . suggested  from  Both i n c r e a s e s  1959)  i n mitochondria  e t a l . (1960) o b s e r v e d chondria  on  increased oxidative  from u n t r e a t e d  a l . 1961), whereas i n h i b i t e d  tissues  that  concentration.  Enhanced m e t a b o l i c  et  effect  isolated  possessed  activity,  to mitochondria  by bean shoot  processes  concentrations  ranges of u n d i s s o c i a t e d a c i d  seedlings sprayed  vitro  the  t e n d e d t o show s t i m u l a t i o n o f r e s p i r a t i o n  Mitochondria  and  on  e m p h a s i s has  the pathways o f  Dugger  been p l a c e d  on  carbohydrate  (1959) c o n c l u d e d  from  their  s t u d i e s t h a t 2,4-D phate c y c l e . Black and  i n c r e a s e s r e s p i r a t i o n v i a the pentose phos-  T h i s view i s strengthened by the f i n d i n g s of  Humphreys  (1962) who  reported  t h a t a d d i t i o n of  to e t i o l a t e d corn s e e d l i n g s  increased  v i a pentose phosphate c y c l e  (as evidenced by  2,4-D  the u t i l i z a t i o n of glucose the  increased  a c t i v i t i e s o f enzymes c a t a l y z i n g the pathway) and  decreased i t  v i a the g l y c o l y t i c pathway.  the same  r e l a t i v e e f f e c t s o f 2,4-D sorghum s e e d l i n g s  and  Reed (1961) r e p o r t e d  on the two  pathways i n e t i o l a t e d  carrot discs.  The  latter investigator  a l s o observed t h a t the r e s p i r a t o r y r a t e s and r e l a t i v e ions of the two  pathways were markedly i n f l u e n c e d by  contributthe nature  of the c a t i o n s i n the r e s p i r a t o r y media. In c o n t r a s t , i t has been claimed the r a t i o of r e s p i r a t o r y "^CC^ 14 to t h a t from g l u c o s e - 1 concentrations.  C,  recovered  from glucose-6-"'"^C  . . increased with increasing  However, 2,4-D  was  that  found to have no  2,4-D such an  14 e f f e c t on  C d i s t r i b u t i o n i n ethanol  e x t r a c t s or  residues  (Fang et a l . 1960). 2.  Nitrogen  metabolism  Extensive r e l a t i o n s h i p between auxin plants.  The  present  the  a c t i o n and n i t r o g e n metabolism i n  l i t e r a t u r e review i s l i m i t e d , o f  to the e f f e c t s of 2,4-D p r o t e i n s and  s t u d i e s have been made on  necessity,  on the contents of f r e e amino a c i d s  a c t i v i t i e s of v a r i o u s  enzyme systems.  and  Emphasis i s  on those r e p o r t s w i t h bean p l a n t s as experimental m a t e r i a l . Free amino a c i d s and p r o t e i n contents Most o f the a v a i l a b l e r e p o r t s increase  i n the p r o t e i n content  show an  o f the 2,4-D t r e a t e d p l a n t s , b u t  the response i s v a r i a b l e w i t h the organ o f the p l a n t and the concentration  o f 2,4-D a p p l i e d .  I n d i v i d u a l amino a c i d s have  been r e p o r t e d  t o be a f f e c t e d to v a r y i n g  degrees.  S e l l e t a l . (1949) f o l l o w e d changes i n p r o t e i n s  quantitative  and f r e e amino a c i d s o f stems o f bean p l a n t s  t r e a t e d w i t h 0.05 ml o f 1000 ppm 2,4-D a t the base o f the primary l e a f b l a d e . that  Analysis  6 days a f t e r the treatment  ( i ) the stems o f the t r e a t e d p l a n t s contained  revealed  approximately  twice as much p r o t e i n as those o f untread ones; and ( i i ) the same t r e n d as noted i n the p r o t e i n content was observed f o r l e u c i n e , i s o l e u c i n e , v a l i n e , phenylalanine, threonine;  h i s t i d i n e , a r g i n i n e and  ( i i i ) the l y s i n e and methionine content o f the stems  of the t r e a t e d p l a n t s was approximately three controls;  times t h a t o f the  ( i v ) the tryptophan and a s p a r a t i c a c i d were only  s l i g h t l y greater  i n t r e a t e d than i n non-treated p l a n t s .  When the  data were expressed as percent amino a c i d i n the crude p r o t e i n , the r e s u l t s i n d i c a t e d t h a t the c h a r a c t e r  o f the p r o t e i n i n the  stems o f the t r e a t e d p l a n t s had changed as compared t o that of the c o n t r o l .  A s i m i l a r accumulation o f p r o t e i n s  i n 2*4-D-  t r e a t e d bindweed p l a n t s has been r e p o r t e d by Smith e t a l . (1947).  Weller 2,4-D  e t a l . (19 57) noted t h a t treatment of bean p l a n t s r e s u l t e d i n a c o n s i d e r a b l e r e d u c t i o n of amino a c i d s  p r o t e i n s i n the r o o t s and proteins. was  I t was  suggested t h a t the i n c r e a s e i n stem p r o t e i n s  the r e s u l t of t r a n s l o c a t i o n of nitrogenous  p a t t e r n of nitrogenous  2,4-D  Clark  (19 55)  when soybean s e e d l i n g s were exposed to 5  remained almost  Soluble  100  and  1000  ppm  the  organic total  unaltered.  observed t h a t s p r a y i n g buckwheat 2,4-D  solutions, increased  t o t a l n i t r o g e n and p r o t e i n contents  and r o o t s .  Leaf n i t r o g e n , however, was  by Smith e t a l . t h a t 2,4-D of the nitrogenous  not a f f e c t e d much.  compounds.  sprays  drawn  b r i n g s about a s h i f t i n t r a n s l o c a t i o n Increases  i n amino a c i d  and p r o t e i n f r a c t i o n s din the r o o t s of Canada T h i s t e l 2,4-D  soluble  i n b o t h stems  T h i s f i n d i n g p o i n t s to the i n v a l i d i t y of the c o n c l u s i o n s  arvense) by  ppm  In t h i s experiment, p r o t e i n  a l s o i n c r e a s e d by the treatment, but  Wort (19 59)  nitrogen,  Freiberg  found to have decreased i n the l e a v e s , but went up  n i t r o g e n content  plants with  the  distribution  compounds were a l s o found by  i n the stems and r o o t s of the t r e a t e d p l a n t s . n i t r o g e n was  m a t e r i a l to  A l t e r a t i o n s i n the  i n the n u t r i e n t s o l u t i o n .  n i t r o g e n was  and  leaves, but an i n c r e a s e i n stem  stem from leaves and r o o t s .  and  with  of low c o n c e n t r a t i o n s  r e p o r t e d by Rasmussen and Lawrence  (1954).  content  (Cirsium  have a l s o been  38. Akers and Pang (1956) found a decrease i n a s p a r t i c and glutamic a c i d s i n stems and r o o t s o f bean p l a n t s t r e a t e d w i t h 2,4-D and 2,4,5-T. vestigators  Further  r e s e a r c h by these i n -  showed t h a t a p p l i c a t i o n o f 50 p.g o f 2,4-D to the mid-  r i b o f one o f the primary leaves  o f young bean p l a n t s  14 i n 3 to 4 times g r e a t e r  incorporation of  resulted  . C i n t o these d i -  c a r b o x y l i c amino a c i d s , b u t the t o t a l amounts and percentages of these a c i d s had been decreased by the treatment.  The con-  c l u s i o n drawn from these r e s u l t s was t h a t i n a d d i t i o n to enhancing the b i o s y n t h e s i s , simultaneous i n c r e a s e  the treatment had a l s o prompted a  i n the o x i d a t i o n r a t e s o f these amino a c i d s . Work o f L i v i n g s t o n e t a l . (1954) and  F u l t s and Payne (1956) a l s o i n d i c a t e s changed amino a c i d metabolism  i n the p l a n t s  t r e a t e d w i t h 2,4-D.  Chromatographic  a n a l y s i s o f amino a c i d s o f sugarbeet r o o t s subsequent to f o l i a r sprays o f 2,4-D and MH, alanine, creased.  showed t h a t 5 days a f t e r the treatment  glutamic a c i d , l y s i n e and t r y o s i n e contents had deHowever, when h a r v e s t e d and chromatographed 60 days  a f t e r the treatments, r o o t s o f the t r e a t e d p l a n t s were r i c h e r i n a l l these amino a c i d s as compared w i t h the c o n t r o l s . changes i n the amino a c i d spectrum of the leaves were  No detectable.  Increases i n p r o t e i n content . o f b a r l e y and wheat  (Dunham, 1951; Pande, 1954) and potato tubers (Payne  et a l . 1953) f o l l o w i n g 2,4-D a p p l i c a t i o n have a l s o been  reported.  39.  The  l a t t e r i n v e s t i g a t o r s r e p o r t e d an i n c r e a s e i n glutamic  but  a decrease i n 11 other f r e e amino a c i d s of potato  developed on p l a n t s sprayed w i t h  0.5  l b 2,4-D  per  acid,  tubers  acre.  Very l i t t l e work has been undertaken to study the e f f e c t s o f 2,4-D-nutrient treatment on the p r o t e i n and amino a c i d metabolism of p l a n t s . nature,  Huffaker  et a l . (1967) s t u d i e d the e f f e c t of  a p p l i c a t i o n of 2,4-D o f wheat and b a r l e y . formulations  In the o n l y work o f  and  field  i r o n supplements on p r o t e i n  The  content::  r e s u l t s showed t h a t of the  t e s t e d , the i s o o c t y l e s t e r o f 2,4-D  this  three  had  the  greatest  e f f e c t i n i n c r e a s i n g the c o n c e n t r a t i o n o f p r o t e i n o f wheat, presence of FeDTPA w i t h the i s o o c t y l e s t e r of 2,4-D the c o n c e n t r a t i o n  The  increased  of p r o t e i n even higher. Other workers have s t u d i e d the  effect  of auxin treatment on the p h y s i c a l p r o p e r t i e s of the p r o t e i n s . Thus, an a l t e r e d heat c o a g u l a b i l i t y o f cytoplasmic of pea  stem (Galston and Kaur, 1959)  p a t t e r n o f p r o t e i n s from potato  and  tops and  as seen on e l e c t r o p h o r e t i c s e p a r a t i o n  proteins  an a l t e r a t i o n i n the tubers, and bean tops,  (Yasuda et a l . 1956)  treatment of the p l a n t s w i t h 2,4-D, have been r e p o r t e d . f a c t t h a t the s h i f t i n e l e c t r o p h o r e t i c p a t t e r n of the i s towards anode i n potato bean t i s s u e s , has  tissues while  l e d to the p r o v o c a t i v e  after  The  proteins  i t i s towards cathode i n suggestion  that  2,4-D  e f f e c t s on the q u a l i t a t i v e c h a r a c t e r s o f the p l a n t p r o t e i n s  are  40.  species s p e c i f i c  (Wort, 1964). Enzymes Inasmuch as enzymes c o n t r o l the b i o -  chemical machinery and t h e r e f o r e growth o f the c e l l ,  i t is  not very s u r p r i s i n g t h a t most o f the r e s e a r c h e r s , attempting t o g a i n i n s i g h t i n t o the mode o f auxin a c t i o n , have centered e f f o r t s on enzyme-auxin i n t e r a c t i o n .  their  T h i s approach has indeed  been v e r y rewarding as i s e v i d e n t from a f a s t growing l i t e r a t u r e . Information  on 2,4-D e f f e c t s on v a r i o u s enzyme systems was f i r s t  reviewed by Wort (1954).  L a t e r r e p o r t s on 2,4-D e f f e c t s on  enzyme systems have been d i s c u s s e d by Woodford e t a l . (1958), H i l t o n e t a l . (1963) and Wort (1964a).  Wort (1964b) has a l s o  summarized a v a i l a b l e l i t e r a t u r e on 2,4-D-mineral e f f e c t s on the enzyme systems. Table  I summarizes more  important  i n f o r m a t i o n on the i n v i v o and _in v i t r o e f f e c t s o f 2,4-D and 2,4-D-minerals on enzyme systems i n p l a n t s . made t o make the l i s t exhaustive; to  the present  No attempt has been  o n l y the r e p o r t s p e r t i n e n t  study on s t i m u l a t o r y e f f e c t s have been i n c l u d e d .  TABLE I THE EFFECTS OF 2,4-D on enzyme a c t i v i t i e s .  Species  Enzyme Amylase Ascorbic Oxidase  Method o f Treatment  acid  Catalase  Cytochrome,. oxidase IAA oxidase  Red kidney bean Wax bean  Add to stem s l i c e s i n homogenate  Barley  in  Sunflower  spray  Cucumber  in  2,4-D concentration 10 ppm 5xl0- M  ie  + 0  4  -  homogenate seedling  Effect  -  0.15%  spray  Wheat  spray  3% 5 and 500  Wheat Sunflower, oat  spray leaves spray s e e d l i n g  Red  apply to e x c i s e d a b s c e s s i o n zone  5,10,20 ppm  in  -  Key  + + +  Alexander (1965) Marre and A r r i g o n : (1954) Black and Humphreys (1962)  Potato  kidney  Cucumber  tops  homogenate  - 5 3  Stem Leaf + +  G a l l (1948) Wagenknecht e t a l , (1951) Miller & Burris (1951) R a t i k i n and Potapova (1955)  5xl0 5xl01.5%  homogenate  to M  Reference  —  ppm  + + +  Cane sugar Pea, l i l i u m  spray tops i n homogenate  5xl0 to 5xl0~ M 0.2 M 5x10-7 M  Corn  treat  lxlO  - 5  Key (1962) S i v o r i and F r a n c i s c o (1950)  Depress natural increase  Wort and Cowie (1953) T s i t o v i c h (1955) R a t i k i n and Potapova (1959) Osborne (1958)  (1962)  3  Hexokinase Glucose-6-Pgenase  seedlings  - 3  M  TABLE I  Enzyme Glyceraldehyde-3-P dehydrogenase  Species i n assay mixture  Sugarcane Corn Isocitric dehydrogenase Nitrate reductase  crystalline enzyme Corn  (Continued)  Method o f Treatment i n assay mixture  2y4-D concentration 100 ppm  spray tops treat etiolated seedling 40 ppm  0.2% lxlO  spray s e e d l i n g  2.25xl0 9xl0~ M 2.25xl0 9xl0 M  - 3  M  Effect* +  0 + —> -  _b  +  - 6  -  3  Cucumber  spray s e e d l i n g  Freed e t a l . (1961) Alexander (1965) Black and Humphreys (1962) Tomizawa and Koike (1954) Beevers e t a l . (1963) I b i d (1963)  - 3  P e c t i n methyl esterase  Peroxidase  Radish  treat roots  Sugar beet  spray tops  Bean  1 drop a t l e a f base a t a b s c i s s i o n zone  Bean  + 0.1% 2,4-Dm i n e r a l dust 1000 ppm . 04 jag  at i n t e r n o d e a t a b s c i s s i o n zone  0.5 7 jug 5xl0~ M  Potato  spray  1.5-3.0%  Wheat  spray top  5 & 500 ppm  spray tops  0.2%  Cane  sugar  5  Y o u s s e l and Wagih (1963)  +, (37-85%) Wort (1964b) + Neeley e t a l . (1950b) Check Osborne (1958) • normal decrease + F e l b e r (1948) Check Osborne (1958) : normal decrease + S i v o r i & Francisco (1950) .0-4 day •f 8 day - Wort & Cowie (1953 + Alexander (1965)  TABLE I  (Continued)  Enzyme Phosphorylase  Species Wheat  Method o f Treatment spray tops  Phosphatase  Tomato Wheat  spray spray top  15 ppm 5 & 500 ppm  Proteinases  Corn & Pea Bean  treat seedlings 1 drop a t l e a f base  1.5 ppm 1000 ppm  Bean  spray  0.5 t o 100 ppm  PEP Carboxylase  (1)  (11)  as above Bean Ribulose-5-P system (Phosphorboisomerase) (Phosphoribulose kinase) (Ribulose-5-Pcarborylase) Transaminase Cane Sugar spray tops '*+: S t i m u l a t i o n ; -: i n h i b i t i o n ; 0: no e f f e c t ;  2,4-D concentration 5 & 500 ppm  2,4-D+Fechelate  as above  0. 2% —> : f o l l o w e d by.  Effect* + + +  Reference Wort & Cowie (1953) Yakashkina (1956) Wort & Cowie (1953) Olsen (1950) Rebstock e t a l . (1952)  + Stem + Leaf & root+ h i g h - Huffaker e t a l . (1962) e s t by 0.5 ppm greater + than 2y4-D alone as above Huffaker e t a l . (1962)  +  Alexander  (1965)  44.  3.  Other metabolic changes Studies have a l s o been made to observe  changes i n phosphorus metabolism (Ormrod and W i l l i a m s ,  1960;  Fang and B u t t s , 1954) and n u c l e i c a c i d metabolism (Rebstock et a l .  1954; Key and Hanson, 1961) as brought about by  a p p l i c a t i o n to p l a n t s .  2,4-D  The b e a r i n g o f these f i n d i n g s on the  present study w i l l be c o n s i d e r e d under ' Discussion-' .  45. i  MATERIALS AND METHODS A.  Plant M a t e r i a l A s i n g l e v a r i e t y o f bushbean  (Phaseolus v u l g a r i s L. v a r .  Top Crop) was used i n a l l the experiments. Seeds o b t a i n e d from B u c k e r f i e l d ' s L i m i t e d , New Westminister, B.C., were sown i n pots (6V'x4V)  c o n t a i n i n g s t e r i l i z e d garden s o i l a t the r a t e o f 6  seeds per p o t .  A f t e r 7 days, s e e d l i n g s were t h i n n e d to 1 or 2  uniform p l a n t s i n each pot, as r e q u i r e d .  The p l a n t s were  i r r i g a t e d r e g u l a r l y w i t h tap water. The growth room p r o v i d e d a 16-hour p h o t o p e r i o d , 75 t o 80°F i n the l i g h t and 65 to 70°F i n the dark p e r i o d ;  light  i n t e n s i t y r a n g i n g from 1200 to 1500 f o o t - c a n d l e s a t the tops of  the p l a n t s , and r e l a t i v e humidity o f 60 t o 70% i n the  l i g h t and 70 t o 80% i n the dark p e r i o d .  The pot l o c a t i o n s were  changed a t a p p r o p r i a t e i n t e r v a l s i n order t o average l o c a l environmental B.  Spray  variability.  Treatments F o l l o w i n g the s u g g e s t i o n made by Wort (1962), p l a n t s  i n a l l experiments were sprayed a t the age o f 14 days, when the primary leaves were f u l l y expanded, b u t the f i r s t t r i f o l i a t e was s t i l l  i n a bud stage. (i) (ii)  sulfate  The f o l l o w i n g treatments were a p p l i e d :  One ppm 2,4-D  solution,  M i c r o n u t r i e n t s o l u t i o n , composed o f f e r r o u s  (FeS04* 7H2O) , manganous s u l f a t e  (MnSO^^O),  cupric  46. sulfate  (CuSO^*5H 0), z i n c s u l f a t e (ZnSG^'VE^O) and b o r i c a c i d 2  -4 (H-jBO^) each a t the c o n c e n t r a t i o n of 5x10  M w i t h r e s p e c t to  each m i c r o n u t r i e n t r e f e r r e d to above. (iii)  2,4-D-nutrient  components as  ( i i ) p l u s 1 ppm  The  2,4-D.  p l a n t s were s e l e c t e d f o r u n i f o r m i t y , arranged  b l o c k s or groups and The  s o l u t i o n , w i t h the same  l a b e l l e d one  day p r i o r to the  spraying.  spray s o l u t i o n s were a p p l i e d as a f i n e mist to r u n - o f f ,  i n g a l l a e r i a l parts o f each p l a n t , i n c l u d i n g upper and leaf surfaces.  The  s o l u t i o n s were allowed  in  cover-  lower  to dry on the  surface  o f the p l a n t f o r about an hour, and t h e r e a f t e r the pots were t r a n s f e r r e d to the growth room. C.  Measurement Times A l l measurements of j u v e n i l e growth, chemical  p o s i t i o n , photosynthesis, were made 5, 10,  and  com-  r e s p i r a t i o n , and enzyme a c t i v i t i e s  15 days a f t e r the treatments.  Pods were  p i c k e d s i x weeks a f t e r the treatments and the seeds were har-^ v e s t e d 11 weeks a f t e r the  spraying.  A l l h a r v e s t s , except where s t a t e d otherwise,  were  r o u t i n e l y made i n morning hours. D.  J u v e n i l e Growth Measurements P l a n t s were f i r s t measured f o r t h e i r stem h e i g h t ,  from s o i l s u r f a c e to the stem apex. the primary p a i r o f l e a v e s , was  The  l e a f number, e x c l u d i n g  then recorded.  The primary l e a f  47. p a i r was  not c o n s i d e r e d i n any f u r t h e r measurements of  juvenile  growth, and stem and p e t i o l e s were c o n s i d e r e d t o g e t h e r . Roots were c a r e f u l l y washed w i t h c o l d running water u n t i l f r e e o f entangled s o i l and humus p a r t i c l e s , d r i e d by p r e s s i n g g e n t l y between paper towels, and weighed. Leaf area measurements, e x c l u s i v e o f the  primary  l e a v e s , were made by p l a c i n g the l e a f b l a d e s i n a semi-dark room over l i g h t s e n s i t i v e o z a l i d papers under c l e a n g l a s s sheets, f l o o d i n g w i t h l i g h t f o r 3-5 minutes and then exposing the to ammonia fumes f o r c o l o r development.  papers  Leaf o u t l i n e s were  marked w i t h a p e n c i l and t h e i r areas measured w i t h a planimeter (G. C o r a d i , Z u r i c h ) . A f t e r o b t a i n i n g the f r e s h weights,  the p l a n t m a t e r i a l s  were oven d r i e d a t 75°C f o r 24 hours and weighed  subsequently.  M o i s t u r e percentages were l a t e r computed. E.  Y i e l d Measurements Pods, 4 cm and l a r g e r , were p i c k e d s i x weeks a f t e r  the  treatments and r e c o r d s were made o f the number o f pods per p l a n t and the weight of f r e s h pods per p l a n t . a t room temperature  The pods were s t o r e d  and weighed again a f t e r f o u r days.  l o s s d u r i n g storage was  subsequently  Moisture  calculated.  P l a n t s a s s i g n e d f o r data on seed p r o d u c t i o n were harv e s t e d e l e v e n weeks a f t e r the treatment when the seeds were f u l l y mature.  The pods were c o l l e c t e d i n separate envelops,  and  :  48. allowed another week of d r y i n g a t room temperature.  The number  and weight o f seeds per p l a n t and weight o f one hundred  seeds  were then determined. F.  Analytical 1.  Procedures Pigments:  C h l o r o p h y l l a and b, and c a r o t e n o i d  contents o f l e a f b l a d e s were determined (Beckman Model B).  spectrophotometrically  The pigments were e x t r a c t e d i n 80% acetone  a c c o r d i n g to the procedure suggested by Frank and Kenny and were p r o t e c t e d from ambient  light.  (1954),  The o p t i c a l d e n s i t y  (O.D.) o f each s o l u t i o n was r e a d a t 663 mp, a g a i n s t 80% acetone as b l a n k i n a 1-cm c e l l .  645 mu. and 440 mp The c o n c e n t r a t i o n s  of c h l o r o p h y l l a and b were c a l c u l a t e d f o l l o w i n g s p e c i f i c abs o r p t i o n c o e f f i c i e n t s o f McKinney (1940) and t h a t o f c a r o t e n o i d s u s i n g the equation of von W e t t s t e i n (1957). expressed as pigment (See Appendix 2.  The r e s u l t s are  c o n c e n t r a t i o n per gram f r e s h l e a f weight  f o r details},, p. 153 ) . Reducing Sugars and Sucrose:  made on d r i e d samples.  Determinations were  Soxhlet e x t r a c t i o n i n e t h a n o l , c l e a r i n g  and d e l e a d i n g o f the e x t r a c t s were done a c c o r d i n g t o the p r o cedure o f Loomis and S h u l l  (1937).  For sucrose d e t e r m i n a t i o n s , 50 ml o f c l e a r deleaded e x t r a c t was s u b j e c t e d to o v e r n i g h t i n v e r t a s e h y d r o l y s i s a t the room temperature.  F i n a l l y , the r e d u c i n g sugars o f both  h y d r o l y z e d and unhydrolyzed s o l u t i o n s were measured.color-  49.  i m e t r i c a l l y u s i n g the d i n i t r o p h e n o l reagent method. A standard  curve was prepared w i t h graded s o l u t i o n s o f glucose  (See Appendix, p.154). hydrolyzed  The d i f f e r e n c e between h y d r o l y z e d  and un-  e x t r a c t , a f t e r m u l t i p l i c a t i o n w i t h the f a c t o r 0.95,  gave the amount o f the sucrose. 3.  Free Amino A c i d s : E x t r a c t i o n o f s o l u b l e amino a c i d s Seventy percent  ethanol  e x t r a c t i o n of  the f r e s h p l a n t m a t e r i a l was employed t o separate the s o l u b l e amino a c i d s from the i n s o l u b l e nitrogenous compounds. end,  harvested  To t h i s  p l a n t m a t e r i a l s were c o a r s e l y chopped w i t h a  sharp r a z o r b l a d e and d u p l i c a t e samples o f e x a c t l y 1 g each were taken out.  These were ground w i t h about 5 ml o f 70% e t h a n o l ,  u s i n g a mortar and p e s t l e . added to c l e a n the mortar.  Another 5 t o 10 ml o f ethanol  was  The i n s o l u b l e m a t e r i a l was sedimented  by c e n t r i f u g i n g a t 2000 X g f o r 5 minutes. decanted and s t o r e d i n a r e f r i g e r a t o r .  The supernatant was  The r e s i d u e was twice  r e e x t r a c t e d w i t h f u r t h e r 10 t o 15 ml o f 70% e t h a n o l .  The  supernatants from the three e x t r a c t i o n s were pooled q u a n t i t a t i v e ly.  The ethanol was then s u b j e c t e d  to r a p i d evaporation  under a  f i n e j e t of a i r and the r e s i d u e r e d i s s o l v e d i n 70% ethanol t o g i v e a volume o f 5 ml.  The s o l u t i o n was r e c e n t r i f u g e d and the  c l e a r supernatant decanted r a p i d l y , adjusted  t o the f i n a l volume  and kept i n a r e f r i g e r a t o r i n a stoppered tube.  50.  Chromatographic  Technique  Two dimensional descending  chromato-  g r a p h i c technique was employed as d e s c r i b e d by Block e t a l . (1955).  F i f t y to 100 lambda e t h a n o l e x t r a c t was s p o t t e d 8 cm  from the two edges o f a 28x28 cm Whatman No. 1 f i l t e r Phenol: water in  paper.  (8:2) was used as the f i r s t d e v e l o p i n g s o l v e n t  the d i r e c t i o n o f the g r a i n o f the f i l t e r paper, w h i l s t the  n-butanol: g l a c i a l a c e t i c a c i d : d i s t i l l e d water  (4:1:5) mixture  (upper phase) was used f o r d e v e l o p i n g i n the second  direction.  The papers were sprayed w i t h 0.1% ninhydrin  (1,2,3-triketohydrindene) i n acetone  (w/v), allowed to  dry i n a hood and then heated i n an oven a t 75& f o r 10 minutes to  develop the c o l o r o f the amino a c i d spots.  isatin  ( 2 , 3 - i n d o l i n e d i o n e ) spray  In some cases,  (0.1% i n acetone  (w/v)) was  a l s o employed t o a s c e r t a i n the i d e n t i t y o f a p a r t i c u l a r amino a c i d , such as p i p e c o l i c a c i d , p r o l i n e , h y d r o x y p r o l i n e e t c . , A l l chromatograms i n one s e t were developed similar conditions.  a t the same time under  The unknown spots were i d e n t i f i e d by com-  p a r i n g w i t h standard Rf values o b t a i n e d w i t h s o l u t i o n s o f known amino a c i d s developed w i t h same reagents under comparable conditions  (See Appendix, p.160). Q u a n t i t a t i v e E v a l u a t i o n o f the Spots The photometric method o f Block e t a l .  (1955), based on the measurement o f the maximum i n t e n s i t y o f the  spot w i t h a P h o t o v o l t Densitometer,  was  employed. However, s i n c e  the backgrounds of the d i f f e r e n t chromatograms were not  found  to be of comparable o p t i c a l d e n s i t y a f t e r the n i n h y d r i n spray, some m o d i f i c a t i o n was  warranted.  To t h i s end,  taken of the f a c t t h a t f r e e p r o l i n e was  absent  advantage  was  i n ethanol  e x t r a c t s of a l l the p l a n t p a r t s , as r e v e a l e d by the p r e l i m i n a r y chromatographic  experiments.  A c o n s t a n t amount (10 lambda) of  a standard p r o l i n e s o l u t i o n  (8mM)  was  t h e r e f o r e s p o t t e d on each  chromatogram together w i t h the p l a n t e x t r a c t under a n a l y s i s . T h i s gave a b r i g h t y e l l o w spot w i t h n i n h y d r i n , w i t h i n - 5% reproducible o p t i c a l densities. chromatogram was  T h i s p r o l i n e spot on each  employed f o r s e t t i n g the background.  Before  measuring unknown spots on a chromatogram, the instrument s e t on o p t i c a l d e n s i t y 0.1  (or another,  p r o l i n e spot under the l i g h t beam. were then r e a d one by one tensity.  The  was  i f r e q u i r e d ) w i t h the spots from p l a n t e x t r a c t s  i n the r e g i o n of t h e i r maximum i n -  The o p t i c a l d e n s i t i e s thus o b t a i n e d w i t h spots o f  p l a n t e x t r a c t s are expressed as percentage  of the O.D.  of the  standard p r o l i n e spot. 4.  Ascorbic Acid Q u a n t i t a t i v e determinations of a s c o r b i c  a c i d contents were l i m i t e d to the f r e s h pods ( w i t h i n 2 hours a f t e r the harvest) and a f t e r 4 days of storage of room The e x t r a c t i o n procedure  employed was  temperature.  e s s e n t i a l l y the same as  52.  d e s c r i b e d by L o e f f l e r and Ponting a c i d was  (1942) except t h a t o x a l i c  used r a t h e r than metaphosphoric  pods t o a c i d was  m o d i f i e d to 1:7.  a c i d i n the e x t r a c t was  Determination o f a s c o r b i c  f o l l o w e d by the indophenol r e d u c t i o n  technique as m o d i f i e d by Schuster colormeter.  a c i d and the r a t i o of  (1950) f o r use w i t h a K l e t t  A standard r e g r e s s i o n l i n e was  drawn from r e a d i n g s  o b t a i n e d w i t h graded s o l u t i o n s o f pure a s c o r b i c a c i d ( N u t r i t i o n a l B i o c h e m i c a l C o r p o r a t i o n , Cleveland) R e s u l t s are expressed as mg G.  a s c o r b i c a c i d per 100 gm  fresh  weight.  Photosynthesis and R e s p i r a t i o n r a t e s Measurement The  same two c o n t r o l or t r e a t e d p l a n t s were used f o r  p h o t o s y n t h e s i s and r e s p i r a t i o n measurement 5,10, after  (See Appendix,p.157.) .  and 15 days  treatment. The r a t e s were determined by the use of a Beckman  i n f r a r e d a n a l y z e r IR 215  and a Heath B u i l t  Servo-Recorder,  Model EUW-20A i n the growth room i n which the p l a n t s were growing.  The p l a n t chamber, i n an open system, was  3-mil polythene  a 20-lb c a p a c i t y  bag.  The pot o f the p l a n t , e n c l o s e d i n a 2-mil polythene bag s e a l e d around  the p l a n t stem to prevent CO2  exchange,  was  s e t c a r e f u l l y i n the chamber and the chamber s e a l e d t i g h t l y around a rubber cork, p r o v i d i n g i n l e t and e x i t f o r the gas  and  f o r the t h e r m i s t o r probe. For p h o t o s y n t h e s i s measurement the chamber was  exposed  53.  to l i g h t o f 1600 f o o t c a n d l e s , and d u r i n g d e t e r m i n a t i o n o f the r a t e o f r e s p i r a t i o n was covered w i t h a b l a c k c l o t h t o exclude l i g h t .  The decrease or i n c r e a s e i n the c o n c e n t r a t i o n  of CO2 l e a v i n g the chamber, compared w i t h the CO2 concent r a t i o n o f the incoming  a i r , was used to compute the volume  o f CC>2 used or produced. A p l a n i m e t e r was used to measure the areas o f l e a f blade tracings.  R e s u l t s were expressed as m i c r o l i t e r s  of CO2 used or formed per dm  2  H.  Determination o f Enzyme Enzyme a c t i v i t i e s  assayed  per hour. Activities  i n r o o t s , stems, and l e a v e s were  5,10, and 15 days a f t e r the treatment.  In a l l cases,  the p l a n t s f o r enzyme assays were h a r v e s t e d between 9 and 10. a.m.  except f o r n i t r a t e reductase f o r which h a r v e s t s were made i n  the a f t e r n o o n between 2 and 3 p.m.  The l e a f b l a d e s were  detached  from the p e t i o l e s , the l a t t e r b e i n g taken t o g e t h e r w i t h the stem.  Roots were thoroughly washed i n c o l d running water,  f r e e d o f r o o t - n o d u l e s and d r i e d g e n t l y between paper b e f o r e use.  Phosphorylase,  c a t a l a s e , transaminase  r e d u c t a s e a c t i v i t i e s were determined from f r e s h l y h a r v e s t e d t i s s u e s .  towels  and n i t r a t e  i n homogenates prepared  For p h o s p h o g l y c e r y l  k i n a s e and s u c c i n i c dehydrogenase, the t i s s u e were s t o r e d i n a refrigerator  (0 t o 4°C) f o r 12 t o 24 hours b e f o r e u s i n g f o r  54.  enzyme e x t r a c t i o n .  For each treatment, 3 p l a n t s were a n a l -  yzed i n d u p l i c a t e samples a t each Details regarding standard  curves, 1.  age.  preparation  e t c . , are given  of s p e c i f i c reagents,  i n Appendix.  Phosphorylase Preparation  C e l l - f r e e homogenate was  of the c e l l - f r e e homogenate:  prepared by b l e n d i n g  one weight of  c o a r s e l y chopped t i s s u e w i t h f i v e volumes of c o l d  distilled  water i n a p r e c h i l l e d b l e n d o r run a t f u l l speed.  The  s u l t i n g s o l u t i o n was cheese c l o t h . period t i l l p a r a t i o n was  I t was  r a p i d l y s t r a i n e d through four l a y e r s of stored i n a r e f r i g e r a t o r for a b r i e f  used f o r the enzyme assays.  T h i s enzyme p r e -  used f o r phosphorylase, transaminase, and  except t h a t i n a s s a y i n g t i l l e d water) was  catalase further d i l u t i o n  found to be necessary i n order  (by  catalase dis-  to b r i n g  the magnitude o f the r e a c t i o n w i t h i n the l i m i t s of manometers.  re-  the  55. Assay procedure:  Sumner's (1950) method, which  i s a m o d i f i c a t i o n o f F i s k e and Subbarow's method (1925),  was  employed to assay the enzyme phosphorylase by r u n n i n g the r e a c t i o n i n the d i r e c t i o n o f s t a r c h s y n t h e s i s and  measuring  the amount of the i n o r g a n i c phosphate  The  liberated.  reaction  mixture c o n s i s t e d o f 1 ml o f enzyme p r e p a r a t i o n and 2 ml o f buffered substrate.  The r e a c t i o n was  c o n t i n u e d f o r 1 hour a t  30°C i n a c o n s t a n t temperature water b a t h . i n c u b a t i o n p e r i o d , the r e a c t i o n was 6.66%  ammonium molybdate.  At the e x p i r y o f the  terminated w i t h 5 ml o f  A d d i t i o n o f 5 ml 7-5  N H2SO4 f o l l o w e d  by 5 ml o f 4% a c i d i c f e r r o u s s u l f a t e developed a deep b l u e c o l o r . F i n a l l y the s o l u t i o n was and O.D.  d i l u t e d w i t h 10 ml o f d i s t i l l e d water  o f each and i t s blank was  equipped w i t h a r e d f i l t e r .  read i n a K l e t t colorimeter  The a c t u a l amount o f phosphate  was  r e a d from a standard curve prepared w i t h known amounts o f phosphate  u s i n g the same reagents as above.  i s d e f i n e d as ,ug i n o r g a n i c phosphate i n - the enzyme.-extract, 2.  per  Specific  activity  l i b e r a t e d per mg  protein,  hour.  Catalase P r e p a r a t i o n o f the c e l l - f r e e homogenate: Same  as f o r phosphorylase. Assay procedure:  C a t a l a s e a c t i v i t y was  by the manometric method as d e s c r i b e d by Umbreit  determined  e t a l . (1957).  The main compartment o f the Warburg f l a s k r e c e i v e d 1.5 ml o f  56  phosphate b u f f e r  (0.1 M,  pH 7.5)  d i l u t e d c e l l - f r e e homogenate. p r e - c h i l l e d hydrogen peroxide. p e r i o d i n the constant  and  The  0.2  ml of a p p r o p r i a t e l y  s i d e arm r e c e i v e r 0.5  ml  of  A f t e r a 10 minute i n c u b a t i o n  temperature bath of the Warburg apparatus  (held at 26°), the manometers were t i p p e d g e n t l y to l e t the hydrogen peroxide manometer was  initiate  i n s t a n t l y c l o s e d and  manometer r e c o r d e d . was  the r e a c t i o n .  of  the  the i n i t i a l r e a d i n g on  the  2.2  stopcock  In the c o n t r o l v e s s e l , 0.2  used i n p l a c e of the homogenate.  contained  The  ml d i s t i l l e d water was  ml d i s t i l l e d water  A thermobarometer which a l s o i n c l u d e d to r e c o r d  change i n temperature of the bath or atmospheric p r e s s u r e . r e a c t i o n was  allowed  to run f o r 3 minutes, a f t e r which the  on each manometer was  recorded.  c a l c u l a t e d using f l a s k  reading  M i c r o l i t e r s of O2 produced were  s p e c i f i c a c t i v i t y i s d e f i n e d as  p r o t e i n per  3.  The  constants.  The produced per mg  any  u l O2  hour.  Transaminase P r e p a r a t i o n of the c e l l - f r e e homogenate:  as f o r  phosphorylase. Assay procedure:  transaminase  (G 0 T) was  Glutamic-oxaloacetic  assayed i n c e l l - f r e e homogenates  f o l l o w i n g the procedure o u t l i n e d by Reitman and F r a n k e l The  Same  assay mixture was  s u b s t r a t e and  0.5  composed of 1 ml  (1957).  -ketoglutarate-aspartate  ml of the enzyme p r e p a r a t i o n .  The r e a c t i o n  was  allowed to proceed f o r e x a c t l y 30 minutes i n a c o n s t a n t temperature water b a t h s e t at 37°C.  The  r e a c t i o n was  terminated  by a d d i t i o n o f 1 ml o f 2 , 4 - d i - n i t r o p h e n y l h y d r a z i n e f o l l o w e d by The  the a d d i t i o n of 10 ml of 0.4  c o l o r was  the O.D.  N sodium hydroxide.  allowed to develop f o r e x a c t l y 30 minutes.  o f each s o l u t i o n , and  K l e t t colorimeter  the use  i t s blank, was  ( o x a l o a c e t i c acid)  Finally,  measured i n a  equipped w i t h a green f i l t e r .  amounts o f the keto a c i d mined by  reagent,  The  actual  formed were  deter-  o f a standard c h a r t prepared w i t h known quan-  t i t i e s of oxaloacetate  u s i n g the c o n d i t i o n s  s p e c i f i e d above  (See Appendix, p. 167) . The oxaloacetate  s p e c i f i c a c t i v i t y i s d e f i n e d as urn  formed per mg 4.  p r o t e i n per hour.  N i t r a t e Reductase Preparation  c e l l - f r e e e x t r a c t was  of the crude enzyme:  prepared by g r i n d i n g one  weight o f  c o a r s e l y chopped f r e s h t i s s u e w i t h four volumes of 0.1  M K HP0 2  thione, (0-4°C).  4  buffer  (pH 7.8),  containing  10~  3  chilled  M reduced g l u t a -  i n a Waring b l e n d o r f o r 3 minutes i n a c o l d room The  homogenate was  o f cheese c l o t h and  r a p i d l y s t r a i n e d through 4 l a y e r s  the f i l t r a t e was  centrifuged,  c e n t r i f u g e , a t 20000 x g f o r 20 minutes a t 0-4°C. supernatant s o l u t i o n , thus obtained, was enzyme.  Crude  in a Sorvall The  cell-free  used f o r assaying  the  58.  Assay procedure:  For the d e t e r m i n a t i o n of  n i t r a t e reductase a c t i v i t y , the procedure (1953) as m o d i f i e d by Yang (1964) was 0.05  to 0.2  o f Evans and Nason  employed.  ml o f the enzyme p r e p a r a t i o n was  mixture c o n t a i n i n g 0.1 ml of 0.1 M KN0 , 0.05 3  0.05  ml o f 2 x l 0 ~  3  ml.  I n c u b a t i o n was  minutes i n a constant temperature and 1 ml o f 1%  2  stop the r e a c t i o n .  (w/v)  spectrophotometer,  determined  M  FAD,  s u l f a n i l a m i d e were added to (w/v)  (complete  the O.D.  N-  (1-Napthyl)-  added and the contents  F i f t e e n minutes were allowed f o r  the development of the c o l o r .  cell.  5  water b a t h a t 30°C, a f t e r which  One ml o f 0.22%  mixed by i n v e r t i n g the tube.  a 1-cm  ml o f 2 x l 0 ~  c o n t i n u e d f o r 30  ethylene diamine h y d r o c h l o r i d e reagent was  its. blank  added to a r e a c t i o n  M DPNH, and 0.1 M phosphate b u f f e r to g i v e  a t o t a l volume o f 0.5  1 ml o f H 0  At zero time,  F i n a l l y , u s i n g a Beckman Model  of each experimental s o l u t i o n  except f o r DPNH) was  DU  and  measured a t 540 mu. i n  The a c t u a l amount of the n i t r i t e formed  was  from a standard curve prepared w i t h known  q u a n t i t i e s of n i t r i t e  (See Appendix, pj.66 ).  The s p e c i f i c a c t i v i t y i s d e f i n e d as mum  of  n i t r i t e formed per mg p r o t e i n per hour. 5.  Phosphoglyceryl  Kinase  P r e p a r a t i o n of the crude enzyme: One h t of the f r o z e n t i s s u e was s u c c i n a t e b u f f e r (pH 6.2)  blended w i t h three volumes of c o l d  i n a Waring b l e n d o r f o r 3 minutes a t  weig-  59.  full  speed.  The  s o l u t i o n was  r a p i d l y s t r a i n e d through 4 l a y e r s  of cheese c l o t h and c e n t r i f u g e d  a t 2000 x g f o r 10 minutes i n  a Sorvall centrifuge  The c l e a r supernatant  a t 0-4°C.  was  drawn o f f and used f o r a c t i v i t y d e t e r m i n a t i o n . Assay procedure: A x e l r o d and Bandurski  (1953) was  The assay method of  employed.  Notwithstanding  the d i f f i c u l t y i n p r e p a r i n g 1,3-diphosphoglycerate, i n v e s t i g a t o r s developed a procedure f o r measuring  these  the enzyme  through the u t i l i z a t i o n  of 3-phosphoglyceric a c i d as s u b s t r a t e  i n the presence of ATP.  The diphosphosphoglycerate i s trapped  w i t h hydroxy1amine, and the anhydride thus formed c o l o r i m e t r i c a l l y by the hydroxamic  i s measured  t e s t of Lippman and  Tuttle  (1945). Reactions were run i n 10-ml  t e s t tubes.  To each tube were added 1 ml o f 0.1 M s u c c i n a t e b u f f e r (pH 6.2),  1 ml of 2 M hydroxylamine  0.5 ml of 0.01 paration,  0.25  The tube was  2 ml o f FeCl3-TCA-  The r e a c t i o n i n the experimental tube was  i n i t i a t e d by the a d d i t i o n of 1 ml o f 0.01 acid  M MgC^., i n  shaken a f t e r the a d d i t i o n of each r e -  The c o n t r o l tube at t h i s time r e c e i v e d  HCl reagent.  (barium s a l t ) .  6.2),  to 0.5 ml of the crude enzyme p r e -  1 drop o f 0.1 M NaF and 1 drop o f 0.01  that order. agent.  M ATP,  h y d r o c h l o r i d e (pH  then  M 3-phosphoglyceric  The r e a c t i o n c o n t i n u e d f o r 1 hour a t 3 0°C  i n a c o n s t a n t temperature water b a t h and was  terminated by the  60. a d d i t i o n o f 2 ml o f FeCl -TCA-HCl reagent to the experimental 3  tubes.  Ten minutes was  g i v e n f o r c o l o r development  e x p i r y of which the s o l u t i o n s were c e n t r i f u g e d  to s e t t l e  p r e c i p i t a t e and the c l e a r brownish s o l u t i o n was The O.D.  a t the any  drawn o f f .  o f the s o l u t i o n from each experimental tube, and i t s  blank, was  determined by a Beckman Model DU  at 430 mu. i n a 1-cm  Spectrophotometer  cell. The s p e c i f i c a c t i v i t y i s d e f i n e d  crease i n O.D. 6.  as the i n -  per mg p r o t e i n per hour. S u c c i n i c Dehydrogenase Preparation  o f the c e l l - f r e e homogenate:  One weight o f f r o z e n t i s s u e was macerated w i t h 2 volumes o f 0.1 M phosphate b u f f e r The s o l u t i o n was  u s i n g a mortar and p e s t l e .  transferred quantitatively into a centrifuge  tube and c e n t r i f u g e d l a y e r was  (pH 7.4)  f o r 10 minutes a t 2 000 x g.  c a r e f u l l y decanted and used f o r the assay. Assay procedure:  of Kun and Abood incubation  (1949) was  p e r i o d was  E s s e n t i a l l y the procedure  f o l l o w e d w i t h the m o d i f i c a t i o n  that  prolonged to 2 0 hours, as suggested by  Isenberg e t a l . (1951). o f 0.1 M phosphate b u f f e r succinate,  The t h i c k upper  Into a 15-ml  tube were p i p e t t e d 0.5  (pH 7.4), 0.5 ml o f 0.2 M  ml  sodium  1 ml o f homogenate and l a s t l y 1 ml of f r e s h l y prepared  0.1% t r i p h e n y l t e t r a z o l i u m c h l o r i d e s o l u t i o n .  A f t e r shaking, the  tubes were stoppered and kept i n a constant temperature water  bath  a t 38°C.  hours a t the tube.  The  cipitate  end  o f w h i c h 7 ml  dissolve  into  off.  Finally,  (# 420)  20  added t o  400  pg  the c l e a r  o f each t e s t  red  pre-  solution  solution,  colorimeter  o f formozan d i s s o l v e d  each  then  a t 80°C f o r 5 m i n u t e s  A s t a n d a r d c u r v e was  and  before  equipped  made u s i n g i n 10 ml  of  (See Appendix,,p.165 ). The  specific  jag o f f o r m o z a n f o r m e d p e r mg 7.  Protein The  To  Appendix), minutes.  was  a s u i t a b l e volume o f t h e enzyme a d d e d 2 ml  e a c h t u b e was  reagent  (Fisher  Spectrophotometer, determined  alkaline  a t 500  then  t h e O.D.  again.  copper  allowed  a d d e d 0.2  Scientific  contents mixed t h o r o u g h l y  foll-  o f a l l t h e enzyme p r e p a r a t i o n s  t h e c o n t e n t s m i x e d , and To  the  hour.  method o f Lowry e t a l . (1951) was  d e s c r i b e d above. ml)  i s d e f i n e d as  Determination:  the p r o t e i n  t o 0.1  activity  p r o t e i n per  owed t o d e t e r m i n e  Phenol  was  for  s o l u t i o n s were  using a Klett  filter.  50,100,200,300, and  (0.05  The  t h e O.D.  ( w i t h homogenate h e a t e d  a blue  acetone  to continue  of pure acetone  the acetone.  a s s a y i n g ) , were d e t e r m i n e d with  allowed  f o r 3 m i n u t e s a t 2000 x g and  decanted  i t s blank  i n c u b a t i o n was  c o n t e n t s were s h a k e n v i g o r o u s l y t o l e t t h e r e d  centrifuged was  The  Co., Using  and  solution  to stand f o r ml  F a i r l a w n , N.J.) a Beckman M o d e l  2 hours,  (see 10  of 1 N F o l i n - .  o f each e x p e r i m e n t a l  mu. b e t w e e n 0.5  preparation  as  and  the  DU  solution  was  suggested  by  62.  Bailey  (1962) .  A t i s s u e blank was  made u s i n g a l l the contents  as above except the F o l i n - P h e n o l reagent, which was  replaced  by an equal volume of d i s t i l l e d water. A standard curve was  prepared w i t h  known amounts of Bovine albumin, which served to e s t a b l i s h the a c t u a l amount of p r o t e i n i n each enzyme p r e p a r a t i o n .  63. RESULTS I  J u v e n i l e Growth 1.  F r e s h weights:  Data on f r e s h weights  of r o o t s ,  stems, and leaves are presented i n Table I I and a l s o r e p r e s e n t e d g r a p h i c a l l y i n F i g u r e 1. Roots: i n c r e a s e d f r e s h weights observation.  The  spray c o n t a i n i n g 2,4-D  o f r o o t s s i g n i f i c a n t l y a t a l l times o f  A d d i t i o n of m i n e r a l s to 2,4-D  the s t i m u l a t o r y a c t i o n of the hormone. caused by 2,4-D  alone  f u r t h e r enhanced  Thus, w h i l e the i n c r e a s e s  alone were 21, 17, and 16% , the corresponding  i n c r e a s e s caused by the mixture were 30, 20, and 18% a t 5, and 15 days,  respectively. Stem:  Stem f r e s h weights  followed c l o s e l y  the p a t t e r n o f s t i m u l a t i o n induced i n the r o o t s . treatments  w i t h the treatment. induced by 2,4-D  alone was  augmented f u r t h e r by the  For example, w h i l e 2,4-D  spray * was 4  varied  As i n the r o o t s , the s t i m u l a t i o n of growth  11%.  inclusion  alone i n c r e a s e d f r e s h  weights by 8% f i v e days a f t e r the treatment, by the MD  A l l three  s t i m u l a t e d growth but the degree of response  of m i n e r a l s .  10,  the i n c r e a s e caused  S t i l l g r e a t e r d i f f e r e n c e s were  *:  A l l percentages are c a l c u l a t e d w i t h r e s p e c t to c o r r e s ponding v a l u e s i n the c o n t r o l p l a n t s .  +:  A b b r e v i a t i o n s used: c = c o n t r o l (no s p r a y ) ; M = m i n e r a l treatment; D = 2,4-D treatment; MD = "mineral p l u s 2,4-D" treatment.  TABLE I I Effect  o f m i n e r a l s , 2,4-D, o r 2 , 4 - D - m i n e r a l s p r a y s on f r e s h and d r y w e i g h t s o f r o o t s , stem, and l e a v e s o f bushbean (Phaseolus v u l g a r i s ) p l a n t s .  weights,  x  Days a f t e r 5 Treatment Freshweight g  weight mg  Stem  Leaves" " 1  15  Roots  Stem  Leaves  Roots  Stem  Leaves  C*  1.09*  2.41  1.33  2.05  3.61  4.16  2.59  5. 57  6. 01  M  1. 10  2.42  1.22  2.10  3.62  4.20  2.61  5.60  6. 27  1.32* 1.42*  2.53 2. 68*  1. 02* 1.14*  2.39* 4.31*; 2.46* 4.48*  4. 89* 5.33*  3.01* 6. 74* 3.05* 6.94*  C  124  149  151  217  484  506  293  831  846  M  126  153  139  228  511  520  315  879  926  D  133  139  105*  245*  527*  537  335*  910*  974*  13 7 *  194*  113*  259*  551 *  538  340*  946*  1060*  D MD  Dry  Roots  treatment  10  MD  7.25 7. 79  A v e r a g e v a l u e p e r p l a n t o f 24 measurements e a c h . E x c l u s i v e o f the p a i r o f primary leaves. I n T a b l e I I t o V I , X t o X I I , XIV t o XV, T u k e y ' s W t e s t has b e e n u s e d t o t e s t t h e s i g n i f i c a n c e o f means. I n t h e s e t a b l e s , any two means c o n n e c t e d b y t h e same v e r t i c a l l i n e do n o t d i f f e r s i g n i f i c a n t l y a t 0.05 l e v e l . Means d i f f e r i n g s i g n i f i c a n t l y (P= 0.05) f r o m t h e r e s p e c t i v e c o n t r o l mean a r e i n d i c a t e d b y *. I n T a b l e s I I t o XV, c = c o n t r o l (no s p r a y ) ; M = m i n e r a l s p r a y ; D = 2,4-D s p r a y ; MD = "2,4-D p l u s m i n e r a l s " s p r a y .  65.  C D M M D C D M M D C D M M D 5 10 15 Days a f t e r treatment F i g . 1: E f f e c t o f 2,4-D (D) , m i n e r a l s (M) , or 2,4-Dm i n e r a l (MD) sprays on f r e s h weights o f r o o t s , stem, and leaves o f bushbean (Phaseolus v u l g a r i s ) p l a n t s , (c = c o n t r o l )  66. measured 10 and  15 days a f t e r Leaves:  2,4-D  and MD  (23 and  by the 10th day.  14%,  treatment. A temporary d e p r e s s i v e e f f e c t  r e s p e c t i v e l y ) a t 5 days was  continued  overcome  At the l a t t e r age both D and MD r e s u l t e d  i n s i g n i f i c a n t l y higher l e a f weights. was  of  This stimulatory e f f e c t  f u r t h e r u n t i l the o b s e r v a t i o n a t 15 days.  t h i s time of measurement i n c r e a s e s by 2,4-D  At  and MD were 21  and  30%, r e s p e c t i v e l y . M i n e r a l s alone caused o n l y s m a l l i n c r e a s e s i n f r e s h weights of r o o t s , stem, and leaves which l a c k e d s i g n i f i c a n c e a t 0.05 2.  l e v e l at a l l times o f o b s e r v a t i o n .  Dry weights:  Data on dry weights of r o o t s , stem,  and leaves are i n c l u d e d i n Table I I and a l s o d e p i c t e d i n F i g u r e 2. Roots:  Sprays c o n t a i n i n g 2,4-D  without  and  w i t h m i n e r a l supplements caused s i g n i f i c a n t i n c r e a s e s i n dry matter p r o d u c t i o n by r o o t s , except when o n l y MD  treatment  brought about a s i g n i f i c a n t i n c r e a s e .  Stem: MD  at the 5-day o b s e r v a t i o n  As i n r o o t s , a t the f i f t h day  only  induced a s i g n i f i c a n t i n c r e a s e i n dry weight of stem.  subsequent o b s e r v a t i o n s , however, both 2,4-D  and MD  to produce s i g n i f i c a n t l y h i g h e r dry matter i n stems. age of o b s e r v a t i o n , i n c r e a s e by MD  surpassed  At  were seen At  every  those by 2,4-D  alone.  67.  LEAF |  |  STEM ROOT  4  _  C D  D M MD  M MD  5  10 Days  Fig.  Effect  of  mineral stem, is)  2,4-D  (MD)  and  after  treatment. (D),  sprays  leaves  plants.  (c  of =  minerals on  (M),  dry weights  bushbean  control).  or  2,4-D-  of  roots,  (Phaseolus  vulgar-  68. Leaves:  Dry weights  c l o s e l y i n p a t t e r n o f response  of leaves  corresponded  to t h a t of t h e i r f r e s h  weights.  The a d d i t i o n of m i n e r a l s gave a c o n s i d e r a b l e 'safening ' e f f e c t 1  at 5 days.  At 15 days the leaves of both D- and MD-treated  p l a n t s possessed increases,  s i g n i f i c a n t l y h i g h e r dry matter  (15 and 2 5%  respectively). Increases i n dry matter of r o o t s , stem, or  leaves caused by m i n e r a l s alone were not s i g n i f i c a n t a t  0.05  level. 3.  Shoot e l o n g a t i o n :  Table I I I and F i g u r e 3 p r e s e n t  data on the e f f e c t o f the treatments  on shoot h e i g h t of the  j u v e n i l e p l a n t s , measured 5, 10, and 15 days a f t e r the  treatment.  S i g n i f i c a n t i n c r e a s e s i n shoot h e i g h t were measured a t the three time i n t e r v a l s .  At the 15-day measurement i n c r e a s e s by D and  were 11 and 18%,  respectively.  m i n e r a l supplements on 2,4-D  MD  The augmentory e f f e c t of the  s t i m u l a t i o n of h e i g h t was  evident.  Increases i n h e i g h t caused by m i n e r a l s alone, however, were not 4. Both 2,4-D  significant.  Leaf number and a r e a :  and MD  complex produced  numbers a t 10 and 15 days.  s i g n i f i c a n t l y higher  leaf  A d d i t i o n of m i n e r a l supplements en-  hanced the s t i m u l a t o r y a c t i o n of 2,4-D i n c r e a s e s caused by 2,4-D  (Table I I I , F i g u r e 4).  alone i n both cases.  a t 10 and 15 days were 14 and  The  23%,  r e s p e c t i v e l y , whereas the corresponding i n c r e a s e s induced by  MD  TABLE I I I E f f e c t o f m i n e r a l s , 2,4-D, or 2,4-D-mineral sprays on p l a n t h e i g h t , l e a f number and l e a f area o f bushbean (.Phaseolus. v u l g a r i s ) p l a n t s .  Days a f t e r 5 Plant Height cm  treatment  10  15  Leaf Ar|a cm  Plant Height cm  Leaf Number  C  18.1  4.5  73.1  24.9  8.6  Leaf Plant Area Height 2 - -. cm cm 195.6 30.1  M  18.3  4.1  66. 3  25.5  9. 0  198.9  D  20.2*  3.5*  55.6*  27. 5*  9. 8*  MD  20.4*  3.6*  56. 7*  28.6*  11.9*  Treatment  Leaf Number  +  Average v a l u e per p l a n t of 24 measurement E x c l u s i v e o f the p a i r o f primary l e a v e s .  each.  x  Leaf Number  Leaf Area 2 cm  14.6  380.6  30. 5  15.6  392.2  201.5  33.9*  18. 0*  401. 8  202.3  ... 35.4*  18. 9*  419.5*  70.  120  115  o S-i -p c o u m o  110  105  100 D  M MD  5 Fig.  3:  D  M MD  10  D  M  MD  15  Days a f t e r t r e a t m e n t E f f e c t o f 2,4-D (D) , m i n e r a l s (M)', o r 2,4-D-mineral (MD) s p r a y s on t h e h e i g h t of bushbean (Phaseolus v u l g a r i s ) p l a n t s .  150  Leaf  140  Leaf  number  Chlorophyll  area  content  130  120 _  110_  i-r  10C D  Fig.  M MD  10 4:  Effect number, leaves.  D  D M MD  15  of  Days  2,4-D  area,  (D),  M MD  10  after  minerals  (M),  D M  D M MD  15  treatment or  and c h l o r o p h y l l content  2,4-D-mineral of  bushbean  (MD)  D  MD  10  sprays  (Phaseolus  on  M MD  15 the  vulgaris)  were 3 8 and  30%. Leaf area, e x c l u s i v e of the p a i r of primary  leaves,  (Table IV)showed the g r e a t e s t response  a t the 15-day o b s e r v a t i o n . 2,4-D  to  treatment  At t h i s age the i n c r e a s e s caused  and by the MD were 5.5  and 10%,  by  respectively.  M i n e r a l s alone d i d not b r i n g about  sig-  n i f i c a n t i n c r e a s e e i t h e r i n l e a f number or i n l e a f area a t any time of o b s e r v a t i o n . II  Chemical  composition  1.  M o i s t u r e content:  Data on the moisture  o f r o o t s , stem, and leaves are g i v e n i n Table IV. moisture content was  content  Although  not a f f e c t e d s i g n i f i c a n t l y by any  treat-  ment, a d e f i n i t e t r e n d of i n c r e a s e i n moisture percentages p l a n t s t r e a t e d w i t h 2,4-D  or 2,4-D  with minerals i s evident.  The i n c r e a s e was most pronounced i n p l a n t s r e c e i v i n g of 2,4-D  alone.  and 88.7% moisture  plants.  moisture  and 89.7%,  i n c o n t r a s t to  88.6,  i n corresponding t i s s u e of the c o n t r o l  S i m i l a r i n c r e a s e s were observed a t 10 and 15  M i n e r a l s alone caused,  of r o o t s ,  p l a n t s were 90, 94.5,  r e s p e c t i v e l y , f i v e days a f t e r treatment, 93.8,  treatment  For example, the moisture percentages  stem, and leaves o f 2,4-D-treated  of  days.  i n most cases, s l i g h t decreases i n the  content. 2.  C h l o r o p h y l l and c a r o t e n o i d c o n t e n t :  (Table V, F i g u r e 4).  TABLE IV E f f e c t o f m i n e r a l s , 2,4-D, or 2,4-D-mineral sprays on moisture content (% moisture). ...of roots., stem, and leaves o f bushbean (Phaseolus v u l g a r i s ) p l a n t s . x  Days a f t e r treatment 5 Treatment  +: x.  Roots  10  15  Stem  Leaves  Roots  Stem  Leaves  Roots  Stem  Leaves  93.8  88.7  89.4  86.6  87.8  88.7  85.1  85.9  C  88.6  M  88.5.  93.7  88.6  89.1  85.9  87.6  87.9  84.3  85.2  D  90.0  94.5  89.7  89.8  87.8  88.9  88.9  86.5  86.6  MD  90.4  92.7  90.1  .89.5  87. 7  88.8  88.8  86.3  86.4  +  None o f the means was s i g n i f i c a n t l y d i f f e r e n t level. Average values o f 24 d e t e r m i n a t i o n s each.  from the r e s p e c t i v e c o n t r o l a t 0.05  TABLE V E f f e c t o f m i n e r a l s , 2,4-D, or 2,4-D-mineral sprays on c h l o r o p h y l l and c a r o t e n o i d content o f bushbean (Phaseolus v u l g a r i s ) l e a v e s . x  Days a f t e r treatment 10  5  15  Treatm  e  n  t  c  " l  t  +  Chi b  C h i a+b  Cart 0. 26  #  Chi a Chi b C h i a+b C a r t mg/gm f r e s h l e a f weight 0. 93 0. 77 1. 70 0.35  Chi a  Chi b  C h i a+b  Cart  1. 19  0.92  2. 11  0.45  c  0. 79  0.65  1.44  M  0. 53*  0. 51*  1. 04* 0. 27  0. 95  0. 79  1. 74  0.36  1. 79*  1.38*  3. 17*  0.46  D  0. 48*  0.45*  0. 93* 0. 26  0. 88  0. 85*  1. 73  0.35  1. 65*  1.39*  3. 04*  0.45  MD  0. 52*  0.49*  1.01* 0. 27  0. 99  0. 85*  1. 84*  0.36  1. 50*  1.23*  2. 73*  0.48  x:  Average values o f 3 determinations  ++:  Chlorophyll  each  Carotenoids  4^  75.  Ten  days a f t e r  treatment  b y MD t r e a t m e n t on had  were s i g n i f i c a n t .  increases  attained significance. alone,  At this  2,4-D a l o n e ,  time  treatments  increases induced by  a n d 2 , 4 - D - m i n e r a l complex were 50,  a n d 29%. C a r o t e n o i d c o n t e n t was n o t a f f e c t e d  by  occasioned  A t t h e 15-day o b s e r v a t i o n ,  t h e o t h e r hand, i n c r e a s e s i n d u c e d b y a l l t h r e e  minerals 48,  only the chlorophyll  any t r e a t m e n t 3.  analyzed  a t any time  of observation.  Sugar c o n t e n t :  Roots,  stem, a n d l e a v e s were  s e p a r a t e l y f o r r e d u c i n g sugars  o f t h e two was t a k e n  significantly  as t h e t o t a l  sugar  and s u c r o s e . content.  A review o f  T a b l e V I shows t h a t i n t h e r o o t s t h e r e was a d e c r e a s e amounts o f b o t h  r e d u c i n g sugars  o f s u g a r s was n o t e d  a t the f i r s t  and sucrose.  The sum  i n the  This depletion  observation five  days  after  treatment  a n d became q u i t e p r o n o u n c e d a t t h e 15-day o b s e r v a t i o n .  A similar  p a t t e r n o f p r o g r e s s i v e decrease  sugars  o f both  reducing  a n d s u c r o s e was e v i d e n t i n stems o f t h e t r e a t e d p l a n t s .  I n t h e l e a v e s , on t h e o t h e r hand, t h e r e was an i n c r e a s e i n t h e reducing  sugar  content  caused  s u c r o s e c o n t e n t was l o w e r e d . content in  by the treatments,  The n e t r e s u l t was a h i g h e r  i n the leaves o f the t r e a t e d p l a n t s .  the sugar 4.  content  o f l e a v e s was b r o u g h t  F r e e amino a c i d s :  e x t r a c t s made i t f e a s i b l e  w h i l s t the sugar  Maximum i n c r e a s e  a b o u t b y MD  treatment.  The a b s e n c e o f p r o l i n e  i n the  t o a d d a c o n s t a n t amount o f p r o l i n e  TABLE VI E f f e c t o f m i n e r a l s , 2,4-D, or 2,4-D-mineral sprays on r e d u c i n g sugars and sucrose content i n r o o t , stem and leaves o f bushbean (Phaseolus v u l g a r i s ) p l a n t s . x  Tissue Root  Treatment C M D MD  Days a f t e r treatment 10 Reducing Reducing Sucrose Total sugars sugars Sucrose Total % o f dry weight 0.40 0. 56 0.62 0.41 1. 03 0.96 0.38 0. 88 0. 50 0.50 0.38 0. 88 0.30* 0.42* 0.40* 0. 72* 0.29* 0.69* 0. 28* 0.68* 0.24* 0.60* 0.40* 0.36*  15 Reducing sugars  Sucrose  Total  0.64 1 0.48 0.34* 0.33*  0. 51 0.36 0.30* 0.32*  1.15 0. 84 0.64* 0.65*  I  Stem C M D MD  2. 50 2.42 2.01* 2.00*  2. 80 2. 70 2. 55 2. 54  5.30 5.12 4. 56* 4. 54*  2.601 2.4o| 1.96* 2. 02*  3.00 2.61* 2. 50* 2.48*  5.60 5.01* 4.46* 4. 50*  2.66 2. 00* 1. 70* 1. 54*  3.21 2.62* 2.11* 2. 00*  5. 87 4.62* 3. 81*1 3. 54*  C M D MD  1. 80 1.90 2. 04* 2.18*  2.02 2.07 1.90 1.80  3. 82 3.97 3.94 3.98  1. 86 1.96 2.30* 2.41*  2.10 2.10 1. 80* 1. 82*  3.96 4.06 4.10 4.23  1.90 2.20* 2.64* 2. 75*  2.251 2. 02 1.66* 1.62*  4.15 4.22 4.30 4.37  I  Leaf  x,  Average v a l u e s o f 3 determinations  each.  I  (10 lambda of 8 mM) gram sheet.  The  to the O.D.  w i t h the p l a n t e x t r a c t to each chromato-  O.D.  of each amino a c i d spot was  of the p r o l i n e b e i n g 100  read r e l a t i v e  on each sheet. In t h i s  v a r i a t i o n i n the background c o l o r was  negated and  the  way-  spot  O.D.  v a l u e s r e f l e c t e d the r e l a t i v e c o n c e n t r a t i o n s of a l l the amino acids  i n a sample. The  i n the  leaves  following  11 amino a c i d s were d e t e c t e d  *X-aminobutyric a c i d ,  (Table V I I ) :  asparagine,  a s p a r t i c a c i d , glutamic a c i d , g l y c i n e , h i s t i d i n e , i s o l e u c i n e , c i n e , methione, s e r i n e , t e c t e d o n l y at the and 1962,  and  tyrosine.  The  p o s i t i v e i d e n t i f i c a t i o n was  "Y-methyleneglutamic a c i d . made, however.  No  treated  Fowden, No diff-  leaves  plants. In the  stem (Table VIII) the amino a c i d s  same as those i n the  glutamic a c i d " was  de-  values  qualitative  observed i n the amino a c i d spectrum o f the  c o n t r o l and  Rf  were s i m i l a r to those g i v e n by  f o r p i p e c o l i c a c i d and  were the  a d d i t i o n a l spots,  15-day h a r v e s t , were observed.  color with ninhydrin  erence was  Two  leu-  not  leaves except t h a t  detected.  " Y -methylene-  Treatments d i d not  l e a d to  any  q u a l i t a t i v e change. In the r o o t s (Table IX).  These i n c l u d e d  o n l y 10 amino a c i d were d e t e c t e d  Y-aminobutyric a c i d , asparagine,  a s p a r t i c a c i d , glutamic a c i d , g l y c i n e , ionine, duced by  s e r i n e , and any  threonine.  No  i s o l e u c i n e , l e u c i n e , meth-  q u a l i t a t i v e v a r i a t i o n was  treatment. Although no q u a l i t a t i v e a l t e r a t i o n  was  in-  TABLE V I I E f f e c t o f m i n e r a l s , 2,4-D, o r 2,4-D-mineral sprays on the r e l a t i v e c o n c e n t r a t i o n s o f the ethano.l-so.lub.le f r e e ..amino a c i d s o f leaves of bushbean (Phaseolus v u l g a r i s ) , x  Days a f t e r treatment 5 Amino a c i d or amide "Y - Aminobutyric a c i d Asparagine Aspartic acid Glutamic a c i d Glycine Histidine Isoleucine Leucine Methionine "Y-Methyleneglutamic acid"** Pipecolic acid"** Serine Threonine *: : **: x  C 210* 190 110 110 96 98 95 95 94  98 120  10  15  M 290 240 120 180 100 100 100 100 98  D 140 130 120 98 96 90 90 90 90  MD 240 180 130 125 98 98 100 100 96  C 200 180 120 120 100 96 100 100 100  M . 320 280 180 240 120 110 120 120 110  D 130 120 100 100 90 90 92 90 90  MD 250 200 140 150 110 100 110 110 95  c  200 170 125 110 100 98 110 110 100  M 340 300 200 260 125 120 135 130 125  D 120 80 90 95 90 88 90 90 85  MD 280 200 160 170 115 110 120 115 120  110 180  -  110 160  -  -  -  -  90 85  100 140  100 96 110 100  220 140 125 220  90 90 85 85  150 114 115 130  100 100  100 no  120 210  Each v a l u e r e p r e s e n t s maximum o p t i c a l d e n s i t y o f the spot r e l a t i v e to the o p t i c a l d e n s i t y of the standard and p r o l i n e spot b e i n g 100. Average v a l u e s o f 2 determinations each. See page 77.  TABLE V I I I E f f e c t of m i n e r a l s , 2,4-D, or 2,4-D-mineral sprays on the r e l a t i v e c o n c e n t r a t i o n s o f the e t h a n o l - s o l u b l e f r e e amino a c i d s of stem o f bushbean (Phaseolus v u l g a r i s ) . x  Days a f t e r treatment 5 Amino a c i d or amide "Y-Aminobutyric Asparagine Aspartic acid Glutamic a c i d Glycine Histidine Isoleucine Leucine Methionine Pipecolic acid Serine Threonine  acid  10  230 340 260 245 115 110 100 100 115  110 130 110 95 90 90 95 95 90  200 220 185 150 105 100 96 96 105  110 115  120 140  100 100  112 125  160 220 170 120 98 100 97 97 94  90 105  98 120  140* 220 130 110 96 98 95 95 94  220 300 230 175 100 100 100 100 98 100 125  96 110  130 200 140 120 100 96 96 96 100  120 160 110 100 92 95 90 90 92  D  -  D  C  M  —  M  MD  C  -  -  15  -  -  MD  -  -  C  M  D  125 190 150 120 98 96 98 98 100 100 105 110  250 360 260 2 80 120 120 115 115 120 140 130 150  100 95 90 92 90 85 86 86 88 96 90 96  MD 180 235 190 165 110 110 100 100 112 120 115 130  See Table VII Average v a l u e s of 2 determinations each.  U3  TABLE IX E f f e c t o f m i n e r a l s , 2,4-D, or 2,4-D-mineral on the r e l a t i v e c o n c e n t r a t i o n s of e t h a n o l - s o l u b l e f r e e amino a c i d s o f r o o t s o f bushbean (Phaseolus v u l g a r i s ) . Days a f t e r 5 Amino A c i d or Amide "Y -Aminobutyric Asparagine Aspartic acid Glutamic a c i d Glycine Isoleucine Leucine Methionine Serine Threonine  acid  C 140* 110 96 100 92 96 96 95 92 110  treatment  10  M  D  160 125 100 125 96 98 98 98 96 120  120 95 96 100 90 95 95 94 90 100  See Table VII Average v a l u e s of 2 determinations each.  MD 150 110 98 110 94 98 98 96 94 110  15  C  M  D  130 100 100 105 100 96 96 100 100 110  180 140 130 130 110 100 100 110 130 135  110 90 95 90 90 90 90 85 90 98  MD 160 120 115 120 110 98 98 110 120 125  C 130 100 105 110 100 100 105 110 110 100  M 200 170 140 130 120 120 120 150 130 140  D  MD  100 85 90 95 85 85 85 82 95 86  165 125 120 115 110 110 110 130 115 120  81.  detectable,  treatments  resulted  These changes r e f l e c t e d organs,  the  i n profound  same g e n e r a l t e n d e n c y  b u t w e r e most marked i n t h e  Table VII r e v e a l s the  profound  the  Treatment w i t h  d e c l i n e became more s t e e p w i t h  d e c l i n e was  gine, aspartic  acid  e x h i b i t e d by and,  aspartic  resulted (b).  acid, but  acid,Y-aminobutyric  i n a course  time.  a l l amino  The  acid,  extent, glutamic  acid,  T h e r e was a t the  caused  by  measured a t the  no  most aspara-  acid. alone  treatment  the  two  glutamic  2,4-D-minerals outlined  i n c r e a s e s were n o t alone.  e x t r e m e s was  in  amino  as g r e a t  This  observed  (a)  as  interi n roots  leaves. (d)  the changes i n such  acid.  15-day o b s e r v a t i o n .  Treatment w i t h  with minerals  asparagine,  glutamic  d e c l i n e i n c o n c e n t r a t i o n o f any  same t i m e  stem, as w e l l as  t h r e o n i n e and  i n t e r m e d i a t e between those  mediate t r e n d between the  and  alone  Treatment w i t h m i n e r a l s  (c)  and  2,4-D  T h o s e s h o w i n g t h e h i g h e s t i n c r e a s e s were  maximum i n c r e a s e was  those  of  a g e n e r a l i n c r e a s e i n the c o n c e n t r a t i o n s of almost a l l  amino a c i d s .  and  three  examination  aminobutyric  to a l e s s e r (b)  The  An  i n the  a g e n e r a l d e c l i n e i n the c o n c e n t r a t i o n of almost  a c i d s and  caused  leaves.  changes.  following: (a)  caused  quantitative  Finally,  i t i s noteworthy t h a t  amino a c i d s as a s p a r a g i n e , " Y - a m i n o b u t y r i c  a c i d were c l e a r l y  d e t e c t a b l e 5 days a f t e r  acid,  treatment.  82.  On the other hand, such amino a c i d s as i s o l e u c i n e , l e u c i n e , g l y c i n e , s e r i n e , methionine showed a delayed change i n t h e i r c o n c e n t r a t i o n , b e i n g d e t e c t a b l e o n l y 10, or i n the most cases 15 days a f t e r the treatments Ill  Photosynthesis  were a p p l i e d .  and R e s p i r a t i o n Rates  The r a t e s o f p h o t o s y n t h e s i s presented  and r e s p i r a t i o n are  i n Table X and a l s o r e p r e s e n t e d  graphically i n  F i g u r e 5. Photosynthesis; photosynthetic  A t the f i v e - d a y o b s e r v a t i o n , the  a c t i v i t y o f the t r e a t e d p l a n t s showed a s l i g h t  d e c l i n e b u t the decreases  were not s i g n i f i c a n t a t 0.05 l e v e l .  In subsequent o b s e r v a t i o n s , the p h o t o s y n t h e t i c r a t e o f the t r e a t e d p l a n t s showed a s i g n i f i c a n t s t i m u l a t i o n . a t t a i n e d a peak a t the 10-day o b s e r v a t i o n .  This stimulation  A t t h i s time o f  measurement, p l a n t s t r e a t e d w i t h m i n e r a l s , 2,4-D, o r 2,4-Dminerals  showed r a t e s o f apparent p h o t o s y n t h e s i s  higher than the c o n t r o l p l a n t s .  16, 26, and 30%  This s i g n i f i c a n t stimulation,  however, tended to fade w i t h time b u t was kept h i g h i n p l a n t s t r e a t e d w i t h 2,4-D-mineral complex.  For example, a t the 15-  day o b s e r v a t i o n , p l a n t s r e c e i v i n g 2,4-D alone showed a photos y n t h e t i c r a t e 14% higher whereas those sprayed w i t h 2,4-Dminerals  s t i l l had a p h o t o s y n t h e t i c r a t e 26% h i g h e r than the  control plants. Rates o f t r u e p h o t o s y n t h e s i s  followed c l o s e l y  TABLE X E f f e c t o f m i n e r a l s , 2,4-D, or 2,4-D-mineral on p h o t o s y n t h e t i c and r e s p i r a t i o n r a t e s o f the a e r i a l p o r t i o n s o f i n t a c t bushbean (Phaseolus v u l g a r i s ) p l a n t s . x  Days a f t e r treatment  Treatment  MD  Apparent Psyn+  5 Respiration  True Psyn  +  10 Apparent R e s p i r True ation Psyn Psyn l e a f surface/hour 3600 1200 4800 +  +  15 Apparent RespirPsyn ation +  True Psyn+  3925  1295  5220  5630*  4310  1390  5690  1495*  6020*  4475*  1490  5965*  1560*  6250*  4875* ,  1660 *  6535*  C  3185  925  4110  M  3050  970  4020  4175*  1455*  D  2880  1000  3880  4525*  2990  1010  4000  4690*  Average v a l u e s o f two d e t e r m i n a t i o n s each. Photosynthesis  03  135 Respiration  Photosynthesis  Photosynthesis  (apparent)  (True)  130  O M  120  4-1  C O U  M-T 0  1330  10C_ D M MD  D M MD  D M MD  D M MD  D M MD  D M MD  D M MD  10  15  D M MD  90 D  10  15  M MD  5 Days  Fig.  5:  10 after  15  treatment  E f f e c t o f 2,4-D ( D ) , m i n e r a l s ( M ) , o r 2 , 4 - D - m i n e r a l (MD) s p r a y s o n r a t e s r e s p i r a t i o n and p h o t o s y n t h e s i s o f a e r i a l p o r t i o n s o f i n t a c t bushbean (Phaseolus v u l g a r i s ) plants.  03  of  the p a t t e r n o f response  o f r a t e s o f apparent  Respiration:  photosynthesis.  Stimulation i n respiration rates of  t r e a t e d p l a n t s was noted from the b e g i n n i n g o f the o b s e r v a t i o n s but the i n c r e a s e s d i d not a t t a i n s i g n i f i c a n c e u n t i l the 10th day.  At the l a t t e r age, p l a n t s t r e a t e d w i t h m i n e r a l s , 2,4-D,  and MD showed r e s p i r a t i o n r a t e s 21, 24, and 30% h i g h e r control plants.  than  As w i t h p h o t o s y n t h e s i s , t h i s s t i m u l a t i o n tended  to be l e s s pronounced a t the 15-day o b s e r v a t i o n i n p l a n t s t r e a t e d w i t h m i n e r a l s alone or 2,4-D alone, b u t was kept a t s i g n i f i c a n t l y h i g h e r l e v e l s p r o v i d e d the p l a n t s were t r e a t e d w i t h 2,4-D-mineral complex. IV  Enzyme A c t i v i t i e s A c t i v i t i e s o f some o f the key enzymes o f carbohydrate  and n i t r o g e n metabolism were determined  i n c e l l - f r e e homogenates  of r o o t s , stem and leaves h a r v e s t e d 5, 10, and 15 days a f t e r the treatment.  These i n c l u d e d phosphorylase,  phosphoglyceryl  k i n a s e , s u c c i n i c dehydrogenase, n i t r a t e r e d u c t a s e , g l u t a m i c o x a l a c e t i c -transaminase,  and c a t a l a s e .  R e s u l t s , expressed as  s p e c i f i c a c t i v i t i e s under the s p e c i f i e d c o n d i t i o n s , a r e presented i n Table XI and X I I . comparative activities.  Table X I I I presents a summary o f  e f f e c t s on s t i m u l a t i o n or s u p p r e s s i o n o f enzyme F i g u r e s 6 to 11 d e p i c t the percentage  values of  enzyme a c t i v i t i e s w i t h r e s p e c t t o c o n t r o l . On a p e r u s a l o f Tables XI and X I I , i t becomes e v i d e n t  86. t h a t both 2,4-D and 2,4-D-minerals,  i n g e n e r a l , had a con-  s i d e r a b l e s t i m u l a t o r y e f f e c t on the a c t i v i t i e s o f a l l the enzymes s t u d i e d .  T h i s s t i m u l a t i o n was, however, not so p r o -  nounced a t the 5-day o b s e r v a t i o n when i n some cases even a s l i g h t r e d u c t i o n i n a c t i v i t i e s o f some enzymes was observed. the passage o f time, b o t h 2,4-D and 2,4-D-minerals significant  brought  With  about  s t i m u l a t i o n s i n a c t i v i t i e s o f a l l the enzymes  assayed 15 days a f t e r the treatment.  Treatment  with minerals  alone had a v a r i a b l e e f f e c t on the enzymes b u t a t 15 days significant  s t i m u l a t i o n was observed o n l y i n a few cases. I t  i s a l s o noteworthy  t h a t s t i m u l a t i o n induced by  2,4-D-mineral  complex, surpassed t h a t by 2,4-D alone i n most cases. 1.  Phosphorylase:  (Table XI, F i g . 6 ) .  a c t i v i t y was s t i m u l a t e d most e f f e c t i v e l y  Phosphorylase  i n the l e a v e s where a l l  the t h r e e treatments r e s u l t e d i n s i g n i f i c a n t l y h i g h e r a c t i v i t y at a l l times o f o b s e r v a t i o n .  A similar trend of stimulation  i s a l s o seen i n stems and r o o t s except t h a t the s t i m u l a t i o n caused by m i n e r a l s alone l a c k e d s i g n i f i c a n c e i n these organs a t 15-day o b s e r v a t i o n . 2. Significant  Phosphoglyceryl kinase:  (Table X i , F i g .  7).  s t i m u l a t i o n i n the a c t i v i t y o f p h o s p h o g l y c e r y l k i n a s e  by both 2,4-D alone and i n combination w i t h m i n e r a l s was noted at the 10- and 15-day o b s e r v a t i o n .  M i n e r a l s alone a l s o  caused  s t i m u l a t i o n o f t h i s enzyme a t these times b u t the i n c r e a s e s were  TABLE XI E f f e c t o f m i n e r a l s , 2,4-D, or 2,4-D-mineral sprays on the a c t i v i t i e s o f phosphorylase, p h o s p h o g l y c e r y l k i n a s e and s u c c i n i c dehydrogenase i n bushbean p l a n t (Phaseolus v u l g a r i s ) .  TreatT i s s u e ment Root C M D MD  Specific activity Phost P.G.K1" 3.80 7.3 3.65 10.1* 3.50 8.6* 3.55 11.2*  Stem  C M D MD  76.5 86. 7* 85.0* 107.0*  2.34 2.20 2.14 2.26  29. 7 33.3* 35.1* 40.0*  Leaf  C M D MD  23.5 33.9* 29.6* 34.6*  6.20 6.00 5.65 5. 78  38.4 31.0* 34.6*1 36.9  + ++. ±. :  x  :  XX. XXX .  +  of S.D.± 31.2 32'." 4 27.6* 30.6  Days a f t e r treatment 10 Specific a c t i v i t y of Phos. P.G.K S.D.# 3.6 4.60 28. 8 4.1* 5.10 33. 0* 5.2*1 5.42* 31.2* 5.4* 6.10* 3 7. 8* x  85.2 96.3* 100.0* 107.4* 20.6 28.1*1 29. 6*1 31.7*  XX  15 Specific activity of Phos. P.G.K. S.D. 2.5 5.20 22.51 2.8 5.68 24.6J I 3.1* 6.25* 26 .,1*1 3.2* 28. 8* 6. 64*  2.52 2.86* 3.05* 3.24*  31.1 40.1* 43.3* 52.4*  87. 5 94.3 105.4* 109.8*  3.34 3.82* 4.16* 4.45*  38. 0 43. 0* 45. 0* 55.5*  7.34 7.95 8. 20* 8.65*  42. 5 50.3* 55. 8* 58.2*  26.0 30. 0* 32.4* 37. 0*  5.40 6. 00 6.11* 7.24*  18. 0 20.0* 20.3* 24.0*  jig i n o r g a n i c phosphate formed/mg p r o t e i n / h o u r Increase i n o p t i c a l density/mg p r o t e i n / h o u r jag formozan formed/mg p r o t e i n / h o u r (Each f i g u r e r e p r e s e n t s computed mean o f 3 r e p l i c a t e s ) Phosphorylase Phosphoglyceryl k i n a s e S u c c i n i c dehydrogenase  % of c o n t r o l s p e c i f i c o o  H O  to  o  to  o  O  activity tn O  HCTi  S O  HUH IPI  ^ Hi O Hi 3 CO CoD rt >d \->rr CO 0 1  Hi  1—  1  1  O O rt  O  o  H Ol  3 MD  in ro  PJ to CO CD 1  3 MD  H O  1-5 0 ^<  o  rt co *•  3  ne  H-  CO rt K CD PJ 3 M  PJ  CO  —'  Qi  o  CD PJ  H  <  to  4^ 1 o O Hi 1 3 C H" C  rt H CD P>  s a O  o  P> 1— 1  H PJ CO CD  01—  CO  o Ul  0  1  he  d rt CO <  3  a  ds  C 0)  rt CD  M Ul  CQ CD CD  3  sd  CD rt  3 o  tm  CD CO  Ul  OJ Hi rt CD i-i  2  P»  a  a  I-" O  s  H ui  a  s  MD  ^  a  so  &>  i—• o •Q  rt p> Hi-i < H- Hco rt  s sa  0  Hi  *88  tr CD PJ Hi 1  Leaf  Stem  Root  140 130  >1 •H  120  >  •H •P  o u  •H M-1 •H O CD  110  CO rH 0  u  -p 100  D M MD  0  D M MD  D M MD  D M  D M MD  MD  D M  MD  o 0  D M  90  MD  D M 10  15  5  MD 10  Days a f t e r Fig.  15  D M MD 5  10  15  treatment.  E f f e c t of 2,4-D (D), m i n e r a l s (M), or 2,4-D-mineral (MD) sprays on the a c t i v i t y of p h o s p h o g l y c e r y l k i n a s e i n r o o t s , stem, and leaves o f bushbean (Phaseolus v u l g a r i s ) p l a n t s .  00  90.  s i g n i f i c a n t o n l y i n the stem t i s s u e s at 10- and  15-day  observation. 3.  S u c c i n i c dehydrogenase:  F i v e days a f t e r  (Table XI, F i g . 8).  treatment s i g n i f i c a n t i n c r e a s e s were found  o n l y i n the stem t i s s u e s .  In the r o o t s and leaves there  was  e i t h e r a s l i g h t i n c r e a s e or a r e d u c t i o n i n the a c t i v i t y .  At  the next o b s e r v a t i o n , a t 10 days, a l l the treatments brought about s i g n i f i c a n t was  maintained  4.  i n c r e a s e s and t h i s s t i m u l a t o r y t r e n d  u n t i l the 15-day o b s e r v a t i o n .  N i t r a t e reductase:  (Table X I I , F i g . 9).  Changes i n the a c t i v i t i e s of enzyme n i t r a t e most p r o f o u n d l y  how  the d i f f e r e n t  to the treatments and a l s o how w i t h time.  exhibited  differently  these changes showed f l u c t u a t i o n s reductase  s t i m u l a t i o n f i v e days a f t e r  At t h i s time of o b s e r v a t i o n ,  by MD  reductase  organs responded  Thus, the a c t i v i t y of n i t r a t e  showed a s i g n i f i c a n t  leaves and  had  i n the r o o t s  treatment.  a c t i v i t y of t h i s enzyme i n the  stems d i d not change s i g n i f i c a n t l y except i n stems  treatment.  hand, both 2,4-D  At the 10- and  15-day o b s e r v a t i o n , on the  and 2,4-D-minerals s i g n i f i c a n t l y  increased  a c t i v i t y o f the enzyme i n the homogenates from r o o t s and as w e l l as l e a v e s . minerals  alone  other  stem,  Furthermore, the s t i m u l a t i o n caused'' by  tended to be  l e s s pronounced by the 15th  whereas the 2,4-D- and 2,4-D-mineral-treated  plants  day,  maintained  TABLE X I I E f f e c t o f m i n e r a l s , 2,4-D, o r 2,4-D-mineral sprays on the a c t i v i t i e s o f n i t r a t e transaminase and c a t a l a s e i n bush bean p l a n t (Phaseolus v u l g a r i s ) .  Days a f t e r  Tissue Root  Stem  Leaf  X.  +. ++.  Treatment  Specific actvity of Nitrate Trans± reductase"*" aminase"*"*" C a t .  x  M D MD  3.7 4. 5*  156 177* 5.6*1 174* 6 . 0*1 180*  49 64* 71* 76*  C M D MD  34 36 37 39*  5.9  681  6.2 6.3  74| 79* 81*  49 1 53 J f 59*  6.0 8.5* 8.1*  68*  9.4*  C M D MD  64 60 58 62  8.4 7.9 7.6 8.2  7.8  *  180 160 166 174  treatment  10 Specific aetvity of Nitrate"*" Trans-++ ± reductase aminase C a t .  33 42* 43* 46*  C,  100 115* 126* 145*  Catalase ityiiri n i t r i t e formed/ mg p r o t e i n / h o u r jum o x a l o a c e t a t e formed/mg p r o t e i n / h o u r mul oxygen liberated/mg p r o t e i n / h o u r (Each f i g u r e r e p r e s e n t s computed mean o f 3 r e p l i c a t e s )  5.8 7.8*  9.0* 10.0*  10.0 11.4* 12.4* 14.2*  reductase,  x  15 Specific actvity of Nitrate"*" Trans-++ ± reductase aminase Cat.  150 181* 195* 201*  56 63 69* 84*  601 67l 71* 74*  531 5?| 62* 70*  140 195*1 216*1  248*  128 1 134 1 147* 167*  8. 8 10. 0 10. 8* 14.2* 6.2 7.4*1 8.1*| 8.8* 21.9 24.0 26.1* 29.4*  91 99 104* 118* 541 57| 60* 67* 108 121. 136* 172 k  Leaf  Stem  Root  170 160  >i  150  •rH  > +> 140 u  rrj  u •rH  130  •rH  g,  120  ro 8 o u 4H O  no 100 D M MD  D M MD  D M MD  D M MD  D M MD  10  15  fJ  D M MD  D M MD  90 D M MD  80  D M  10  15 Days  Fig.  8:  Effect  of  2,4-D  (D),  a c t i v i t y of succinic (Phaseolus v u l g a r i s )  minerals  after  MD  10  5  15  treatment.  to  (M),  or  2,4-D-mineral  dehydrogenase plants.  in  roots,  stem,  (MD)  sprays  and l e a v e s  of  on  the  bushbean  160  Leaf  Stem  Root  150  140 •A >  •A -P CJ  130  m o m  120  w  110  •A v <u  o o S-l -p c o o m o  100 D M MD  D M MD  D M  MD  D M MD D M MD  D M  90  MD  D M MD  D 10  15  5 Days a f t e r  F i g . 9:  10  15  M MD 5  10  D M  MD  15  treatment.  E f f e c t o f 2,4-D (D) , m i n e r a l s (M) , or 2,4-D-mineral (MD) sprays on the a c t i v i t y o f n i t r a t e reductase i n r o o t s , stem, and leaves of bushbean (Phaseolus v u l g a r i s ) p l a n t s .  CO  94. higher a c t i v i t i e s u n t i l the end o f o b s e r v a t i o n 5.  Transaminase:  time.  (Table XII, F i g . 10).  A c t i v i t i e s o f g l u t a m i c - o x a l a c e t i c transaminase ran p a r a l l e l those of n i t r a t e reductase. both 2,4-D  At 10 and  and 2,4-D-minerals had  i n the a c t i v i t y o f the enzyme.  15 days a f t e r  induced  The  pronounced i n r o o t s where the treatment w i t h  10-, 6.  and  Catalase:  72,  and  61%  (Table X I I , F i g . 11).  observed o n l y i n r o o t s and  o b s e r v a t i o n s , both 2,4-D significantly  most  15-day o b s e r v a t i o n .  F i v e days a f t e r treatment, a s i g n i f i c a n t a c t i v i t y was  increases  2,4-D-mineral  complex i n c r e a s e d the transaminase a c t i v i t y by 62, at the 5-,  treatment,  significant  s t i m u l a t i o n was  to  alone and  stimulation i n catalase stem.  At  subsequent  i n combination w i t h  s t i m u l a t e d the a c t i v i t y i n a l l t i s s u e s .  alone brought about s l i g h t i n c r e a s e s t h a t l a c k e d  minerals Minerals  significance  a t 10-day o b s e r v a t i o n i n r o o t s and a t 15-day o b s e r v a t i o n i n all V  organs. Yield Data on y i e l d are i n c l u d e d i n Table XIV  and a l s o shown  g r a p h i c a l l y i n F i g . 12. 1.  Pod f r e s h weight:  Weights of green pods  s i x weeks a f t e r the treatment were i n c r e a s e d s i g n i f i c a n t l y by treatment w i t h 2,4-D  alone or i n combination w i t h  The maximum i n c r e a s e , 2 0%,  was  harvested both  micronutrients.  produced by the 2,4-D-mineral  170  -p  Leaf  Stem  Root  -  160  -rH  > •rH  •P  150  O  rd  u •rH  MH  140  •H  U  d)  0,  130  03 MH  0  O SH •P  C o  MH  o  120 110 100  D M MD  D M MD  nd.  D M MD  D M MD  D M MD  LP  D M MD  90  D M MD  D M MD  D M MD  5  15  10  Days Fig.  10:  Effect  of  activity of  2,4-D of  bushbean  (D),  10 after  minerals  15  10  treatment. (M) ,  glutamic-oxalacetic (Phaseolus  5  15  vulgaris)  or  2,4-D-mineral  transaminase plants.  (MD)  in roots,  sprays  stem,  and  on  the  leaves  vo  180 170  4J •H  >  •rH -P  u rd u  •H M-l -H U  CD &  O  160 150 140 130 120  o  u +J  110  IH  100  c o o  o  D M MD  D M MD  D M MD  D M MD  D M MD  D M MD  D M MD  D M MD  90 D M MD  10  15  5 Days  Fig.  11:  E f f e c t o f 2,4-D (D) , activity of catalase vulgaris) plants.  10 after  15  5  10  15  treatment.  m i n e r a l s (M) , o r 2 , 4 - D - m ' i n e r a l (MD) s p r a y s o n t h e i n r o o t s , stem, and leaves o f bushbean (Phaseolus  KD en  TABLE X I I I Comparative e f f e c t s o f m i n e r a l , 2,4-D, o r 2,4-D-minerals on enzymes of bean p l a n t (Phaseolus v u l g a r i s ) . 5  Tissue  Enzyme  M  D  MD  Days a f t e r treatment 10 Effect of treatment w i t h M D MD M  15  D  MD  + 0 +* + + 0 + + Phosphorylase + 0 0 0 0 + + + 0 + Phosphoglyceryl k i n a s e 0 0 0 0 0 + 0 + + S u c c i n i c dehydrogenase + + + + + + 0 + + N i t r a t e reductase + + + + + + 0 + Transaminase 0 + + 0 + 0 + + Catalase + 0 0 + 0 + 0 + Stem Phosphorylase + + 0 0 0 + 0 + 0 + + Phosphoglyceryl k i n a s e 0 + + + + + + 0 + S u c c i n i c dehydrogenase 0 + 0 + 0 + + 0 + N i t r a t e reductase 0 0 0 + + + + + + Transaminase + + 0 0 0 + + + + Catalase + + + + + + + + Leaf Phosphorylase + 0 0 0 0 + 0 0 0 + Phosphoglyceryl k i n a s e + 0 0 + + 0 + + S u c c i n i c dehydrogenase 0 0 0 + 0 + + + + N i t r a t e reductase 0 0 0 0 + + 0 + + Transaminase 0 0 + 0 + + 0 + + Catalase * The symbols +, 0, and - r e f e r to enzyme s t i m u l a t i o n , no e f f e c t and s u p p r e s s i o n , r e s p e c t i v e l y . The c r i t e r i a f o r s t i m u l a t i o n or suppression were e s t a b l i s h e d a t 15% d e v i a t i o n o f mean v a l u e s of the s p e c i f i c a c t i v i t i e s from the mean c o n t r o l v a l u e f o r each enzyme (Alexander, 1965). Root  98. treatment whereas an i n c r e a s e o f 18% r e s u l t e d from w i t h 2,4-D. was  treatment  M i n e r a l s alone produced an i n c r e a s e o f 6% which  not s i g n i f i c a n t l y d i f f e r e n t from the c o n t r o l a t 0.05 2.  Pod number:  Numbers of pods, 4-cm  level.  and above,  h a r v e s t e d 6 weeks a f t e r the treatment, were a l s o i n c r e a s e d by the three treatments.  The percent i n c r e a s e s by 2,4-D  m i n e r a l s alone and 2,4-D-minerals were 15.5, The  4.5,  alone,  and  20%.  i n c r e a s e s t h a t f o l l o w e d the a p p l i c a t i o n of the m i n e r a l  solution lacked s i g n i f i c a n c e . 3.  Seed y i e l d :  Seed y i e l d was  measured i n terms of  number of f u l l y mature seeds and weight o f a i r - d r y seeds per p l a n t and the weight o f 100 a i r - d r y (a)  seeds.  Number o f seeds:  S i g n i f i c a n t increases  i n the number o f seeds were produced by a l l the The percent i n c r e a s e s by m i n e r a l s alone, 2,4-D m i n e r a l s were 12,  17, and (b)  was  alone, and 2,4-D-  18%. Weight o f seeds:  also s i g n i f i c a n t l y increased.  by the 2,4-D-mineral treatment (c)  treatments.  Seed weight per p l a n t  Maximum i n c r e a s e was  produced  (23%).  Weight of 100  seeds:  Seed weight  was  a l s o i n c r e a s e d by the treatments, but the i n c r e a s e s were not significant. VI  Q u a l i t y of Pods Table XV and F i g . 13. present data on a s c o r b i c a c i d  TABLE XIV E f f e c t o f m i n e r a l s , 2,4-D, o r 2,4-D-mineral on y i e l d o f bushbean p l a n t (Phaseolus v u l g a r i s ) .  Pod weight" "/ p l a n t (g) 1  Treatment  +.  Number pods/plant  Number seeds/plant  Weight d r y seeds/plant  (?)  Weight 100 seeds (q)  c  16.1  4.5  15.3  6.1  40. 0  M  17.1  4.7  17.2*  7.1*  41.2  D  1900*  5.2*  17.9*  7.4*  41. 3  MD  20. 5*  5.4*  18.1*  7.5*  41. 5  4 cm and longer as h a r v e s t e d 6 weeks a f t e r  treatment.  CD CD  TABLE XV E f f e c t o f m i n e r a l s , 2,4-D, or 2,4-D-mineral on v i t a m i n C content, moisture content, and t h e i r l o s s d u r i n g 4 days storage i n bean (Phaseolus v u l g a r i s ) pods.  Treatment  *: **:  at harvest mg/100 g*  Ascorbic acid after 4 days storage mg/100 g*  loss during storage %  at harvest  Moisture after 4 days storage  %  loss during storage 6/**  %  C  13.4  11. 5  14.2  92.1  72.9  20. 8  M  16.2*  13.8*  14.8  91.6  73.4  19.9  D  17. 8*  15.4*  13.5*  90.4  75.2*  16. 8*  MD  19. 0*  16.6*  12.6*  90. 0  75.2*  16.4*  Fresh weight Of o r i g i n a l content  i— o o  1  101.  130  _ F r e s h pod weight  Pod number  Seed weight  Seed number  129 _  120 •-I  o u -p c o 115 o CW  o  110 .  105  100 ... D  M  F i g . 12:  MD  M  MD  M  MD  D  E f f e c t o f 2,4-D (D), m i n e r a l s (M) , or 2,4-D-mineral (MD) sprays on the y i e l d o f bushbean (Phaseolus v u l g a r i s ) p l a n t s .  M  MD  145  Ascorbic  acid  140 _  130 _ Ascorbic acid loss  25 cn U O -P  120 _  Moisture  loss  20 _  03  o u c o u U0 _ m o  £> 15 •H  -P  "°  to  CO O  ^ 100  10 5 0  D  M  F i g . 13:  DM  J D  M  MD  C  D  M  MD  E f f e c t o f 2,4-D (D), m i n e r a l s (M), or 2,4-D-mineral (MD) sprays on a s c o r b i c a c i d content o f f r e s h bean (Phaseolus v u l g a r i s ) p o d s , and l o s s of a s c o r b i c a c i d and moisture d u r i n g 4 days storage of the pods.  o to  103.  content of the f r e s h pods, and a s c o r b i c a c i d and moisture l o s s by pods d u r i n g 4 days storage a t room temperature. 1. content The  (mg/100 gm  A s c o r b i c a c i d content; f r e s h pod weight) was  i n c r e a s e s by the M-,  D-,  The  ascorbic acid  increased  significantly.  and MD-treatment were 21,  34,  and  A s c o r b i c a c i d l o s s d u r i n g storage:  At  the  42%. 2.  end o f 4 days storage, c o n t r o l pods l o s t 14.2% a s c o r b i c a c i d , whereas the pods from 2,4-D p l a n t s l o s t o n l y 12.6%.  of the  original  plus mineral-treated  T h i s amounts to a r e d u c t i o n of  11%  i n the v i t a m i n l o s s i n the pods from t r e a t e d p l a n t s . 3.  Moisture  l o s s during storage:  A l l three  treatments a f f o r d e d p r o t e c t i o n a g a i n s t moisture l o s s d u r i n g the 4-day storage p e r i o d . 2,4-D-mineral-treated  Pods produced by 2,4-D-treated  and  p l a n t s l o s t 19% and 22% l e s s water than  d i d the pods from u n t r e a t e d p l a n t s .  The  e f f e c t of the  mineral  treatment a l s o r e s u l t e d i n l e s s moisture l o s s than c o n t r o l but the d i f f e r e n c e l a c k e d  significance.  104. DISCUSSIONS The  r e s u l t s presented h e r e i n are c o n s i s t e n t w i t h the view  t h a t when a p p l i e d i n a p p r o p r i a t e l y low c o r r e c t ontogenetic c o n d i t i o n s , 2,4-D  p e r i o d and  under a p p r o p r i a t e  l e a d to improved y i e l d  t h a t t h i s may  (Wedding e t a l . 1956;  the  climatic  can indeed b r i n g about s i g n i f i c a n t  i n j u v e n i l e growth o f bean p l a n t s , and  Further,  doses to p l a n t s a t  stimulation finally  Wort 1964a).  the data s t r o n g l y suggest t h a t i n c l u s i o n o f compounds  of m i c r o n u t r i e n t s ,  Cu,  Fe,  Zn, Mn,  and B, augments the  u l a t o r y a c t i o n of the hormone (Wort 1964b). compatible w i t h the data of the present  stim-  T h i s view i s a l s o  investigation  such p h y s i o l o g i c a l processes as p h o t o s y n t h e s i s ,  concerning  respiration,  and a c t i v i t i e s o f enzyme systems i n the t r e a t e d p l a n t s . We  n o t i c e from the data t h a t as the s t i m u l a t i o n i n growth  progresses w i t h time, so do the r a t e s o f carbon a s s i m i l a t i o n , r e s p i r a t i o n , and  enzyme a c t i v i t y .  In a d d i t i o n , the data  q u a n t i t a t i v e changes i n amino a c i d s , as s h a l l be a greater  discussed  d e t a i l l a t e r , s t r o n g l y suggest an i n c r e a s e d  protein synthesis  under the i n f l u e n c e of 2,4-D.  w i l l be b r i e f l y d i s c u s s e d available literature.  and  in  de novo  These aspects  evaluated below, i n the l i g h t  of  F i n a l l y , an attempt w i l l be made to  incorporate these v a r i e d r e s u l t s i n t o a g e n e r a l i z e d t h a t may  on  e x p l a i n data of the present  o f f e r meaningful e x p l a n a t i o n s  hypothesis  i n v e s t i g a t i o n and may  f o r the r e s u l t s obtained by  also other  105. i n v e s t i g a t o r s i n t h i s area o f hormone r e s e a r c h .  Practical  s i g n i f i c a n c e w i l l be c o n s i d e r e d . 1.  J u v e n i l e growth:  Although commendable a t t e n t i o n  has been p a i d i n the p a s t few years to the use o f aqueous sprays and dusts w i t h low 2,4-D doses to o b t a i n g r e a t e r y i e l d s o f s e v e r a l crop s p e c i e s , the study o f immediate e f f e c t s o f these treatments  on growth, and p h y s i o l o g i c a l and b i o c h e m i c a l s t a t u s  o f p l a n t s , s t i l l remains i n an i n f a n t stage.  Studies o f such  a l t e r a t i o n s induced i n a r e l a t i v e l y s h o r t p e r i o d a f t e r  treat-  ment have the twin advantage o f p e r m i t t i n g one to c o r r e l a t e the growth responses w i t h b i o c h e m i c a l changes, and make i t f e a s i b l e t o study the i n t e r a c t i o n o f hormonal e f f e c t s w i t h other p l a n t and environmental v a r i a b l e s .  Both these a r e b a s i c  to the understanding o f the mechanism o f a c t i o n o f growth substances . E a r l i e r r e p o r t s on s t i m u l a t i o n o f v e g e t a t i v e growth by low 2,4-D c o n c e n t r a t i o n s a r e those o f T a y l o r (1964a, b) and Swanson (1946).  Of s p e c i a l i n t e r e s t i n r e l a t i o n to the  f i n d i n g s o f p r e s e n t i n v e s t i g a t i o n on j u v e n i l e growth, a r e the r e c e n t o b s e r v a t i o n s made by M i l l e r e t a l . (1962 a ) .  Their  r e s u l t s a l s o showed s i g n i f i c a n t i n c r e a s e s i n shoot e l o n g a t i o n , t o t a l l e a f area, shoot and r o o t d r y matter  and r o o t n o d u l a t i o n as  measured 14 days a f t e r a p p l i c a t i o n o f 0.5 and 1 ppm aqueous 2,4-D sprays t o young bean s e e d l i n g s .  106. 2.  Yield:  S e v e r a l i n v e s t i g a t o r s have r e p o r t e d i n  the past on the s i g n i f i c a n t i n c r e a s e s o b t a i n e d caused by  a p p l i c a t i o n of f o l i a r  low  c o n c e n t r a t i o n s w i t h or without m i c r o n u t r i e n t  2,4-D  S p e c i a l mention may  sprays  (Wedding e t a l . 1956), sugar beet (Wort,1959), c o t t o n  (Wort, 1966), corn and  ( A r l e , 1954a,b), beans  salts. these  potato  ( M i l l e r et a l .  (Huffaker  e t a l . 1967).  I n t e r a c t i o n between 2,4-D and m i c r o n u t r i e n t s i n improving growth and y i e l d : E a r l i e r i t was  micronutrient  containing  (Wort, 1953), lima beans  1962b; Wort, 1966), and b a r l e y and wheat 3.  dusts  be made o f the i n c r e a s e s o b t a i n e d by  treatments i n the y i e l d of buckwheat  tubers  and  i n crop y i e l d s  s a l t s i n 2,4-D  p o i n t e d out t h a t i n c l u s i o n o f  sprays  augmented the  a c t i o n of the hormone on growth and y i e l d .  stimulatory  I t seems o f  interest  to examine t h i s s t i m u l a t o r y r o l e of microelements more c l o s e l y . Table XVI  g i v e s percentage data on f r e s h and  2,4-D- and plants.  2,4-D-mineral-treated  I t may  p l a n t s w i t h r e s p e c t to c o n t r o l  be noted t h a t the dry weight o f stem a t 15  f o r example, i s i n c r e a s e d by The  dry weights of  corresponding  10% w i t h 2,4-D  i n c r e a s e by m i n e r a l s  of about one-and-a-half times h i g h e r . mentory r o l e of m i n e r a l cases of f r e s h and  ions on 2,4-D  days,  treatment.  p l u s 2,4-D  i s o f the  S i m i l a r pronounced  order  aug-  a c t i o n i s e v i d e n t i n most  dry weights as w e l l as on the  yield.  TABLE XVI Comparative e f f e c t s o f treatment o f bushbean (Phaseolus v u l g a r i s ) p l a n t s w i t h 2,4-D alone and i n combination w i t h m i c r o n u t r i e n t s on f r e s h and dry weights o f r o o t s , stems and l e a v e s . Days a f t e r treatment 10  5 Treatment  Roots  15  Stem  Leaves  Roots  Stem  Leaves  Roots  Stem  Leaves  121*  105  77  117  119  117  116  121  120  MD  130  111  86  120  124  128  118  125  130  D  107  93  70  113  109  106  114  110  115  MD  111  130  75  117  114  106  116  114  125  Fresh Weight  D  +  Dry Weight  Percent o f c o n t r o l v a l u e s . For t r a n s l a t i o n o f codes see Table I I .  108.  4.  Chemical changes: a.  Water c o n t e n t:  An i n i t i a l r i s e i n water  content o f 2,4-D t r e a t e d p l a n t s , as observed i n the p r e s e n t study, has a l s o been r e p o r t e d i n bean stems n a s t u r t i u m and soybean p l a n t s  (Brown, 1946),  ( F r e i b e r g and C l a r k , 1952).  (1953) observed a r i s e i n water content o f buckwheat between  0.5 t o 8 days a f t e r treatment w i t h 2,4-D.  Wort  plants  This  i n i t i a l r i s e i n water content a c c o r d i n g to Bradbury and Ennis (1952) may be the consequence o f p a r t i a l c l o s u r e o f stomata by' 2,4-D sprays. A l t e r n a t i v e l y , t h i s may a l s o r e s u l t from a d i r e c t i n c r e a s e i n water uptake under the i n f l u e n c e o f 2,4-D,  In f a c t ,  there i s an impressive body o f data t h a t s t i m u l a t o r y c o n c e n t r a t i o n s of  auxins s t i m u l a t e water uptake by e x c i s e d t i s s u e s  ( C l e l a n d , 1961).  From the data p r e s e n t e d i n Table IV i t i s observed t h a t 2,4-D treatment i n c r e a s e s water content o f b o t h tops and r o o t s , and that water content o f r o o t s was i n c r e a s e d a t l e a s t as much as those o f tops.  I t should t h e r e f o r e be concluded t h a t the i n -  creased water c o n t e n t i s a consequence o f i n c r e a s e d water uptake through hormone a c t i o n .  T h i s view i s f u r t h e r strengthened by the  observed s t i m u l a t i o n i n p h y s i o l o g i c a l p r o c e s s e s , which, o f course, would r e q u i r e g r e a t e r q u a n t i t i e s of water uptake. b. of  C h l o r o p h y l l c o n t e n t:  While higher doses  2->-4-D have been r e p o r t e d to i n h i b i t c h l o r o p h y l l s y n t h e s i s or  cause i t s d e s t r u c t i o n  (Osborne and Hallaway, 1961; Wedding e t a l .  1954), l i t t l e work has been done t o study the response o f low,  109. stimulatory concentrations  on c h l o r o p h y l l content.  ment w i t h the r e s u l t s o f the present c h l o r o p h y l l content observation, of 1 ppm  f i n d i n g s o f an  increased  i n t r e a t e d leaves a t the 10- and  are the r e s u l t s o f C h i  2,4-D  In agree-  15-day  (1965) , t h a t a p p l i c a t i o n  sprays i n c r e a s e d c h l o r o p h y l l content  of bean  leaves by 23% w i t h i n 7 days. A p o s s i b l e e x p l a n a t i o n f o r the  inhibited  c h l o r o p h y l l s y n t h e s i s a t the 5-day o b s e r v a t i o n i n the study can be  found i n the work o f T u l l i n  a t o r observed t h a t i n j e c t i o n o f 2,4-D  (1962).  alone  young wheat s e e d l i n g s caused c h l o r o s i s .  present  This  i n t o endosperms o f  However, when 2,4-D  i n j e c t e d together w i t h M g , the p l a n t s remained green. ++  mation of a 2,4-D-Mg complex was t h i s behavior.  Mg  may  study.  decomposition of t h i s complex may  accumulation.  then l e a d to r a p i d c h l o r o p h y l l  treatment  f o r i n c r e a s e d i o n uptake  (Cooke, 1957).  The view presented  above a l s o g i v e s  e x p l a n a t i o n f o r the p r o t e c t i v e a c t i o n of m i c r o n u t r i e n t on c h l o r o p h y l l s y n t h e s i s  through  a l s o b r i n g about r a p i d c h l o r o p h y l l  There i s some evidence  by r o o t s f o l l o w i n g 2,4-D  have  Subsequent  A l t e r n a t i v e l y , i n c r e a s e d uptake o f Mg  r o o t s i n l a t e r stages, may  For-  An i n i t i a l b i n d i n g of endogenous  t e m p o r a r i l y impair c h l o r o p h y l l s y n t h e s i s .  biosynthesis.  was  proposed as the e x p l a n a t i o n f o r  I t seems t h a t a s i m i l a r phenomenon may  o c c u r r e d i n the present  investig-  (Table V).  an ions  In t h i s case, p a r t of  110. 2,4-D  may be complexed w i t h some o f the microelement  i o n s , thus  s p a r i n g the M g t o p a r t i c i p a t e i n c h l o r o p h y l l b i o s y n t h e s i s . + +  c.  Sugar content:  S e v e r a l i n v e s t i g a t o r s i n the  p a s t have r e p o r t e d d e p l e t e d sugar and carbohydrate content of 2,4-D-treated p l a n t s Rasmussen, 1947; 1951;  ( M i t c h e l l and Brown, 1945;  S e l l e t a l . 1949;  W e l l e r e t a l . 1950;  Hofmann and Schmelling, 1953).  Wort,  Most of the quoted  i n v e s t i g a t o r s used h i g h , l e t h a l doses o f periments, hence, i t i s d i f f i c u l t  Smith et a l . 1947;  2,4-D  i n their  ex-  to decide whether these  r e s u l t s r e f l e c t decreases due to breakdown of sugar or i m p a i r ment o f p h o t o s y n t h e s i s .  Secondly, no one has attempted  the changes i n d i f f e r e n t p l a n t organs, as was present i n v e s t i g a t i o n . of  undertaken  For these reasons, a d i r e c t  to study i n the  comparison  the p r e s e n t data w i t h above-mentioned r e p o r t s would not seem  very v a l i d . The noteworthy  f e a t u r e o f changes i n sugar  content i n the p r e s e n t study i s the o p p o s i t e response  observed  i n r o o t s (decrease i n sugars) and l e a v e s ( i n c r e a s e i n s u g a r s ) . Inasmuch as the t r e a t e d p l a n t s showed an i n c r e a s e d p h o t o s y n t h e t i c e f f i c i e n c y , i t cannot be expected t h a t the sugar c o u l d have t r a n s l o c a t e d from r o o t s to the l e a v e s .  In f a c t , under these  cir-  cumstances one would expect an i n c r e a s e d supply o f sugars to the roots.  111. In view of a v e r y c l o s e c o r r e l a t i o n between carbohydrate and amino a c i d metabolism, an e x p l a n a t i o n f o r these changes may be sought i n amino a c i d content. d.  Amino a c i d content:  A steady d e c l i n e i n  q u a n t i t y o f s e v e r a l amino a c i d s , n o t a b l y i n asparagine, a s p a r t i c acid, V  - a m  i °kutyric n  a c i d and glutamic a c i d , i n the 2,4-D-treated  p l a n t s corresponds c l o s e l y to the o b s e r v a t i o n made by Payne e t al.  (1952).  In the l a t t e r study 11 amino a c i d s were found to have  decreased i n q u a n t i t y i n the tubers f o l l o w i n g 2,4-D a e r i a l p a r t s o f the potato p l a n t s .  Similar observations of  decreases i n f r e e amino a c i d by 2,4-D by F a l u d i and D a n i e l (1958), Kamal (1958), and R a k i t i n and Zemskaya worthy t h a t i n c o n t r a s t to 2,4-D  sprays to the  treatment have been r e p o r t e d  (1960), Menoret and Morel  (1958).  F u r t h e r , i t i s note-  action, treatment w i t h m i n e r a l s  alone caused an i n c r e a s e i n the p o o l o f f r e e amino a c i d s .  The  p l a n t s t r e a t e d w i t h 2,4-D p l u s m i n e r a l s showed c o n c e n t r a t i o n i n t e r m e d i a t e between these two trends.  C o n s i d e r i n g the con-  c e n t r a t i o n of asparagine as an i n d i c a t o r o f the r e l a t i v e p o o l s i z e , and c a l c u l a t i n g the percent i n c r e a s e or decrease i n t h i s amino a c i d by the treatments, the r e s u l t s are o b t a i n e d as d e p i c t e d i n F i g u r e 14. amino a c i d s .  The same type o f curves can be o b t a i n e d f o r other From t h i s f i g u r e i t becomes a t once e v i d e n t t h a t  the drainage on the amino a c i d p o o l as caused by 2,4-D r e p l e n i s h e d i n treatment w i t h 2,4-D-plus m i n e r a l s .  action i s  CN  rH  60 J  |  I  I  |  I  I  I  5  10  15  5  10  15  5  Days a f t e r Fig.  14:  I  I 10  15  treatment.  R e l a t i v e c h a n g e s i n t h e c o n c e n t r a t i o n o f a s p a r a g i n e as i n d u c e d b y 2•,4-.D (•-—•) minerals .) , o r 2 , 4 - D - m i n e r a l ( ~ • ~ ) , s p r a y s i n b u s h b e a n (Phaseolus x  vulgaris)  x  leaves. #  . x-.-x  ,  Minerals  alone  .  2,4-D-alone Minerals plus  2,4-D  113.  Considering  the changes i n sugars and amino a c i d s  the a l t e r a t i o n s may  be  e x p l a i n e d as f o l l o w s .  caused a g r e a t e r s y n t h e s i s of p r o t e i n and amino a c i d c o n c e n t r a t i o n alone  f a v o r e d a higher  e l i c i t i n g any  treatment  hence a d e c l i n e i n  i n the pool; treatment w i t h  minerals  s y n t h e s i s of the amino a c i d s without  increase i n protein synthesis, therefore free  amino a c i d s accumulated. minerals,  2,4-D  together,  In p l a n t s t r e a t e d w i t h 2,4-D  plus  the s y n t h e s i s o f amino a c i d s and of p r o t e i n were both  stimulated. T h i s theory may to the content it  e x p l a i n other observed f a c t s i n a d d i t i o n  o f sugars and amino a c i d s .  i s i n accordance w i t h  In the f i r s t  place,  the r e l a t i v e i n c r e a s e s i n growth  y i e l d brought about by the three treatments.  and  Secondly, i t a l s o  e x p l a i n s the d i f f e r e n t i a l p a t t e r n of sugars i n leaves and In the leaves an i n c r e a s e d r a t e of photosynthesis provided  have  s u f f i c i e n t sugars to be u t i l i z e d f o r amino a c i d  p r o t e i n s y n t h e s i s and occur.  may  roots.  and  t h e r e f o r e no d e c l i n e i n sugar l e v e l  should  In the r o o t s on the other hand, t h i s i n c r e a s e d p r o t e i n  s y n t h e s i s , might have taken p l a c e a t the expense o f s t o r e d sugars. The v a l i d i t y of t h i s view, of course,  r e s t s on the  assumption  that the amino a c i d s f o r p r o t e i n s y n t h e s i s are d e r i v e d from p o o l of f r e e amino a c i d s .  the  That t h i s i s so has been e s t a b l i s h e d  beyond doubt f o r m i c r o b i a l and mammalian systems.  And more and  114. more evidence i s forthcoming to demonstrate t h a t the same i s true f o r p l a n t s .  Clark  (1958) demonstrated amino a c i d a c t i v a t i o n  i n d i a l y z e d e x t r a c t s o f acetone powders o f spinach oleracia) leaves.  He a l s o r e p o r t e d  (Spinacia  the same a c t i v i t y  associated with soluble f r a c t i o n s of several plant c e l l  species  i n c l u d i n g Avena c o l e o p t i l e , corn s e e d l i n g s , r y e g r a s s , and pea epicotyls.  Davis and N o v e l l i (1958) i s o l a t e d a p r o t e i n f r a c t i o n  from pea t h a t c a t a l y z e d an ATP-pyrophosphate exchange which was s t i m u l a t e d by 11 amino a c i d s . p r o v i d e d by s e v e r a l other  S i m i l a r evidence has a l s o been  i n v e s t i g a t o r s u s i n g p l a n t t i s s u e ex-  t r a c t s , as summarized by Mans  (1967).  concluded from these evidences t h a t  Indeed, Webster  " I t i s now w e l l  (1961)  established  t h a t enzymes t h a t c a t a l y z e the a c t i v a t i o n o f amino a c i d s occur i n c e l l s o f many d i f f e r e n t p l a n t s indeed u n l i k e l y t h a t the order cell  (animals,  and m i c r o b e s ) .  of biochemical  systems l e a d i n g to a p o l y m e r i z a t i o n  It i s  events i n p l a n t  o f a g i v e n number o f  s p e c i f i c amino a c i d s i n t o a p r e c i s e l y predetermined sequence t o form a p a r t i c u l a r p o l y p e p t i d e ,  d i f f e r s from the order  i n m i c r o b i a l and mammalian systems".  established  115. 4.  Respiration S e v e r a l i n v e s t i g a t o r s i n the past have r e p o r t -  ed an i n c r e a s e d r a t e of r e s p i r a t i o n of i n t a c t p l a n t s f o l l o w i n g 2,4-D  a p p l i c a t i o n (Brown, 1946;  Corns, 1950;  Rasmussen, 1947).  However, a l l these experiments were performed w i t h h i g h , doses o f 2,4-D  and  t h e r e f o r e these r e s u l t s cannot be  lethal  expected  to have b e a r i n g on the f i n d i n g s o f the present i n v e s t i g a t i o n . Of s p e c i a l s i g n i f i c a n c e i n r e l a t i o n to i n c r e a s e d r e s p i r a t i o n and growth observed i n the present two  recent reports.  Switzer  (1957) observed t h a t  i s o l a t e d from soybean p l a n t s sprayed with  low  t r a t i o n s possessed an i n c r e a s e d o x i d a t i v e and activity.  S i m i l a r l y , Key  2,4-D  concen-  phosphorylative  et a l . (1960) have r e p o r t e d  i s o l a t e d from 2,4-D-treated soybean  Inasmuch as mitochondria production,  increased  r e s p i r a t i o n and p o i n t to one  i n the l i g h t o f  increased  growth observed i n the present i n v e s t i g a t i o n ,  effect. Photosynthesis Higher doses o f 2,4-D  a p p l i e d to  intact  p l a n t s have r e s u l t e d i n decreased r a t e s o f photosynthesis 1949,  1950;  ATP  p o s s i b l e s i t e i n metabolism where auxin might e x e r t  i t s stimulatory 5.  i n mito-  hypocotyls.  are the most important s i t e s of  these r e p o r t s c o n s i d e r e d  study are  mitochondria  s w e l l i n g and enhanced r a t e o f o x i d a t i v e p h o s p h o r y l a t i o n chondria  the  L o u s t a l a t and Muzik, 1953;  R a k i t i n and  (Freeland,  Potapova, 1959).  116.  Wedding e t a l . (1954) observed  i n h i b i t i o n o f photosynthesis  when c i t r u s l e a f d i s k s were impregnated  w i t h 2,4-D s o l u t i o n s .  However, the authors p o i n t e d out t h a t "The s h o r t p e r i o d o f exposure and the u n n a t u r a l c o n d i t i o n s p r e v a i l i n g d u r i n g the treatment  and d u r i n g the measurement o f p h o t o s y n t h e s i s do not  allow d i r e c t comparison w i t h f i e l d a p p l i c a t i o n s o f the growth regulator". Indeed, the f a c t t h a t low 2,4-D doses under f i e l d c o n d i t i o n s have g i v e n s i g n i f i c a n t i n c r e a s e s i n d r y matter and y i e l d o f s e v e r a l crops has been taken to s i g n i f y a consequence o f enhanced p h o t o s y n t h e s i s  (Wort, 1962).  The s t i m u l a t i o n i n p h o t o s y n t h e s i s by low 2,4-D c o n c e n t r a t i o n s as o b t a i n e d i n t h i s study, was a l s o e v i d e n t i n the work o f C h i (1965).  Furthermore,  the l a t t e r r e -  p o r t a l s o emphasized the v a r i a b i l i t y o f e f f e c t w i t h the c o n c e n t r a t i o n o f hormone a p p l i e d .  Thus, 0.5 and 1 ppm 2,4-D  s t i m u l a t e d p h o t o s y n t h e t i c a c t i v i t y much as i n the p r e s e n t i n vestigation.  However, 5 ppm 2,4-D sprays were found t o i n h i b i t  photosynthesis. Other p i e c e s o f evidence, such as t h a t o f Huffaker e t a l . (1962), a l s o i n d i c a t e the s t i m u l a t i o n o f p h o t o s y n t h e s i s as o b t a i n e d i n the study 10 and 15 days a f t e r the a p p l i c a t i o n o f 2,4-D.  117.  Using  c<  ^2  these i n v e s t i g a t o r s demonstrated t h a t dark CO2 f i x -  }  a t i o n , c a t a l y z e d by phosphoenolpyruvate carboxylase  and the  ribulose-5-phosphate r e a c t i o n systems showed i n c r e a s e d at t h a t l e v e l o f 2,4-D treatment which s t i m u l a t e d yield.  As the c o n c e n t r a t i o n  of the c a r b o x y l a t i o n 6.  stimulation  disappear.  Enzyme A c t i v i t i e s (2)  Physiological basis for stimulation of enzyme a c t i v i t i e s An  concerning  growth and  o f 2,4-D was i n c r e a s e d ,  system tended to  activity  impressive  body o f l i t e r a t u r e  the e f f e c t o f 2,4-D on s e v e r a l enzyme systems e x i s t s .  Some o f the p e r t i n e n t l i t e r a t u r e has a l r e a d y been summarized i n Table I.  Of s p e c i a l s i g n i f i c a n c e i n r e l a t i o n t o s t i m u l a t i o n  of enzymes by 2,4-D i s a r e c e n t r e p o r t by F l o o d e t a l . (1967). These i n v e s t i g a t o r s r e p o r t e d  t h a t treatment o f a r t i c h o k e  tuber  d i s c s f o r 3 days w i t h 10"^ M 2,4-D r e s u l t e d i n a s e v e r a l - f o l d i n c r e a s e i n the a c t i v i t y o f i n v e r t a s e and h y d r o l a s e .  The  a c t i v i t y o f i n v e r t a s e was a l s o i n c r e a s e d but not that o f hydrolase  i n chicory roots. Several hypotheses have been proposed  to e x p l a i n the s t i m u l a t i o n o f enzyme a c t i v i t y under 2,4-D i n fluence.  The f a c t t h a t s t i m u l a t i o n or suppression  a c t i v i t y i s dependent on the c o n c e n t r a t i o n  o f enzyme  o f 2,4-D has l e d  Freed e t a l . (1961) to propose t h a t t h i s ^ b e h a v i o r c o u l d be  118.  explained  on the b a s i s o f an a d s o r p t i o n  s u r f a c e s by p h y s i c a l f o r c e s . theory,  that adsorption  o f 2,4-D on the p r o t e i n  I t i s proposed, a c c o r d i n g  to t h i s  o f small q u a n t i t i e s o f 2,4-D might  i n c r e a s e the e f f i c i e n c y o f the c a t a l y s t , b u t as a d d i t i o n a l molecules a r e adsorbed the p r o t e i n s t r u c t u r e i s a l t e r e d so t h a t a loss of c a t a l y t i c property suggest no e x p l a n a t i o n  results.  However, these authors  as to how a p h y s i c a l a d s o r p t i o n  change the k i n e t i c s o f enzyme r e a c t i o n .  might  Moreover, no e f f o r t has  been made t o e x p l a i n the r e v e r s i b l e nature o f these r e g u l a t o r e f f e c t s on enzyme systems, as observed i n t h i s study as w e l l as r e p o r t e d by s e v e r a l other Alexander, 1965).  i n v e s t i g a t o r s (Wort and Cowie, 1953;  A t the same time, the l i t e r a t u r e on such  e f f e c t s suggests the e x i s t e n c e  o f a common b a s i s f o r these  phenomena. Wort,(1962) advanced the suggestion s t i m u l a t o r y e f f e c t o f low c o n c e n t r a t i o n s  that  o f 2,4-D may a r i s e from  the i n c o r p o r a t i o n o f the hormone i n t o a s u b s t r a t e - r e g u l a t o r enzyme complex w i t h a lower energy o f a c t i v a t i o n than t h a t o f the substrate-enzyme complex. to t h i s theory,  I t i s f u r t h e r proposed,  according  t h a t 2,4-D s a t u r a t i o n o f enzyme and s u b s t r a t e  s e p a r a t e l y c o u l d e x p l a i n the slowing 2,4-D c o n c e n t r a t i o n s .  of reaction with  higher  The noteworthy f e a t u r e o f t h i s h y p o t h e s i s  i s t h a t s i n c e i t proposes the formation  o f a complex i n which both  119.  r e g u l a t o r and  s u b s t r a t e p a r t i c i p a t e , the r e g u l a t o r can move  out of the complex once the s u b s t r a t e has been converted to the products.  Thus i t allows f o r the v a r i a b i l i t y w i t h time on  e f f e c t of the r e g u l a t o r on the same enzyme a c t i v i t y .  the  Secondly,  s i n c e formation of a complex should i n v o l v e some s o r t of chemical l i n k a g e or b i n d i n g , t h i s theory a l s o accounts s p e c i f i c i t y of r e g u l a t o r e f f e c t .  f o r the  Only those enzymes or  s u b s t r a t e s which p r o v i d e s u i t a b l e s i t e f o r b i n d i n g or r e g u l a t o r , would be a f f e c t e d .  Indeed t h a t such a s p e c i f i c i t y does e x i s t  i s e v i d e n t i n the p r e s e n t r e s u l t s and i s a l s o r e p o r t e d by s e v e r a l other i n v e s t i g a t o r s .  Wort and Cowie (19 53) r e p o r t e d  marked d i f f e r e n c e s i n the e f f e c t o f 2,4-D  on. phosphorylase  -amylase a c t i v i t y and t h e i r f l u c t u a t i o n w i t h time.  A recent  r e p o r t of F l o o d e t a l . (1967) shows t h a t while treatment 2,4-D  with  i n c r e a s e d the a c t i v i t y of i n v e r t a s e i t had no e f f e c t  hydrolase enzyme i n c h i c o r y r o o t s .  A s i m i l a r response  and  on  i n enzyme  a c t i v i t y i s e v i d e n t from the r e s u l t s presented i n Table XI and X I I . The r e s u l t s of enzyme a c t i v i t i e s o b t a i n e d may  t h e r e f o r e be b e s t e x p l a i n e d on the b a s i s of the theory  by Wort  (1964) t h a t 2,4-D  after entering into a plant participates  i n the formation of a substrate-enzyme-regulator an a c t i v a t i o n energy enzyme complex.  proposed  complex w i t h  l e v e l d i f f e r e n t from t h a t of s u b s t r a t e -  F u r t h e r support f o r t h i s comes from the r e s u l t s  120.  t h a t m i n e r a l ions which are known to p a r t i c i p a t e i n enzyme a c t i v i t y , a f f e c t e d the a c t i o n of 2,4-D T h i s w i l l be  considered (b)  on enzyme s t i m u l a t i o n .  i n more d e t a i l i n the next s e c t i o n . Mechanism o f the e f f e c t of micron u t r i e n t s a p p l i e d i n the p r e s e n t study. In the present i n v e s t i g a t i o n , the  m i n e r a l spray c o n t a i n e d ments.  f i v e o f the e s s e n t i a l m i c r o n u t r i e n t  These i n c l u d e d manganese, z i n c , copper, i r o n , and  a l l as s u l f a t e s except boron which was  ele-  boron,  used as b o r i c a c i d .  The  e f f e c t of these on growth must have been mediated through t h e i r e f f e c t s on metabolism. micronutrients  malic  pinpointed  these metal i o n s .  enzyme, and  some of the enzymes a c t i v a t e d i n  Thus M n  + +  has been shown to a c t i v a t e  i s o c i t r a t e dehydrogenase (Anderson and  1956) ; phosphoenolpyruvic carboxy k i n a s e 1957)  and  e s t a b l i s h e d t h a t these  a f f e c t the a c t i v i t y of s e v e r a l p l a n t enzymes.  Recent evidence has p l a n t s by  Indeed i t i s now  glutathione  reductase  (Mazelis  (Humphries, 1955).  and  Evans,  Vennesland,  Zinc  a c t i v a t e s s e v e r a l enzymes i n c l u d i n g NAD-dependent dehydrogenases (Nicholas 1957).  and Maybey, 1960)  and hexokinase  Iron p a r t i c i p a t e s i n the s y n t h e s i s  activates c h l o r o p h y l l synthesis port chain.  There i s l i t t l e  and  (Medina and  Nicholas,  of p o r p h y r i n ;  functions  i n electron  it trans-  doubt as to the s i g n i f i c a n c e o f  copper f o r normal p l a n t metabolism. component o f cytochrome oxidase and  I t has been shown to be also participates in  the  a  121  r e a c t i o n s of a s c o r b i c a c i d oxidase,  and phenolases.  Boron  has been suggested to a c t i v a t e carbohydrate m e t a b o l i z i n g The brief, summary emphasizes widespread and  c r i t i c a l r o l e played by  m e t a l l i c ions i n p l a n t metabolism. s u r p r i s i n g that a r e l a t i v e l y of these m i c r o n u t r i e n t s ,  these  the  micronutrient  I t i s therefore,  low c o n c e n t r a t i o n  as i n the present  enzymes.  not  of exogenous  study, may  supply  l e a d to  an enhancement of metabolism by a c t i v a t i n g many of the above mentioned enzyme systems, i n p a r t i c u l a r such energy y i e l d i n g r e a c t i o n s as c a t a l y z e d by dehydrogenases, k i n a s e s enzymes of the e l e c t r o n t r a n s p o r t  might a f f e c t 2,4-D  to b r i e f l y consider metabolism.  the  chain.  P r i o r to c o n s i d e r i n g micronutrients  and  the ways i n which the  a c t i o n , i t seems p e r t i n e n t  the mechanism of t h e i r a c t i o n on  plant  Dixon and Webb (1964) have enumerated s e v e r a l  p o s s i b l e ways i n which metal ions can a c t i v a t e an enzyme: (i) p a r t of the a c t i v e center  The metal may of the enzyme.  (ii) l i n k between the enzyme and and  so h o l d i n g  form an e s s e n t i a l  the s u b s t r a t e (iii)  can produce a c t i v a t i o n i s by  The metal i o n may substrate,  a c t as a b i n d i n g  combining w i t h both  at the a c t i v e center Another way  of the enzyme.  i n which metal ions  changing the  equilibrium  122.  constant o f the enzyme r e a c t i o n .  In cases, f o r example, where  the e q u i l i b r i u m c o n s t a n t i s u n f a v o r a b l e f o r i t s occurrence, a change o f the c o n s t a n t i n a f a v o r a b l e d i r e c t i o n may  enable  the r e a c t i o n t o take p l a c e and so produce an apparent  activation.  T h i s c o u l d be achieved e i t h e r by c o n c e n t r a t i n g the s u b s t r a t e metal complex or by removing the product o f the r e a c t i o n by combining  with i t . (iv)  I t i s also possible that  r e a c t i o n s may be a f f e c t e d by a change o f s u r f a c e charge on the enzyme p r o t e i n brought  about by the charge on t h e c a t i o n . (v)  enzymes i n many i n d i r e c t ways: (a)  The metal ions may  'activate  1  They may remove an  i n h i b i t o r present i n the enzyme environment by making a complex with i t ;  (b) they may d i s p l a c e i n e f f e c t i v e ne t a l i o n s from  combination w i t h the a c t i v e center o f the enzyme or w i t h the f u n c t i o n a l group o f the s u b s t r a t e ; (c) a metal may produce a c t i v a t i o n by d i s p l a c i n g the t r u e a c t i v a t i n g metal, thus  setting  it  (d) metal  f r e e t o combine w i t h the e f f e c t i v e s i t e ; and f i n a l l y ,  ions may s t a b i l i z e the more a c t i v e form o f the enzyme as does Zn  + +  w i t h the a c t i v e , t e t r a m e r i c form o f a l c o h o l dehydrogenase. Thus i t may be r e a l i z e d t h a t the t r e a t -  ment o f p l a n t s w i t h a mixture o f f i v e a c t i v a t i n g m e t a l l i c i o n s , as i n the p r e s e n t i n v e s t i g a t i o n , may have changed the a c t i v i t y  123.  o f s e v e r a l enzymes i n some of the above mentioned ways. Consideration the e f f e c t o f anions, SO4 enzymes.  must a l s o be given  to  i n t h i s case, on the a c t i v i t y  of  There i s c o n s i d e r a b l e  information  i n the  literature  i n d i c a t i n g e f f e c t s of i o n i c environment on the a c t i v i t y o f b o t h p a r t i c u l a t e and instance,  s o l u b l e enzymes.  I t has been r e p o r t e d ,  for  t h a t i n o r g a n i c s a l t s o f both u n i v a l e n t and b i v a l e n t  c a t i o n s markedly i n f l u e n c e the a c t i v i t y o f cytochrome oxidase ( M i l l e r and Evans, 1956).  Massey (1953) r e p o r t e d  a d d i t i o n to s e v e r a l other  a 2-to  s e v e r a l anions.  and Evans (1960) r e p o r t e d fumarase had  may  Thus, H i a t t  t h a t the same anions t h a t a c t i v a t e d  no e f f e c t on,  enzyme. The  be  be  about e f f e c t s o f anions on the enzymes i s the v a r i a -  b i l i t y of response observed w i t h d i f f e r e n t enzymes.  malic  , in  3 - f o l d a c t i v a t i o n of m a l i c  Another i n t e r e s t i n g f e a t u r e to considered  4  anions, a c t i v a t e d s a l t - f r e e fumarase.  H i a t t and Evans (1960) r e p o r t e d dehydrogenase by  that S 0  or decreased the a c t i v i t y o f  the  authors emphasized t h a t the e f f e c t s o f anions  enzyme s p e c i f i c . The  o f f e r a biochemical  considerations  explanation  o u t l i n e d not  f o r the v a r i a b l e e f f e c t s of  mineral  treatments on s e v e r a l enzyme systems obtained  present  i n v e s t i g a t i o n , they a l s o o f f e r an answer to the  observed w i t h the organ and  only  the age  o f the p l a n t .  the  i n the variability  Translocation  124.  of m i n e r a l  ions from one  p a r t to another, t h e i r d i l u t i o n  with  the newly formed compounds, or t h e i r b i n d i n g w i t h i n an organ to d i f f e r e n t  degrees, may  response, as was  l e a d to the v a r i a b i l i t i e s i n the  found i n t h i s (c)  study.  Possible biochemical a c t i o n between, 2,4-D ions The  nature o f i n t e r and m i c r o n u t r i e n t  f o l l o w i n g seem to be  the more  probable ways i n which the metal ions might have i n t e r a c t e d and m o d i f i e d the f u n c t i o n i n g of the enzyme-regulator complex: (i) metallic  ions to i n t e r a c t w i t h  i t conceivable  t h a t i t may  The  s t r i k i n g capacity of  s e v e r a l l i g a n d s at a time, make  serve to b i n d the enzyme-regulator  complex on one hand and the s u b s t r a t e on the other. p i c t u r e , the metal i o n may  be  looked upon as an  In t h i s  intermediate  link. (ii) the formation  The metal ions may  facilitate  o f enzyme-regulator complex by p r o v i d i n g a s u i t -  able charge on the p r o t e i n s u r f a c e f o r the b i n d i n g of the r e gulator. indirect  The metal ions i n t h i s mechanism w i l l p l a y o n l y  an  role. (iii)  a particular  The metal i o n s may  stabilize  form of the enzyme-regulator complex r e s p o n s i b l e f o r  the maximal c a t a l y t i c  efficiency.  125.  (iv)  A l t e r n a t i v e l y , the metal  ions may combine w i t h the r e g u l a t o r or p r e c i p i t a t e w i t h i t , thus b l o c k i n g i t s e n t r y i n t o the complex.  T h i s mechanism probably  seems t o operate when 2,4-D i s a p p l i e d i n l e t h a l doses.  This,  to some extent, may e x p l a i n the 'safening' or ' p r o t e c t i v e ' a c t i o n assigned  to such metals as i r o n (Wort, 1962). F i n a l l y , i t may be p o i n t e d out  t h a t there w i l l be s e v e r a l i n d i r e c t ways i n which these micron u t r i e n t ions might i n f l u e n c e the f u n c t i o n i n g o f 2,4-D.  Mention  may be made o f such matters as i n f l u e n c e on the metabolism o f the r e g u l a t o r i t s e l f ,  on i t s t r a n s l o c a t i o n out o f and b i n d i n g  w i t h i n c e l l p a r t i c u l a t e f r a c t i o n s , and e f f e c t s on other  growth •  r e g u l a t o r s w i t h which 2,4-D might be i n t e r a c t i n g , such as g i b b e r e l l i n s and k i n i n s . the f i n a l  I t must be concluded, t h e r e f o r e ,  that  e f f e c t must have been determined by a v a r i e t y o f  f a c t o r s. 7.  Proposed mechanism f o r s t i m u l a t o r y e f f e c t s o f 2 4-D minerals on growth and y i e l d . T  C e n t r a l t o the mechanism b e i n g proposed i s the s t i m u l a t i o n observed f o r s e v e r a l key enzymes under the i n f l u e n c e of 2,4-D and 2,4-D-minerals, as seen i n t h i s study as w e l l as r e p o r t e d by other  investigators.  In the f i r s t p l a c e , phosphory-  l a s e , one o f the enzymes r e s p o n s i b l e  f o r making a v a i l a b l e f r e e  126.  sugars from s t o r e d s t a r c h , showed c o n s i d e r a b l y  increased  activity.  Amylases, the enzymes which p a r t i c i p a t e i n the h y d r o l y s i s o f starch reserves  to s o l u b l e sugars, have a l s o been r e p o r t e d  be a c t i v a t e d c o n s i d e r a b l y 1953). Recent r e p o r t s  i n 2,4-D-treated p l a n t s  (Flood e t a l . 1967)  to  (Wort and Cowie,  have a l s o i n d i c a t e d  increased h y d r o l y t i c a c t i v i t y i n tissues treated with l O ^ M -  2,4-D.  From these c i t a t i o n s as w e l l as from the r e s u l t s of the  present  study, i t may  be  deduced t h a t one  treatments w i t h s t i m u l a t o r y 2,4-D  o f the e f f e c t s of  doses i s an i n c r e a s e d  formation  of r e a d i l y a v a i l a b l e sugars. The as the u l t i m a t e  formation  of photosynthate which  source o f sugars c a t a b o l i z e d d u r i n g r e s p i r a t i o n ,  as w e l l as a source o f carbon supply processes, and  was  a l s o found to be  15-day o b s e r v a t i o n s .  supply  for a l l biosynthetic  s t i m u l a t e d by  treatments at  T h i s i n c r e a s e d photosynthate may  necessary sugars f o r i n c r e a s e d r e s p i r a t i o n and  amino a c i d  serves  10well  thence  synthesis. Two  s t u d i e d i n the p r e s e n t  key  enzymes of r e s p i r a t o r y metabolism  i n v e s t i g a t i o n , namely,  kinase  and  10 and  15 days a f t e r treatment w i t h 2,4-D  phosphoglyceryl  s u c c i n i c dehydrogenase, a l s o showed i n c r e a s e d  enzymes are important f o r the generation During c o n v e r s i o n  activity  or 2,4-D-minerals. of u t i l i z a b l e  of 1,3-diphosphoglyceric a c i d to  energy.  Both  12 7.  3-phosphoglyceric a c i d , the r e a c t i o n c a t a l y z e d by phoglyceryl catalyzes  kinase,  ATP  conversion  i s synthesized.  S u c c i n i c dehydrogenase  o f s u c c i n i c a c i d to fumaric a c i d w i t h  mation of reduced FAD.  The  l a t t e r may  l e a d i n g to the f o r m a t i o n of ATP.  enter  i n t o the ETS  Alexander, 1965), and  s t i m u l a t e d by  chain  hexokinase,  cytochrome oxidase o f the e l e c t r o n  ( R a k i t i n and  for-  There i s independent evidence  t h a t s e v e r a l other enzymes of g l y c o l y s i s (e.g.,  port chain  phos-  Potapova, 1959)  treatments w i t h low  2,4-D  are a l s o doses.  trans-  considerably I t seems obvious,  thus, t h a t 2,4-D-treated p l a n t s m a i n t a i n a h i g h e r r e s p i r a t o r y a c t i v i t y by v i r t u e o f enzyme s t i m u l a t i o n and The  net r e s u l t o f t h i s  u t i l i z a b l e energy, and from the TCA  should be  a greater  an i n c r e a s e  available  production  substrate. of  i n supply o f carbon  cycle, for biosynthetic  skeletons  processes.  In the p r e s e n t i n v e s t i g a t i o n , the a c t i v i t y n i t r a t e reductase and  glutamic-oxalacetic  received considerable  s t i m u l a t i o n by  n i t r a t e reductase a c t i v i t y may which can be used f o r s y n t h e s i s Glutamic a c i d can  treated plants.  action.  Increased  quantity  o f glutamic a c i d by  then l e a d to i n c r e a s e d  synthesis  transaminase a l s o  supply a g r e a t e r  amino a c i d s through t r a n s a m i n a t i o n . suggest i n c r e a s e d  2,4-D  The  of  synthesis  of N H 3  amination. of  several  present data a l s o  of p r o t e i n i n 2,4-D- and  Payne et a l . (1953) have r e p o r t e d  2,4-D-mineral increased  1218.  p r o t e i n content i n potato tubers formed on •plants. 2,4-D  S i m i l a r r e p o r t s of i n c r e a s e d  mented carbon and amounts of new growth.  concentrations -^C  p r o t e i n content under  a c t i o n have been p u b l i s h e d by other i n v e s t i g a t o r s . F i n a l l y , i t may  new  2,4-D-treated  energy supply may  protoplasm and  be  s t a t e d t h a t the aug-  l e a d to formation of l a r g e r  c e l l wall material,  In f a c t , i t has been r e p o r t e d of 2,4-D  that  do l e a d to an i n c r e a s e d  essential for stimulatory  incorporation  i n t o c e l l w a l l f r a c t i o n s (Wightman and Neish, 1959).  o v e r a l l r e s u l t of the above may productivity.  be  s t i m u l a t i o n to growth  of  The and  These ideas are summarized i n F i g . 15 which shows  the l o c i o f measurements made i n t h i s i n v e s t i g a t i o n .  129.  2H  CO 1  P h o t o s y n t h e s i s measured C0 uptake  as  7  Cellulose^-  — Starch  Siigars^  etc.  \,  Phosphorylase  i i 1,3-Dip"hosphoglyceric acid ADP  > 3  Phosphoglyceryl  kinase  ATP<r3-Phosphoglyceric acid  Pyruvate Pyridine  1  nucleotides  s 7  I . . Aspartic ac i°- N  Glutamic  Glutamate  apid/^ / oxalo-  R e s p i r a t i o n measured as COg evolution  \  transaminase y a c e t a t i c '• acid Fumardic' ..  citrate \  f  FADH;^  4  CO.  \  k,CO.  racid  Succinic 6 dehydrogenase FADSuccinic. . acid.  oC-Ketfoglutar.ic y acid  A  CO,  Glutamic acid NEL /Ts I I  N0 ~ 9  Nitrate reductase  N0Fig.  15:  B i o s y n t h e t i c scheme t o show t h e p o i n t s where s t i m u l a t i o n b y 2,4-D, o r 2 , 4 - D - m i n e r a l s was o b s e r v e d i n t h e i n v e s t i g a t i o n .  130.  CONCLUSIONS  On the b a s i s o f the r e s u l t s obtained, and t h e i r comparison w i t h e x i s t i n g l i t e r a t u r e on the s u b j e c t ,  the f o l l o w i n g con-  c l u s i o n s may be drawn: (1)  When a p p l i e d i n a p p r o p r i a t e l y  low doses and a t  the c o r r e c t o n t o g e n e t i c time, f o l i a r sprays o f 2,4-D l e a d to s t i m u l a t i o n i n j u v e n i l e growth of bushbean p l a n t s .  (Phaseolus  v u l g a r i s ) , as measured i n terms o f f r e s h and dry weights o f r o o t s , stem and l e a v e s ; (2)  l e a f number and area;  and p l a n t  height.  The s t i m u l a t i o n o b t a i n e d by p l a n t s i n v e g e t a t i v e  growth l e d t o improved y i e l d as measured by number and f r e s h weight o f pods; number and weight o f dry seeds, and the weight o f i n d i v i d u a l seed. vitamin  The pods from t r e a t e d p l a n t s contained  C content a t the time o f h a r v e s t  a c i d and m o i s t u r e , d u r i n g not o n l y i n c r e a s e s (3)  storage.  and l o s t l e s s  higher  ascorbic  Thus, the treatments  evoked  i n y i e l d b u t a l s o improvements i n the q u a l i t y .  Inclusion of micronutrients  Fe, Cu, B, Zn, and  -4 Mn, i n low c o n c e n t r a t i o n stimulatory  (5x10  M) f u r t h e r augmented the  a c t i o n o f the hormone.  This was apparent i n j u v e n i l e  growth and i n y i e l d as w e l l as i n m e t a b o l i c processes and enzyme activities. (4)  The treatments induced c o n s i d e r a b l e  alterations  i n the chemical composition o f p l a n t s b u t the magnitude v a r i e d  131.  w i t h the p l a n t organ.  Sugar contents of leaves were i n c r e a s e d  but those of stem and r o o t s were decreased; content was  the amino a c i d  i n c r e a s e d by m i n e r a l s but decreased by 2,4-D  p a r t i c u l a r l y i n leaves.  In p l a n t s t r e a t e d w i t h 2,4-D  m i n e r a l s , the amino a c i d content was r e s u l t i n g from e i t h e r alone. together suggested (5)  alone,  plus  i n t e r m e d i a t e between those  Changes i n sugar and amino a c i d  a g r e a t e r s y n t h e s i s of p r o t e i n .  A c l o s e p a r a l l e l was  observed between s t i m u l a t i o n  of v e g e t a t i v e growth, p h y s i o l o g i c a l processes  (respiration  and  photosynthesis) and a c t i v i t i e s of some of the key enzymes of carbohydrate  (phosphorylase, p h o s p h o g l y c e r y l k i n a s e ,  s u c c i n i c dehydrogenase) and n i t r o g e n metabolism r e d u c t a s e , and (6)  and  (nitrate  transaminase). For each treatment,  the time e l a p s e d between the  a p p l i c a t i o n of the chemical and the hour o f o b s e r v a t i o n , found to be a v e r y important (growth,  f a c t o r i n determining the  was  response  chemical composition, or p h y s i o l o g i c a l processes  and  enzyme a c t i v i t i e s ) . (7) i n response t h a t 2,4-D  The  s t i m u l a t i o n of enzymes by 2,4-D  and  fluctuations  seem to be b e s t e x p l a i n a b l e on the b a s i s o f hypothesis p a r t i c i p a t e s i n the formation of a  r e g u l a t o r complex w i t h a c t i v a t i o n energy enzyme-substrate  complex.  substrate-enzyme-  lower than t h a t of  M i n e r a l i o n s , by v i r t u e of t h e i r  a b i l i t y to p a r t i c i p a t e i n s e v e r a l enzyme a c t i v a t i o n s , may  further  132.  modify the nature o f t h i s complex. (8) minerals  The  s t i m u l a t o r y a c t i o n o f 2,4-D  and 2,4-D-  can be e x p l a i n e d on the f o l l o w i n g p h y s i o l o g i c a l and  biochemical  basis: (1)  The s t i m u l a t e d r a t e o f photosynthesis produced a l a r g e r amount of photosynthate which c o u l d be u t i l i z e d i n the b i o s y n t h e s i s of a l l c e l l c o n s t i t u e n t s or serve as s u b s t r a t e f o r r e s p i r a t i o n .  (2)  The s t i m u l a t e d r a t e of r e s p i r a t i o n and a c t i v i t y o f such enzymes as phosphog l y c e r y l k i n a s e and s u c c i n i c dehydrogenase r e s u l t e d i n an i n c r e a s e d supply of a v a i l a b l e energy, as ATP and reduced n u c l e o t i d e s , f o r b i o s y n t h e s i s , and i n l a r g e r amounts o f keto a c i d s which p r o v i d e the carbon s k e l e t o n s of amino acids.  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The Macmillan Co.,  Weyer, E.M.,  (Ed.). 1967. P l a n t growth r e g u l a t o r s . N.Y. 144(1): pp. 382.  Ann.  N.Y.  N.Y.  pp.  249.  Acad. S c i . ,  Wightman, F. , and A . C Neish. 1959. The i n f l u e n c e of p l a n t growth r e g u l a t o r s on c e l l - w a l l metabolism. Proc. 9th I n t e r n a t i o n a l Bot. Congr., Montreal, Canada, p. 430. Williams,  G.,  and S. Dunn. 1961. R e l a t i o n of l i g h t q u a l i t y to e f f e c t s of 2,4-D on c h l o r o p h y l l and C0 exchange. Weeds. 9:243-250. 2  Wittwer, S.H.  and, F.G. Teubner. 1959. F o l i a r a b s o r p t i o n of m i n e r a l n u t r i e n t s . Rev. P l a n t P h y s i o l . 10:13-32.  Ann.  Wittwer, S.H.,  and A.E. Murneek. 1946. F u r t h e r i n v e s t i g a t i o n s on the value o f hormone sprays and dusts f o r green bush snap beans. Proc. Amer. Soc. Hort. S c i . 47:285-293.  Wittwer, S.H.  1964. F o l i a r a b s o r p t i o n of p l a n t n u t r i e n t s . P l a n t S c i . 8:161-182.  Adv.  Frontiers  150.  Wolf, D.E., G. V e r m i l l i o n , A. Wallace, and G.H. A h l g r e n . 1950. E f f e c t o f 2,4-D on carbohydrate and n u t r i e n t element content and on r a p i d i t y o f k i l l o f soybean p l a n t s growing a t d i f f e r e n t n i t r o g e n l e v e l s . Bot. Gaz. 112:188-197. Woodford, E.K., H o l l y , K., and C.C. McCready. 1958. H e r b i c i d e s . Ann. Rev. P l a n t P h y s i o l . 9:311-358. Wort, D.J. 1950. The e f f e c t o f some chemical growth r e g u l a t o r s on the v i t a m i n content o f p l a n t s . West. Canada. Weed Contr. Confr. Proc. 4:45-46. Wort, D.J. 1951 E f f e c t s o f n o n - l e t h a l c o n c e n t r a t i o n s o f 2,4-D on Buckwheat. P l a n t P h y s i o l . 26:50-58. Wort, D.J. 1953. F o l i a r a p p l i c a t i o n o f sprays and dusts, c o n t a i n i n g major and minor elements w i t h and without 2,4-D. West. Canada Weed Contr. Confr. Proc. 7:93-101. Wort, D.J., and L.M. Cowie. 1953. The e f f e c t s o f 2,4-dichlorophenoxyacetic a c i d on phosphorylase, phosphatase, amylase, c a t a l a s e , and peroxidase a c t i v i t y i n wheat. P l a n t P h y s i o l . 28:135-139. Wort, D.J. 1954. I n f l u e n c e o f 2,4-D on enzyme systems. 3(2):131-135.  Weeds.  Wort, D.J. 1956. The f o l i a r a p p l i c a t i o n o f minor elements w i t h 2,4-D. 9th Meeting West S e c t i o n , N a t i o n a l Weed Committee, Canada. 9:22-28. Wort, D.J. 1957. The f o l i a r a p p l i c a t i o n o f minor elements w i t h 2,4-D. 10th Meeting West S e c t i o n , N a t i o n a l Weed Committee, Canada. 10:52-56. Wort, D.J., and Shrimpton, D.M. 1958. E f f e c t o f f o l i a r a p p l i e d amino t r i a z o l e on the p h o t o s y n t h e s i s and r e s p i r a t i o n o f leaves o f bean and wheat. 11th Meeting West S e c t i o n , N a t i o n a l Weed Committee, Canada. 11:124.  151.  Wort, D.J.  1959. The f o l i a r a p p l i c a t i o n of 2,4-D and other growth r e g u l a t o r s w i t h and without minor elements. Proc. i n : I n t e r n a t i o n a l Cong. Crop P r o t e c t i o n , Hamburg, 1957. 52-56.  Wort, D.J.  1961. E f f e c t s on the composition and metabolism of the e n t i r e p l a n t . i n : Handbuch der P f l a n z e n p h y s i o l o g i e , W. Ruhland, Ed. 14:140-1136.  Wort, D.J.  1962. The a p p l i c a t i o n of s u b l e t h a l c o n c e n t r a t i o n s of 2,4-D and i n combination w i t h m i n e r a l n u t r i e n t s . World Review Pest C o n t r o l . 1(4):6-19.  Wort, D.J.,  and LaBerge, D.E. 1963. E f f e c t s o f 2,4-D n u t r i e n t dusts on the growth and y i e l d of beans and sugar b e e t s . Quoted i n Wort, 1964b.  Wort, D.J.  1964a. E f f e c t s of h e r b i c i d e s on p l a n t composition and metabolism, i n : The P h y s i o l o g y and B i o c h e m i s t r y of H e r b i c i d e s . Audus, L . J . Ed. Acad. P r e s s , N.Y. p. 291-334.  Wort, D.J.  1964b. Responses of p l a n t s to s u b l e t h a l c o n c e n t r a t i o n s of 2,4-D, without and w i t h added m i n e r a l s . i n : The P h y s i o l o g y and B i o c h e m i s t r y of H e r b i c i d e s . Audus, L . J . Ed. Acad. Press, N.Y. 335-342.  Wort, D.J.  1966. E f f e c t s of 2,4-D-nutrient dusts on the growth and y i e l d of beans and sugarbeets. Agron. Jour. 58:27-29.  Yakushkina,  N.I. 1948. P h y s i o l o g i c a l and b i o c h e m i c a l changes induced i n growth by the a p p l i c a t i o n of growth substances. Dokl. Akad. Nauk. SSSR (Engl. T r a n s l . ) 61:939-942.  Yang, K.J.  1964. Diphosphopyridine n u c l e o t i d e - n i t r a t e reductase i n Beta v u l g a r i s L. Ph.D. T h e s i s , Univ. B r i t i s h Columbia, pp. 140.  152. Y a s u d a , A.K., M.G. Payne, a n d J . L . F u l t s . 1956. E f f e c t 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 and m a l e i c h y d r a z i d e on p o t a t o p r o t e i n s a s shown by paper e l e c t r o p h o r e s i s . Nature (London). 176: 1029-1030. Y o u s s e l , E . , a n d E. Wagih. 1963. The 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 the a b s o r p t i o n and a s s i m i l a t i o n o f p o t a s s i u m n i t r a t e by r a d i s h r o o t s l i c e s . Jour. Bot. U n i t e d Arab Repub. 6:61-73. Yurkevitch,  I.V. 1963. E f f e c t o f m i c r o e l e m e n t s on t h e a c t i o n o f t h e T.U. (2,4,5-T) s t i m u l a t o r . F i g i o l . R a s t e . ( P l a n t P h y s i o l . ) . 10:90-97.  Zimmerman, P.W., and A.E. H i t c h c o c k . 1942. S u b s t i t u t e d phenoxy and b e n z o i c a c i d growth substances and t h e r e l a t i o n o f s t r u c t u r e t o physiological activity. C o n t r . B o y c e Thompson I n s t . 13:313-322.  153.  APPENDIX I ANALYTICAL PROCEDURES A.  Spectrophotometries determination o f amounts o f c h l o r o p h y l l a, c h l o r o p h y l l b and c a r o t e n o i d s in leaf material. 1.  f i n e and mix  Harvest  the leaves and chop the b l a d e m a t e r i a l  thoroughly. 2.  Weigh out 1 g o f l e a f m a t e r i a l .  3.  G r i n d thoroughly  i n c o l d mortar w i t h a small  amount o f c l e a n sand and CaC03. 4. thoroughly.  Now add 'ca 10 ml 80% c o l d acetone and g r i n d  Decant the acetone i n t o a small f l a s k .  Repeat  g r i n d i n g and e x t r a c t i o n u n t i l the l e a f m a t e r i a l becomes white. Pool a l l the e x t r a c t s . 5.  C e n t r i f u g e the e x t r a c t f o r 3 to 5 minutes  a t 2000 r.p.m. 6.  Decant the supernatant  and make to mark w i t h acetone.  i n t o a 50 ml v o l u m e t r i c  Mix by i n v e r t i n g .  Pour i n t o  f l a s k , stopper and keep i n dark f o r a b r i e f time t i l l are ready  a l l samples  f o r reading. 7.  i f necessary)  Read the o p t i c a l d e n s i t y ( d i l u t e w i t h  acetone,  i n a Beckman Spectrophotometer Model B a g a i n s t 80%  acetone a t 663, 645 and 440.5 mu.. 8.  F i n d the c o n c e n t r a t i o n o f c h l o r o p h y l l a and  154.  chlorophyll b using efficients  the f o l l o w i n g s p e c i f i c  o f McKinney Ca  =  a b s o r p t i o n co-  (1940):  (12.3 D  - 0.86 D  6 6 3  ) V  6 4 5  d x 1000 x W Cb  =  (19.3 D  - 3.6 D  6 4 5  6 6 3  ) V  d x 1000 x W where:  9. the e q u a t i o n  C  -  concentration  a  -  chlorophyll a  b  =  chlorophyll b  D  «  O p t i c a l d e n s i t y a t wave l e n g t h i n d i c a t e d  d  =  length o f l i g h t path  V  =  f i n a l volume o f e x t r a c t  W  =  f r e s h weight o f l e a f m a t e r i a l used i n extraction  i n cm  Find the concentration o f carotenoids  of von Wettsfein Cc  i n mg/g f r e s h w e i g h t  =  4.695 D  4  using  (1957): 4  0  >  5  - 0.268  C  (  a  +  b  )  where: c 10. B.  =  concentration of carotenoids  Convert  carotenoid content  R e d u c i n g Sugar D e t e r m i n a t i o n Method. DNP  i n mg/1  t o mg/g f r e s h w e i g h t .  by Dintrophenol  (DNP)  Reagent: S o l u t i o n A:  D i s s o l v e 7.145 g s o d i u m  155.  2,4-dinitrophenol  i n 230 ml 5% NaOH.  on water b a t h u n t i l pletely.  Heat  the DNP d i s s o l v e s com-  Then add 2.5 g: phenol.  Heat  more, i f the s o l u t i o n does not remain c l e a r . S o l u t i o n B:  Dissolve  100 g sodium potassium  t a r t a r a t e i n Ca 500 ml d i s t i l l e d water.  Mix  S o l u t i o n A and B, t r a n s f e r to a 1 l i t e r  volumetric  flask.  S o l u t i o n A and B.  Rinse out c o n t a i n e r s o f Make the v o l u m e t r i c  up t o  mark. Determination:  Into  3 l a r g e t e s t tubes p i p e t t e  e x a c t l y 6 ml DNP Reagent.  Into tube 1 p i p e t t e  e x a c t l y 2 ml d i s t i l l e d water, i n t o tube 2 and 3 p i p e t t e 2 ml o f c l e a r deleaded e x t r a c t . the tubes i n c o l d , running ^ 0 are ready.  till  Keep  a l l samples  Stopper a l l tubes l o o s e l y w i t h  g l a s s wool and p l a c e e x a c t l y 6 minutes.  i n b o i l i n g water f o r Transfer  the tubes i n  c o l d running water and read between 3 t o 2 0 minutes.  Read the absorbance o f each s o l u t i o n  i n a K l e t t colorimeter 1 i s blank.  using r e d f i l t e r .  Tube  Refer a l l readings to the standard  156.  2.0  157.  curve prepared i n the same way w i t h d i f f e r e n t c o n c e n t r a t i o n o f glucose (see F i g . 16), and express sugar content on C.  d r y weight b a s i s .  Colorimetric (1)  determination of ascorbic  a c i d i n pods.  Chemicals: (i)  0.05% s o l u t i o n o f o x a l i c a c i d .  Make from  a stock 5% s o l u t i o n which keeps i n definitely, (ii)  Standard dye (Na-2,6-dichlorophenol indophenol) s o l u t i o n made by d i s s o l v i n g 14 mg dye i n warm d i s t i l l e d water, tered  and made to 1 l i t e r w i t h c o l d  t i l l e d water. (iii)  (2)  dis-  Store i n f r i g e ,  Standard s o l u t i o n s 0.5%  fil-  of ascorbic  acid i n  1 ml standard a s c o r b i c  acid i n  oxalic  acid.  Calibration; (i)  Pipette  duplicate (ii)  i n c o l o r i m e t e r tubes,  Blow i n 9 ml dye s o l u t i o n from a r a p i d delivery pipette, within  . (iii)  i n v e r t to mix and read  20 seconds.  Repeat the procedure i n d u p l i c a t e f o r each standard a s c o r b i c  acid  solution.  J  10 F i g . 17:  I  I  I  20 30 40 A s c o r b i c a c i d (mg/1). Standard c h a r t f o r a s c o r b i c a c i d .  I  50  159.  (iv)  F i n a l l y , prepare b l a n k by mixing 1 ml 0.5% o x a l i c a c i d , 9 ml dye  solution  and a t i n y c r y s t a l of pure a s c o r b i c  acid.  Read b l a n k . (v)  S u b s t r a c t b l a n k from each.  (vi)  P l o t s c a l e r e a d i n g s on o r d i n a t e a g a i n s t c o n c e n t r a t i o n o f a s c o r b i c a c i d on abscissa.  Obtain b e s t f i t t i n g l i n e by  l e a s t square method. (3)  Determination i n bean pods: (i)  Blend 1 weight f i n e l y chopped pods w i t h 7 volumes o f 0.5% o x a l i c a c i d i n a Waring b l e n d o r run a t f u l l  speed f o r  3 minutes. (ii)  Decant and e x t r a c t through 4 l a y e r s o f cheese c l o t h and a d j u s t t o volume,  (iii)  Repeat  the process as i n ' C a l i b r a t i o n '  u s i n g 1 ml of e x t r a c t i n p l a c e o f standard ascorbic a c i d (iv)  solution.  Refer a l l r e a d i n g s to c a l i b r a t i o n c h a r t ( F i g . 17) to f i n d the c o n c e n t r a t i o n of ascorbate i n the  (v)  sample,  Express data as mg f r e s h weight.  a s c o r b i c acid/100 g  160.  D.  Amino A c i d  Identification  Standard p o s i t i o n s the  investigation.  1  are i n c l u d e d  o f amino a c i d spots o b t a i n e d i n i n F i g . 18.  23  Rf 18:  0.6 Dimension ^1  0.9  A schemetic r e p r e s e n t a t i o n o f two-dimensional chromatogram showing standard p o s i t i o n s of spots o b t a i n e d w i t h known amino a c i d s .  162.  TABLE XVII Key t o amino a c i d numbers  Number  Amino a c i d o r amide  used i n F i g . 18.  Number  Amino a c i d o r amide  1.  Cysteine  13.  cC-Alanine  2.  Cystine  14.  & -Alanine  3.  Asparagine  15.  4.  Lysine  16.  5.  Histidine  17.  Y  6.  Arginine  18.  Pipecolic  7.  Aspartic  19.  Proline  8.  Serine  20.  Methionine  9.  Glycine  21.  Valine  10.  Glutamine  22.  Phenylalanine  11.  Glutamic  23.  Isoleucine  12.  Threonine  24.  Leucine  acid  acid  Tyrosine ct -aminobutyric -aminobutyric acid  acid acid  163.  APPENDIX I I  ENZYME ASSAYS (i) (ii)  For standard c h a r t s r e f e r to F i g . 19 to 22. P r o t e i n d e t e r m i n a t i o n i n enzyme e x t r a c t s S o l u t i o n A:  Prepare 2% Na C03 i n O.lN NaOH.  S o l u t i o n B:  Prepare 0.5% CUSO4 i n 1% sodium-  2  potassium S o l u t i o n C:  tartarate.  Mix 50 ml o f S o l u t i o n A w i t h 1 ml o f S o l u t i o n B. in  Stopper, keep  frige.  F o l i n - P h e n o l reagent: source.  Obtain from (Nutritional  commercial  Biochem).  d i l 1:1 w i t h d i s t i l l e d water b e f o r e use. Procedure: (1)  To 0.1 (or 0.2) ml enzyme p r e p a r a t i o n add 2 ml o f s o l u t i o n C.  Mix and a l l o w  to stand f o r 10 minutes. (2)  Add 0.2 ml IN F o l i n - p h e n o l reagent and a l l o w a t l e a s t 3 0 minutes  f o r the c o l o r  development. (3)  Read each and i t s blank a t 500 mu, between 0.5 and 2 hours.  164.  *  cu rH  £  2 00  CU  Microgram Fig.  19:  phosphate/2 8 ml  Standard c h a r t f o r phosphorylase.  165.  Standard c h a r t f o r s u c c i n i c hydrogenase (Acc. to Kun and Abood)  50  100  150  200  Microgram formozan/10 F i g . 20:  Standard c h a r t f o r s u c c i n i c  250  ml.  dehydrogenase.  166.  5  10  15  m micro mole • n i t r i t e F i g . 21:  Standard c h a r t f o r n i t r a t e r e d u c t a s e .  20  167.  

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