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The auxin-like properties of potassium naphthenates and their effect on indole-3-acetic acid biosynthesis… Loh, John Wai-Choong 1972

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THE AUXIN-LIKE PROPERTIES OF POTASSIUM NAPHTHENATES AND THEIR EFFECT ON INDOLE-3-ACETIC ACID BIOSYNTHESIS AND DEGRADATION. by John Wai-Choong Loh Dip. A g r i c , (Malaysia), 1965. B.Agr.Sc. (Hons), (Malaysia), 1970. A thesis submitted i n p a r t i a l f u l f i l l m e n t of the requirements f o r the degree of MASTER OF SCIENCE i n the Department of BOTANY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA, VANCOUVER, BRITISH COLUMBIA, CANADA. 1972 June In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Botany The University of British Columbia Vancouver 8, Canada Date 22nd August, 1972 i ABSTRACT The auxin-Tike properties of potassium naphthenates (KNap), and the e f f e c t of these compounds on indoleacetic acid (IAA) biosynthesis and degradation were examined. Chapter I. The auxin-like properties of KNap. Cucumber seeds were treated with. 1000 ppm KNap and a s i g n i f i c a n t (at the 0.05 level) i n h i b i t i o n of root growth (91%) , compared with control seedlings was obtained. The e f f e c t s of KNap and indolebutyric acid on the i n i t i a t i o n of roots by stem cuttings of Phaseolus vulgaris L. were examined. The treatments with 10 and 100 ppm of both compounds s i g n i f i c a n t l y stimulated root i n i t i a t i o n . Root i n i t i a t i o n of azalea stem cuttings was s i g n i f i c a n t l y augmented by 10, 100, and 1000 ppm KNap. The elongation of dark-grown Alaska pea stem segments was stimulated by 1.0 ppm KNap (279% over the c o n t r o l ) . Surprisingly, t h i s stimulation did not d i f f e r s i g n i f i c a n t l y from that caused by 0.1 ppm IAA (339% over the c o n t r o l ) . KNap at 100 and 1000 ppm, applied to the d i s t a l end of debladed peti o l e s , did not a f f e c t abscission. The times required for 50% abscission of petioles treated with 10 and 1000 ppm cyclohexanecarboxylic acid were s i g n i f i c a n t l y greater than that for abscission of control p e t i o l e s , but not from that required by pet i o l e s treated with 100 ppm naphthalene-acetic acid. Chapter II. The e f f e c t of KNap on IAA biosynthesis and degradation. When the seeds' of Phaseolus vulgaris L. were soaked for 12 hours i n a solution of 100 ppm KNap immediately p r i o r to sowing, there was a s i g n i f i c a n t increase (140% over control plants) i n the content of IAA i n the a p i c a l 5-8 cm of the stems of 14-day-old plants. The immersion of the root systems of 13-day-old dark-grown bean plants i n a solution of 100 ppm KNap for 24 hours resulted i n a s i g n i f i c a n t stimulation (4% over the control) of the a c t i v i t y of the IAA oxidase system. The evidence presented i s interpreted as supporting the view that KNap has some auxin-like properties. The v a l i d i t y of t h i s i n t e r p r e t a t i o n i s discussed. i i i TABLE OF CONTENTS page Abstract i Table of contents i i i Abbreviations v i L i s t of tables v i i L i s t of figures v i i i L i s t of plates . i x Dedication x Acknowledgements . x i Introduction . x i i Naphthenic acids 1 CHAPTER I. INVESTIGATION OF THE AUXIN-LIKE PROPERTIES OF POTASSIUM NAPHTHENATES. 8 a) Growth of i n t a c t roots of cucumber seedlings. LITERATURE REVIEW 9 MATERIALS AND METHODS 13 RESULTS 15 DISCUSSION 18 i v page b) Adventitious root i n i t i a t i o n i n succulent  and woody stem cuttings. LITERATURE REVIEW 20 MATERIALS AND METHODS 23 i . Succulent stem cuttings using bean plants. 23 i i . Woody stem cuttings using azalea plants. 26 RESULTS 28 i . Rooting of succulent stem cuttings of bean. 28 i i . Rooting of woody stem cuttings of azalea. 33 DISCUSSION 37 c) Elongation of pea stem segments. LITERATURE REVIEW 39 MATERIALS AND METHODS 42 RESULTS 44 DISCUSSION 47 d) Abscission of debladed pe t i o l e s of bean  plants. LITERATURE REVIEW 49 MATERIALS AND METHODS 58 RESULTS 61 DISCUSSION 63 V page CHAPTER II. THE EFFECT OF POTASSIUM NAPHTHENATES ON IAA BIOSYNTHESIS AND DEGRADATION. 65 a) The e f f e c t of KNap on IAA biosynthesis. LITERATURE REVIEW 66 MATERIALS AND METHODS 70 RESULTS 75 DISCUSSION 77 b) The e f f e c t of KNap on IAA degradation. LITERATURE REVIEW 79 MATERIALS AND METHODS 84 RESULTS 88 DISCUSSION 90 Conclusions 92 Bibliography 93 vi'. ABBREVIATIONS HNap Naphthenic acids KNap Potassium naphthenates CHCA Cyclohexanecarboxylic a c i d Sh-8 ' A compound of the cyclohexylbutanol c l a s s NAA Naphthaleneacetic a c i d IAA Indoleacetic a c i d IBA Indolebutyric a c i d 2,4-D 2,4-dichlorophenoxyacetic a c i d 2,4,5-T 2,4,5-trichlorophenoxyacetic acid v i i LIST OF TABLES Table Page I Response of i n t a c t roots of cucumber to KNap. 16 II Rooting response of bean stem cuttings to KNap and IBA treatments. 30 III Response of stem cuttings of azalea to KNap. 34 IV Response of dark-grown Alaska pea stem segments to exogenously applied IAA, CHCA, and KNap. 46 V Abscission of debladed p e t i o l e s of bean (Phaseolus v u l g a r i s L.) i n response to KNap, CHCA, and NAA treatments. 62 VI IAA content of the 5-8 cm t i p s of ep i c o t y l s of 14-day-old dark-grown Phaseolus vulgaris L. seedlings following treatment with 100 ppm KNap. 76 VII S p e c i f i c a c t i v i t y of the enzymes i n the conversion of tryptophan to IAA i n the epi c o t y l s of dark-grown bean plants. 76 VIII The a c t i v i t y of the IAA oxidase system i n the 14-day-old dark-grown bean epic o t y l s following treatment with 100 ppm KNap. 8 9 Figure LIST OF FIGURES Page 1 Growth response of i n t a c t roots of cucumber. 17 2 Stimulation of root i n i t i a t i o n of bean stem cuttings by KNap and IBA treatments. 31 3 Stimulation of root i n i t i a t i o n i n azalea stem cuttings by KNap treatments. 35 LIST OF PLATES Plate Page 1 Rooting response of bean stem cuttings to KNap and IBA treatments. 32 2 Rooting response of azalea stem cuttings to KNap treatments. 36 3 Determination of abscission of a pe t i o l e by the use of a f i v e gramme pressure applicator. 60 X DEDICATION This t h e s i s i s d e d i c a t e d t o my w i f e , Trudy, f o r her devotion, a s s i s t a n c e , and p a t i e n c e t h a t have made my study p o s s i b l e . x i ACKNOWLEDGEMENTS G r a t e f u l t h a n k s a r e ex t e n d e d t o my a d v i s o r , P r o f e s s o r D.J. Wort, f o r c o u n s e l , g u i d a n c e , and c r i t i c a l r e v i e w o f the e n t i r e m a n u s c r i p t ; t o D r s . R.F. S c a g e l , I.E.P. T a y l o r , W.B. S c h o f i e l d , and R. J. B a n d o n i , f o r a s s i s t a n c e and a d v i c e ; t o Mr. S.M. Sm i t h o f t h e F a c u l t y o f F o r e s t r y , f o r a d v i c e i n s t a t i s t i c a l a n a l y s i s ; and t o Mr. D.R. P e i r s o n , f o r t e c h n i c a l a s s i s t a n c e i n making p h o t o g r a p h i c p r i n t s . INTRODUCTION The p u b l i c a t i o n o f Went's c l a s s i c a l p a p e r " W u c h s s t o f f und Wachstum" i n 1928 i n w h i c h he d e s c r i b e d a q u a n t i t a t i v e method o f u s i n g Avena s e e d l i n g s f o r a s s a y i n g p l a n t g r o w t h s u b s t a n c e s , opened a new e r a o f r e s e a r c h . K o g l e t a_l i n 1933 i s o l a t e d a c r y s t a l l i n e g r o w t h - i n d u c i n g s u b s t a n c e f rom human u r i n e . They a l s o d e t e r m i n e d t h e c h e m i c a l n a t u r e o f t h e g r o w t h s u b s t a n c e , and, t o g e t h e r w i t h t h e l a t e F.A.F.C. Went, c o i n e d t h e t e r m " a u x i n " (from Greek " a u x e i n " , t o g r o w ) . The f r e q u e n t use o f t h e t e r m " a u x i n " b y r e s e a r c h e r s r e s u l t s i n t h e l a c k o f u n i f o r m i t y i n n o m e n c l a t u r e , and l e a d s t o c o n f u s i o n . Many w o r k e r s use t h e t e r m " a u x i n " t o r e f e r t o a s i n g l e s u b s t a n c e , i n d o l e - 3 - a c e t i c a c i d ( I A A ) . To add t o t h e c o n f u s i o n , IAA i s t h e s u b s t a n c e r e f e r r e d t o as " h e t e r o a u x i n " b y many i n v e s t i g a t o r s i n R u s s i a and B u l g a r i a . Tukey e t a l (1954)recommended t h e t erm " a u x i n " be used as a g e n e r i c t e r m o r group-name f o r "compounds c h a r a c t e r i z e d b y t h e i r c a p a c i t y t o i n d u c e e l o n g a t i o n i n s h o o t c e l l s " . The name " a u x i n " used h e r e i s t a k e n i n t h e g e n e r i c s e n s e . x i i i The d e f i n i t i o n of auxin by Tukey et a l (1954) requires a r e v i s i o n i n view of s i m i l a r c h a r a c t e r i s t i c s exhibited by other compounds such as gibberellic:. acids. A good d e f i n i t i o n for auxin i s lacking. The general b e l i e f that Went's Avena c o l e o p t i l e c curvature t e s t (Went, 1928) i s a conclusive bioassay for auxin i s questioned by the finding of Huseinov (1960) that "hybberellic acid" (probably g i b b e r e l l i c acid) caused bending of the Avena c o l e o p t i l e , suggesting polar movement of g i b b e r e l l i c acid. Huseinov's finding has been supported by Jacobs and Kaldeway (1970) who demonstrated polar move-ment of g i b b e r e l l i c acid (GA^) i n young pe t i o l e s of Coleus. The lack of polar movement of IAA i n some plants has been shown by Eschrich (1968) using radioactive IAA on V i c i a faba. The bioassays commonly used to demonstrate auxin a c t i v i t y are: 1. Wents Avena c o l e o p t i l e curvature t e s t (Went, 1928). 2. S p l i t pea stem curvature t e s t (Went, 1934). 3. Root growth t e s t (Moewus, 1948; Audus, 1949, 1951; Ready et a l , 1947; Aberg, 1957). x i v 4. Straight growth t e s t , using Avena c o l e o p t i l e segments (Bonner, 1933, 1949); using pea stem segments (Galston and Hand, 1949; Galston and Baker, 1951, 1953; Christiansen and Thimann, 1950). 5. Petiole abscission t e s t (Luckwill, 1956; M i t c h e l l et a l , 1968). 6. Adventitious root i n i t i a t i o n t e s t (Libbert, 1956; M i t c h e l l et a l , 1968). The detection of IAA complexes such as IAA-glucose (Klambt, 1961; Zenk, 1964), and IAA-aspartate (Andreae and Good, 1955; Zenk, 1964) i s an important contribution towards the understanding of the mechanism of IAA action i n plants. IAA complexes may escape detection because they do not give t y p i c a l indole colour reactions with the Salkowski and E h r l i c h reagents which are commonly used by investigators i n auxin research. While workers are becoming increasingly aware that free IAA accounts for only a portion of the auxin a c t i v i t y i n plants, and that bound IAA i s of "physiological s i g n i f i c a n c e " , the time has come for a turning point i n auxin research. Future research should be geared to i s o l a t i o n , i d e n t i f i c a t i o n , and quantitative determination of IAA complexes. The fact that a p p l i c a t i o n of naphthenic acids. (HNap) to plants results i n increased growth, and the report that petroleum growth substances caused bending of the Avena c o l e o p t i l e •,2>y Huseinov (1960), r a i s e s the question of the q u a l i f i c a t i o n joi -HNap and t h e i r s a l t s as auxin. In t h i s i n v e s t i g a t i o n , some of the auxin-like properties of naphthenates were deter-mined, as were t h e i r e f f e c t s on the biosynthesis and degradation of IAA. !Phe f i r s t phase of the present i n v e s t i g a t i o n deals with the evaluation of the auxin-like properties of potassium naphthenates (KNap), u t i l i s i n g the following bioassays: 1. Growth of i n t a c t roots of cucumber (Cucumis sativa L) 2. Adventitious root i n i t i a t i o n i n succulent and woody stem cuttings. 3. Elongation of pea stem segments. 4. Abscission of p e t i o l e s of bean (Phaseolus vulgaris) plants. The second phase of the present i n v e s t i g a t i o n i s concerned with the e f f e c t of KNap on IAA biosynthesis and degradation. In the pages that follow, the term " s i g n i f i c a n t " implies s t a t i s t i c a l s i g n i f i c a n t difference at the 0.05 l e v e l , unless otherwise stated. 1 NAPHTHENIC ACIDS The name, n a p h t h e n i c a c i d , was f i r s t s u g g e s t e d b y M a r k o v n i k o f f and O g l o b i n (1883) f o r t h e C,,H 0 a c i d s o f unknown s t r u c t u r e 11 20 2 w h i c h H e l l and M e d i n g e r (1874) r e c o v e r e d from Rumanian c r u d e o i l . The p l a n t g r o w t h s t i m u l a t o r s o b t a i n e d from p e t r o l e u m have been named and a b b r e v i a t e d d i f f e r e n t l y b y v a r i o u s r e s e a r c h e r s namely: n a p h t h e n i c g r o w t h s u b s t a n c e s (N.G.S.), p e t r o l e u m growth s u b s t a n c e s (P.G.S.), n a p h t h e n i c a c i d s (HNap), p o t a s s i u m n a p h t h e n a t e s (KNap), o i l hormone s u b s t a n c e s (O.H.S.), n a p h t h a growth m a t t e r , o i l g r o w t h m a t t e r , p e t r o l e u m n u t r i e n t , s i n t o v i t (an o x i d i s e d p e t r o l e u m p r o d u c t ) , Sh-8 (a c y c l o b u t a n o l ) , p e t r o l e u m o r g r o w t h - h e l p i n g s u b s t a n c e s (P.R.V. o r N.R.V.), H.P.B., Kh.T.I., H.T.I. The l a t t e r two a r e used b y i n v e s t i g a t o r s i n B u l g a r i a . I n t h e R u s s i a n language, t h e a b b r e v i a t i o n s R.V. mean " g r o w t h - h e l p i n g s u b s t a n c e " . N a p h t h e n i c a c i d s a r e i s o l a t e d from p e t r o l e u m b y a c i d -b a s e e x t r a c t i o n , and from d i e s e l o i l by a d s o r p t i o n . N a p h t h e n i c a c i d s a r e known t o be a complex m i x t u r e o f a l k y l a t e d a l i c y c l i c m o n o c a r b o x y l i c a c i d s , some o f w h i c h a r e d e r i v a t i v e s o f c y c l o p e n t a n e , c y c l o h e x a n e , and c y c l o h e p t a n e . J o l l y (1967) n o t e d t h a t c y c l o p e n t y l d e r i v a t i v e s p r e d o m i n a t e i n t h e n a p h t h e n i c a c i d m i x t u r e , f o l l o w e d b y c y c l o h e x y l compounds. R e c e n t l y , Kazanis (1971) by mass spectrometries nuclear magnetic resonance and i n f r a r e d analyses, detected methyl 2-DL-4D-dimethyl heptanoate, methyl octanoate, methyl nonanoate, and c i s 1,3-dimethylcyclohexycarboxylate i n the naphthenic acid mixture i s o l a t e d from crude petroleum. The molecular weights and b o i l i n g points of HNap vary with the source of the acids. HNap with an average molecular weights of 214 (Cason and Khodair, 1966); 211, 239, and 303 (Tanchuk, 1971); 216 (Nametkin, 1971) have been reported. A b o i l i n g point range from 30° to 150° C was reported by Kazanis (1971), and 45° to 170° by Artaraonov (1971). The commercially a v a i l a b l e naphthenic acids, used i n the present in v e s t i g a t i o n s , have an average molecular weight of 230. The carboxyl group of most i n d i v i d u a l members of the naphthenic ac i d mixture i s not attached d i r e c t l y to the a l i c y c l i c r i n g , but i s separated from the r i n g by an a l i p h a t i c side chain containing one to f i v e or more methylene groups. According to J o l l y (1967), a general formula may be written as RtC^^COOH, where R i s an a l i c y c l i c nucleus composed of one or more rings. An exception to t h i s general rule i s cyclohexanecarboxylic acid and i t s K s a l t which have been shown to e x h i b i t b i o l o g i c a l a c t i v i t y (Wort and Patel, 1 9 7 0 ; Severson, 1 9 7 1 ; Padmanabhan, 1 9 7 2 ; Peirson, 1 9 7 2 ) and also i n the present i n v e s t i g a t i o n s . The naphthenic acids, obtained from Baku have an acid number of 2 5 9 mg KOH/g (Nametkin, 1 9 7 1 ) . Naphthenic acids have a c h a r a c t e r i s t i c rubbery odour which varies with the aci d source, degree of refinement, and content of phenolic and suphur compounds. The acids are r e a d i l y soluble i n non-polar solvents, and the lower molecular weight members, such as cyclohexanecarboxylic a c i d and cyclopentanecarboxylic a c i d are sparingly soluble i n water. In low concentrations ( 5 0 0 0 ppm or l e s s ) , naphthenates have been found to promote vegetative and reproductive growth (e.g. Severson, 1 9 7 1 ) . There are also reports that when applied at high concentrations naphthenates act as herbicides (Mailov, 1 9 6 8 ; Zhukova, 1 9 6 5 ) . In t h i s respect, the behaviour of naphthenates i s s i m i l a r to that of '2,4-D. The mode of action by which applied naphthenates influenc plant growth and metabolism i s obscure. Wort ( 1 9 6 9 ) suggested 4 the anion, naphthenate was responsible f o r the stimulation of vegetative and reproductive growth i n beans. The glucose ester and aspartic acid amide of naphthenic acids rather than the free a c i d were responsible for the stimulation of glucose uptake and i t s metabolism i n bean root t i p s according to Severson (1972). At t h i s time, i t i s not possible to designate one or more naphthenic acids as ..specifically responsible fo r stimulation of plant growth and development but some s t r u c t u r a l c h a r a c t e r i s t i c s of e f f e c t i v e acids have been suggested (Wort and Patel, 1970). Following t h e i r a p p l i c a t i o n to bean plants, naphthenates stimulate many p h y s i o l o g i c a l and biochemical processes. The stimulation of photosynthesis and dark r e s p i r a t i o n (Fattah, 1969; Wort et a l , 1970), protein synthesis (Severson, 1972; Wort et aJL, 1971), and the s p e c i f i c a c t i v i t i e s of numerous enzymes in crude extracts (Chu, 1969; Fattah, 1969; Fattah and Wort, 1970; Wort et al_, 1971) suggest that naphthenate stimulation of plant growth i s the r e s u l t of the action of the chemicals or t h e i r metabolites at the genetic and metabolic l e v e l s (Wort et a l , 1971). A method for quantitative determination of sodium naphthenates i n plants was given by Guseinov (1970). 5 Responses to naphthenate treatments, including greater vegetative and reproductive growth have been reported for maize, tomato, potato, winter cereals tgrapes, and other crop plants (Popoff and Boikov, 1966). The e f f e c t s of naphthenates on plant growth, y i e l d and composition were summarised by Severson (1971). Petroleum growth substances are v e r s a t i l e i n causing stimulation of growth and metabolism. The stimulatory e f f e c t s of P.G.S.. are not only observed i n plants but also i n animals. P.G.S. when administered o r a l l y at 1.0 mg/kg body weight of castrated rams, increased the serum protein l e v e l by 3.64-7.2% with a maximum a f t e r 40 days, and increased serum albumin^ l e v e l by 12.92% (Mekhtiev, 1970). Gorshkova (1970) reported p r o l i f e r a t i o n of both i n t e r and i n t r a lobular tissues i n the l i v e r of rabbits 10 days a f t e r administration of 20 mg P.G.S. per kg body weight. Thirty-day doses of 5 mg/kg increased rabbit body weight by 18-20%, while 20 mg/kg decreased the body weight by 20-22% (Loshmanova, 1970). The non-biological uses of naphthenic acids and t h e i r s a l t s are varied. They are used as lub r i c a n t s i n r e f r i g e r a t i o n and air-conditioning, d r i e r s i n the paint industry, c a t a l y s t s preservatives, emulsifiers ( J o l l y , 1967), and as lead naphthenate, a constituent i n a mixture that i s r e s i s t a n t to s a l t water corrosion. They are also used as copper naphthenates i n f u n g i c i d a l preparations. The information -accumulated thus far i s s u f f i c i e n t to j u s t i f y the i n c l u s i o n of naphthenic acids and t h e i r s a l t s as one of the plant growth substances along with the so-c a l l e d "established" plant growth regulators such as: indole-3-acetic acid, g i b b e r e l l i c acids, cytokinins, 2,4-D, 2,4,5-T, naphthaleneacetic acid, 2,3,6 tri-iodobenzoic acid, maleic hydrazide, cycocel, and phosfon. Naphthenic acids and t h e i r s a l t s may be superior to other plant growth regulators due to t h e i r dual stimulatory e f f e c t s on plants and animals. 7 NAPHTHENIC ACIDS CO OH COOH cyclopentanecarboxylic a c i d : C6 H10°2 -, molecular weight 114 cyclohexanecarboxylic a c i d C 7 H 1 2 0 2 molecular weight 128 C O O H 3-methyl-cyclopentyl-n-propionic acid 9 16 2 molecular weight 156 CHAPTER I INVESTIGATION OF THE AUXIN-LIKE PROPERTIES OF POTASSIUM NAPHTHENATES. 9 ia) Growth of i n t a c t roots of cucumber seedlings. LITERATURE REVIEW A survey of the l i t e r a t u r e on the influence of naphthenates on root -growth indicates the scanty nature of research i n t h i s area. The prevalent response of i n t a c t roots to exogenous auxin i s retardation of elongation. Roots have been reported to respond to low concentrations of in d o l e a c e t i c acid (IAA) by s l i g h t increase i n growth rate (e.g. Burstrom, 1951; Larpen, 1961). The use of root measurements of red clover to determine the presence of 2,4-D i n s o i l was described i n a paper by Nutman et a l (1945). They found that 2,4-D at concentrations of 1.0, 10, and 100 ppm were i n h i b i t o r y to root growth. Investigations on the e f f e c t of auxins on root growth have shown that, i n general, substances which cause growth promotion i n the Avena c o l e o p t i l e , cause i n h i b i t i o n of root growth (e.g. Bentley and Bi c k l e , 1952). Moewus i n 1949 who devised the cress root t e s t as a quantitative method of estimating growth substances, found 10 that 2.4 x 10 M IAA was necessary f o r 50% i n h i b i t i o n of root growth. Audus (1949) obtained 50% i n h i b i t i o n of root -7 growth an cress, radish, garden pea, and maize i n 6.8 x 10 M 2,4-D i n so l u t i o n c u l t u r e at pH 6.8. On the e f f e c t of IAA on root growth using Moewus (1949) cress t e s t , Bentley (1951) showed that IAA i n h i b i t e d root growth i n Lepidium sativum over a range of concentrations from 1-0 to 10 mg/1. A s l i g h t growth promotion at lower concentrations, to a maximum -7 "8 , of ca 30% at 10 to 10 mg/1 was also found. Ready and Grant (1947) found seedlings of Cucumis sativus to be s e n s i t i v e to 2,4-D. The growth of primary roots was i n h i b i t e d to 50% -7 by 2.1 x 10 M so l u t i o n of 2,4-D. Using seedlings of'Avena s a t i v a grown on f i l t e r paper moistened with solutions of —6 growth substances, Lane (1936) found that 1.7 x 10 M IAA was necessary for 50% i n h i b i t i o n of root growth, while Bonner -7 -6 and K o e p f l i (1939) reported 3 x 10 M IAA and 7 x 10 M NAA were required to br i n g about the same i n h i b i t i o n . A reduction of ca 10% i n growth of the root system following treatment with sprays of 0.005% P.G.S. on.eggplants was demonstrated by Ali-Zade and Guseinov (1965). 11 B e n t l e y (1958) found an a v e r a g e maximum s t i m u l a t i o n o f 20% -3 w i t h IAA a t 5.7 x 10 M u s i n g Moewus r o o t t e s t (Moewus, 1948, 1949). The e f f e c t o f t e m p e r a t u r e on r o o t g r o w t h was d e s c r i b e d i n a paper by P i l e t and Went (1956). They o b s e r v e d t h a t -4 IAA a t 10 M r e t a r d e d g r o w t h o f o l d r o o t s o f Lens c u l i n a r i s , and t h e degree o f r e t a r d a t i o n i n c r e a s e d m a r k e d l y w i t h t e m p e r a t u r e , whereas t h e growth o f young r o o t s was a c c e n t u a t e d s l i g h t l y a t low t e m p e r a t u r e s and i n h i b i t e d a t h i g h t e m p e r a t u r e s . -8 A t 1.0 x 10 M IAA, g r o w t h o f b o t h o l d and young r o o t s was s t i m u l a t e d a t a l l t e m p e r a t u r e s (5-27° C) . .-H u s e i n o v (1960), u t i l i s i n g s i m i l a r c o n c e n t r a t i o n s o f n a p h t h e n i c growth s u b s t a n c e s (N.G.S.) and IAA ( h e t e r o a u x i n ) (0.1, 0.01, 0.001, and 0.0001%) o b t a i n e d s i m i l a r t r e n d s i n r e s p o n s e o f r o o t g r o w t h o f wheat i . e . i n h i b i t o r y a t h i g h c o n c e n t r a t i o n (0.1%) and s t i m u l a t o r y a t weak c o n c e n t r a t i o n s (0.0001% N.G.S., 0.001% I A A ) . He a l s o employed d i f f e r e n t p e r i o d s o f seed soak (6, 12, and 24 h o u r s ) i n 0.005, 0.01, and 0.05% N.G.S., and f o u n d t h a t r o o t l e n g t h s o f w i n t e r wheat were g r e a t e r t h a n t h a t o f t h e c o n t r o l s . Seed w e t t i n g w i t h 12 weak solutions of N.G.S. (0.004 and 0.0004%) p r i o r to sowing, enhanced the root lengths of cotton, onion, cucumber, and winter wheat. The growth of cotton roots was stimulated by soaking the seeds i n solutions of 10 mg/1 HNap (Babaev, 1966). 13 MATERIALS AND METHODS Seeds of cucumber (Cucumis sativus L.) were washed with tap water and f i f t e e n uniform si z e seeds were placed i n each p e t r i dish (diameter 9 cm) containing three pieces of Whatman No. 1 f i l t e r paper. Eight ml volumes of each of the following t e s t solutions were separately to each dish: KNap 1000 ppm KNap 100 ppm KNap 10 ppm KNap 1.0 ppm D i s t i l l e d water served as co n t r o l . The p e t r i dishes were covered and placed i n an incubator at a constant temperature of 20° C for 72 hours. At the end of the incubation period, the lengths of the primary roots were determined. The average length of the roots was used to compute the percent i n h i b i t i o n of root elongation: Average control root length minus average treated root length Average control root length 14 Each treatment was repeated four times, and the whole experiment was l a i d down i n a randomised complete block design. The r e s u l t s of the experiment were subjected to s t a t i s t i c a l analysis and Duncan's New M u l t i p l e Range Test (Duncan, 1955). Preparation of potassium naphthenates . (KNap) aqueous so l u t i o n from naphthenic acids (HNap) Seventeen ml of a 12.3% (w/v) KOH s o l u t i o n (2.1 g KOH i n 17 ml d i s t i l l e d water) was added to an Erlenmeyer f l a s k containing 5 g HNap ( P r a c t i c a l grade, average molecular weight 230> Eastman Organic Chemicals, Rochester, New York). The f l a s k was shaken for 10-15 minutes and the s o l u t i o n was made to a volume of 25 ml with d i s t i l l e d water. The solu t i o n thus prepared was the stock s o l u t i o n containing 250 mg of KNap per ml. By d i l u t i n g 1.0 ml of the stock solu t i o n with 49 ml of d i s t i l l e d * the f i n a l concentration of KNap was -2 2 x 10 M (5000 ppm or 0.5%). The pH of the d i l u t e d s o l u t i o n was adjusted to ca 10 with 1.0 N HC1. RESULTS Low KNap concentrations of 1.0 and 10 ppm treatment of cucumber seeds stimulated root growth (ca 3 and 4% respectively),. However, t h i s -stimulatory e f f e c t of KNap was not s i g n i f i c a n t . High KNap concentrations (100 and 1000 ppm) had an i n h i b i t o r y e f f e c t on root growth (ca 21 and 91% r e s p e c t i v e l y ) . Only the i n h i b i t o r y e f f e c t of 1000 ppm was s i g n i f i c a n t (Table I ) . The r e s u l t s are presented g r a p h i c a l l y i n figure 1. 16 Table I. Response of i n t a c t roots of cucumber to KNap. Concentration Average length of roots (ppm) from 60 determinations (mm) 0 (control) 93.18 a 1.0 96.32 a 10 97.37 a 100 73.30 a 1000 8.37 ** Values followed by the same l e t t e r do not d i f f e r s i g n i f i c a n t l y from one another, according to Duncan's New Mult i p l e Range Test (Duncan, 1955). ** Value d i f f e r s s i g n i f i c a n t l y from the c o n t r o l value at the 0.01 l e v e l . 17 18 DISCUSSION The trend i n response of cucumber roots to KNap i s t y p i c a l of auxin e f f e c t , i . e . prevalent retardation of elongation i n high concentrations and s l i g h t promotion i n low concentrations (e.g. Burstrom, 1951;" Larsen, 1961). The i n h i b i t i o n of cucumber root growth by 1000 ppm KNap i s s i m i l a r to that reported by Huseinov (1960) using N.G.S. i n wheat. In the present experiment, the treatments with low concentrations of KNap (1.0 and 10 ppm) on cucumber resulted i n a s l i g h t promotion of root growth. Similar increases have been reported by Huseinov (1960) u t i l i s i n g 5, 10, and 50 ppm N.G.S. seed soak of wheat. The f a i l u r e to obtain s i g n i f i c a n t stimulation of root growth i n 1.0 and 10 ppm KNap concentrations suggests that the e f f e c t i v e property of KNap i s predominantly that of i n h i b i t i o n of root growth which i s c h a r a c t e r i s t i c of auxin (e.g. Burstrom, 1951; Larsen, 1961). The retardation of root growth caused by IAA i s exhibited i n much lower concentrations than KNap, for instance, at ca 0.3 ppm on Avena s a t i v a (Lane, 1936); at a range from 1.0 to 10 ppm on Lepidium sativum - 7 (Bentley, 1951); at 10 M on wheat root (Burstrbm, 1942). The fact that i t requires a r e l a t i v e l y high concentration of KNap (1000 ppm) to bring about a s i g n i f i c a n t retardation of root .growth as compared to IAA (e.g., 1.0 to 10 ^ ppm, Bentley, 1951) suggests that the degree of effectiveness of KNap i s less than that of IAA. 20 ib) Adventitious root i n i t i a t i o n i n succulent and woody stem cuttings. LITERATURE REVIEW The auxin-induced formation of roots on stem and l e a f cuttings, and on i n t a c t plants provides a good bioassay for auxin a c t i v i t y . That the formation of adventitious roots on a c u t t i n g always occurs at the p h y s i o l o g i c a l base, was f i r s t shown i n willow by H. Vochting i n 1878. An extensive c r i t i c a l review of the r o l e of hormones i n the formation of adventitious roots was published by Libbert (1957a). The o r i g i n a l q uantitative method was developed by Went (1934b), u t i l i s i n g e t i o l a t e d Pisum stem cuttings. Later, Went's method was modified by other i n v e s t i g a t o r s for the use with d i f f e r e n t species of plants (e.g., Libbert, 1957b; Luckwill, 1956). The p a r t i c i p a t i o n of auxin i n c o n t r o l l i n g root branching was amply demonstrated by Thimann (1936) i n experiments 21 t r e a t i n g the roots of Avena and Pisum with IAA. The app l i c a t i o n of indolebutyric acid i n concentrations of 12.5 and 25 mg/1 to the base of root cuttings of Taraxacum and Cichorium stimulated abundant root production (Warmke and Warmke, 1950). Polikarpova (1963) reported the use of P.G.S. during root formation i n green cuttings of cherries, gooseberries, and blackcurrants, favourably affected the formation of roots and subsequent development of the cuttings. The best concentration was found to be 0.01% (100 ppm) P.G.S. The p h y s i o l o g i c a l a c t i v i t i e s of sodium s a l t s of low and high molecular weight naphthenic acids obtained from d i f f e r e n t sources were investigated with respect to root formation (Porutskii et aJL, 1963) . They found that root formation decreased with increasing molecular weights of HNap obtained from d i f f e r e n t sources as indicated i n t h i s order: d i e s e l f u e l < transformer o i l < l u b r i c a t i o n o i l < motor o i l . Root formation was stimulated by P.G.S. i n cuttings of cranberry, phlox, vine, p r i v e t , and the leaves of p e r i l l a , but no stimulation i n cuttings of spindle-tree and poplar was observed (Yusufov, 1963). The physiology of root primordia i n i t i a t i o n has been extensively discussed following the introduction of the rh i z o c a l i n e hypothesis by Bouillenne and Went i n 1933. 22 The name, rhi z o c a l i n e , was f i r s t suggested by Bouillenne and Went i n 1933 for the rooting substance produced by the leaves i n the presence of l i g h t . This substance i s stored i n the cotyledons and buds, and transported b a s i p e t a l l y . According to the r h i z o c a l i n e hypothesis (Cooper, 1936; Went, 1938), the ba s i p e t a l transport of auxin causes i t to accumulate at the base of a cutting, and the r e s u l t i n g auxin gradient causes a downward movement of r h i z o c a l i n e to the base of the cu t t i n g . Auxin then "activates" or "reacts" with r h i z o c a l i n e to bring about root formation. The existence of r h i z o c a l i n e or r h i z o c a l i n e - l i k e substances was amply demonstrated (Bouillenne and Bouillenne, 1952; Galston, 1948; Hess, 1962). The l a t e s t modification of the r h i z o c a l i n e hypothesis by Bouillenne (1964) describes r h i z o c a l i n e as a complex of three factors, namely 1) A highly s p e c i f i c , mobile factor with orthodiphenolic groups which i s synthesised i n the leaves. 2) Auxin. 3) An oxygen-requiring enzyme located i n s p e c i f i c c e l l s and tis s u e s . Hess (1957) and Kawase (1964) added the concept of cofactors. The cofactors were considered to be endogenous substances capable of acting s y n e r g i s t i c a l l y with IAA i n the rooting of cuttings of mung bean and b r i t t l e willow. Haissig (1971) suggested that RNA i s necessary for the i n i t i a t i o n of root primordia. MATERIALS AND METHODS i) Succulent stem cuttings using bean plants. Uniform size seeds of bush bean plant, Phaseolus  •vulgaris L. c u l t i v a r Top Crop (Buckerfield's Ltd., New Westminster, B.C.) were sown on steam-sterilised composted s o i l i n s i x wooden f l a t s (30 x 47 x 7 cm). Uniformity of plants was achieved by periodic c u l l i n g of runts. The plants were grown i n a greenhouse with a 14-hour photoperiod under Sylvania Gro-Lux phototube which gave 10,000 lux l i g h t i n t e n s i t y at the top of the plants at 25° + 1° C. The stems of 14-day-old seedlings were cut at ca cm above s o i l l e v e l and washed with running tap water. One-half of each of the two primary leaves was removed to reduce t r a n s p i r a t i o n a l loss of water. The bases of the stem cuttings thus prepared were soaked for s i x hours to a depth of four cm i n one of the following solutions: 1) D i s t i l l e d water served as contr o l . 2) KNap 10 ppm 3) KNap 100 ppm 4) Indolebutyric acid 10 ppm 5) Indolebutyric acid 100 ppm A c i r c u l a r Pyrex glass container (diameter 27 cm) s u f f i c i e n t to hold one l i t r e of solution, was placed i n a wooden f l a t . Care was exercised not to allow the leaves to come i n contact with the solution. At the conclusion of the soak period, the cuttings were removed from the solution, and the basal portions of t h e i r stems inserted to a depth of four cm i n evenly spaced rows i n the rooting medium kept i n the propagation chamber. The rooting medium consisted of 1:1 (v/v) of fine sand and peat. The sand was sieved successively through %-inch mesh and 1/8-inch mesh sieves, and then washed thoroughly. 25 The peat was wetted with tap water and screened through %-inch mesh sieve. The rooging medium was s t e r i l i s e d at 248° C f o r 15 minutes at a pressure of 15 l b per square inch. The propagation chamber was covered with a polythene l i d to allow a d a i l y 14-hour l i g h t of i n t e n s i t y 5200 lux to reach the cuttings. The chamber had a i r temperatures of 20.5 + 1° C, and a r e l a t i v e humidity of 90 ± 5%. Indole-3-butyric acid, a rooting hormone, was included i n t h i s experiment to serve as a standard for comparison (Mitchell et a l , 1968). The stem cuttings were allowed to root for f i v e days, then the root systems were thoroughly washed i n running water. The number and length of roots were determined. The experiment was conducted i n a randomised complete block design with three blocks. Each treatment consisted of 24 cuttings, with eight cuttings randomly placed i n each block. The r e s u l t s were subjected to analysis of variance and Duncan's Multi p l e Range Test (Duncan, 1955). 26 i i ) Woody stem cuttings using azalea plants. Stem cuttings of "Chichibu", an evergreen Japanese azalea of the Wadai group (Rhododendron indicum var. eriocarpum x kaempferi, according to Lee et al_, 1952), were c o l l e c t e d on 20th August, 1971 from several bushes i n the Nursery on campus. Uniform stem cuttings were taken from the a p i c a l regions (12-15 cm) of the plants. The stem cuttings c o l l e c t e d from the f i e l d , were washed and were cut under water below the second node. Three to four leaves were l e f t on each cutting. The prepared stem cuttings were s u r f a c e s s t e r i l i s e d by immersing i n 1.0% Captan fungicide for 15 minutes. Thereafter, the cuttings were soaked for s i x hours i n one of the following solutions i n the greenhouse at a l i g h t i n t e n s i t y of 6000 lux, and a i r temperatures of 25° + 1° C: 1) KNap 1000 ppm 2) KNap .100 ppm 3) KNap 10 ppm 4) D i s t i l l e d water served as contro l . 27 Following the six-hour soak period, the basal ends o f the cuttings were inserted into s t e r i l i s e d rooting medium,consisted of 1:1 (v/v) f i n e sand and peat prepared as described i n ( i ) , contained i n wooden f l a t s . The cuttings were allowed to root i n a greenhouse. The greenhouse provided a 16-hour photoperiod, a i r temperatures o f 22 + 1° C, and a l i g h t i n t e n s i t y of 4,400 lux. The.relative humidity o f the experimental area was maintained at ca 100% by means of an overhead water spray operated by an automatic humidity sensing device. The experiment was set up i n accordance to the randomised complete bloc3c design comprising four treatments arranged i n four blocks with ten cuttings of each treatment per block. The stem cuttings remained i n the rooting medium for a t o t a l of 35 days 4 The rooted cuttings were then c a r e f u l l y removed, washed, and the number and. length of the roots determined. An analysis of variance and Duncan'-s New M u l t i p l e Range Test (Duncan, 1955) were performed on the r e s u l t s obtained. : RESULTS i) Rooting of succulent stem cuttings of bean. The average number of roots on a stem cutting i s used to represent the degree of root i n i t i a t i o n . This i s based on the assumption that the number of roots i s analogous to the number of root primordia. In a preliminary experiment, logarithmic d i l u t i o n s of KNap concentrations of a range from 1000 to 0.0001 ppm were used to determine the e f f e c t of d i f f e r e n t concentrations of KNap on root i n i t i a t i o n and growth, and to acertain the optimal concentrations for subsequent investigations. It was found that 1000 ppm KNap was injurious to stem cuttings, and caused a profound i n h i b i t i o n of root i n i t i a t i o n . The portion of the stem cuttings immersed i n 1000 ppm KNap gave a "water soaked" appearance. The greatest stimulatory e f f e c t was observed with 10 and 100 ppm. 29 The i n d u c t i o n o f r o o t s , and t h e p r o m o t i o n o f r o o t g r o w t h f o l l o w i n g t r e a t m e n t s w i t h KNap and i n d o l e b u t y r i c a c i d (IBA) were s i g n i f i c a n t l y d i f f e r e n t from t h a t o f c o n t r o l p l a n t s a t t h e 0.01 l e v e l . The t r e a t m e n t s w i t h 10 and 100 ppm KNap gave i n c r e a s e s i n r o o t i n i t i a t i o n o f 46.35 and 152.94% r e s p e c t i v e l y o v e r c o n t r o l p l a n t s . The c u t t i n g s • i n 10 and 100 ppm IBA had i n c r e a s e s o f 164.54 and 297.41% r e s p e c t i v e l y o v e r c o n t r o l p l a n t s . The s t i m u l a t i o n o f r o o t i n d u c t i o n (152.94%) b y 100 ppm KNap d i d n o t d i f f e r s i g n i f i c a n t l y f r o m t h e s t i m u l a t i o n (164.54%) b y 10 ppm IBA ( T a b l e I I ) . G r e a t e r s w e l l i n g a t t h e b a s e s o f K N a p - t r e a t e d stem c u t t i n g s o v e r c o n t r o l c u t t i n g s were o b s e r v e d , b u t no d e t e c t -a b l e d i f f e r e n c e s between KNa p - t r e a t e d . and I B A - t r e a t e d c u t t i n g s . The s t i m u l a t i o n o f r o o t i n i t i a t i o n as p e r c e n t a g e o f t h e c o n t r o l , i s r e p r e s e n t e d s c h e m a t i c a l l y i n f i g u r e 2. The e f f e c t s o f t h e t r e a t m e n t s on t h e stem c u t t i n g s can be seen i n P l a t e 1. 30 Table II . Rooting response of bean stem cuttings to KNap and IBA treatments. Treatment +Average Increase +Average Increase number of over t o t a l length over .roots1; per cont r o l of roots c o n t r o l c u t t i n g (%) per c u t t i n g (%) D i s t i l l e d 28.20 „ 231.33 H 20 (control) KNap 10 ppm 41.27 46.35 400.00 72.91 KNap 100 ppm 71.33 a 152.94 507.40 119.34 IBA 10 ppm 74.60 a 164.54 722.67 212.40 IBA 100 ppm 112.06 297.41 1257.33 443.52 +Value from 15 stem cuttings. Values followed by the same l e t t e r do not d i f f e r s i g n i f i c a n t l y from each other, according Duncan's New Multi p l e Range Test (Duncan, 1955). 31 40d n 300 • Average number of 2 0 0 " roots per c u t t i n g (% control) 100 • c o n t r o l KNap KNap' IBA IBA 10 ppm 100 ppm 10 ppm 100 ppm Figure 2. Stimulation of root i n i t i a t i o n of bean stem cuttings by KNap and IBA treatments. Plate 1. Rooting response of bean stem cuttings KNap and IBA treatments. Legend: 1 Cutting i n 2 Cutting i n 3 Cutting i n 4 Cutting i n 5 Cutting i n d i s t i l l e d water. 10 ppm KNap. 100 ppm KNap. 10 ppm IBA. 100 ppm IBA. 33 i i ) Rooting of woody stem cuttings of azalea. The treatments of azalea stem cuttings with KNap (10, 100, and 1000 ppm) stimulated both induction and growth of roots. High concentrations of KNap (100 and 1000 ppm) resulted i n a s i g n i f i c a n t increase i n number of roots (ca 50 and ca 134% respectively) over the con t r o l cuttings at the 0.01 l e v e l . However, the low concentration (10 ppm) of KNap had n o . s i g n i f i c a n t stimulation of root i n i t i a t i o n over c o n t r o l values. A l l concentrations of KNap (10, 100, and 1000 ppm) s i g n i f i c a n t l y augmented root growth (ca 36, 66, and 220% respectively) at the 0.01 l e v e l . The treatments with 10 and 100 ppm KNap. did not d i f f e r s i g n i f i c a n t l y from each other. A s l i g h t "scorching" of the portion of the stems immersed i n 1000 ppm KNap was observed. The r e s u l t s of t h i s i n v e s t i g a t i o n are given i n Table i n and the stimulatory e f f e c t of the treatments can be seen i n Plate 2. Root i n i t i a t i o n c a l c u l a t e d as percentage of co n t r o l values are represented i n a histogram i n - f i g u r e 3. 34 Table I I I . Response o f stem c u t t i n g s o f a z a l e a t o KNap. Concentration Average number Increase Average t o t a l Increase o f KNap (ppm) of r o o t s per over len g t h o f over c u t t i n g + c o n t r o l r o o t s per c o n t r o l (%) c u t t i n g + (%) 0 ( c o n t r o l ) 29.62 a — 279.75 — 10 33.37 a 12.66 382.17 b 36.61 100 44.46 50.10 464.12 b 65.90 1000 69.29 133.93 894.58 219.77 + Value from 24 stem c u t t i n g s . Values f o l l o w e d by the same l e t t e r do not d i f f e r s i g n i f i c a n t l y from each other, according t o Duncan's New M u l t i p l e Range Test (Duncan, 1955). 250 i 200 150 Average number of roots per cutt i n g {% control) 100 50 con t r o l 10 ppm 100 ppm 1000 ppm Figure 3. Stimulation of root i n i t i a t i o n i n azalea stem cuttings by KNap treatments. Plate 2. Rooting response ,of azalea stem cuttings KNap treatments. The concentrations of KNap employed as indicated. 37 DISCUSSION L The rooting of stem cuttings i n d i s t i l l e d water i s evidence of the presence of endogenous auxin, and the predominance of i t s b a s i p e t a l transport. The average number of roots formed per treated stem c u t t i n g i s taken as a measure of root-forming a c t i v i t y of KNap and IBA treatments. The marked increases i n the average number of roots per cuttin g i n KNap- and IBA-treated cuttings suggest that these compounds possess high root-forming a c t i v i t y . The greater number of roots i n KNap treated cuttings may be the r e s u l t of accelerated mitosis which leads to an increased rate of i n i t i a t i o n of root meristems. The stimulation of root i n i t i a t i o n and the increased swellings at the extreme t i p s of the bases of KNap-treated stem cuttings over that of the controls may be accounted for on three bases: 1) The possible augmentation of endogenous auxin by KNap. 2) The r e l a t i v e l y low mobility, and greater chemical s t a b i l i t y of KNap following a p p l i c a t i o n to stem cuttings which res u l t e d i n the accumulation and retention of KNap near the s i t e of a p p l i c a t i o n . 3) A mobilization of root-forming substances or cofactors 38 f o r example r h i z o c a l i n e , to the s i t e of KNap a p p l i c a t i o n . Chemical s t a b i l i t y and low mob i l i t y are two important properties of root-forming substances (e.g., Audus, 1959). The stimulation of rooting of azalea cuttings i s i n agreement with the observations of Polikarpova (1963) using 100 ppm P.G.S. on woody cuttings of cherries, gooseberries, and blackcurrants. In very high KNap concentration (1000 ppm), a to x i c e f f e c t on bean cuttings set i n and o f f s e t stimulation r e s u l t i n g i n a "water soaked" appearance i n the treated stem portions. The optimum root-forming property of KNap was found to be i n the concentrations of 100 ppm i n succulent bean cuttings, and 1000 ppm i n woody azalea cuttings. The increases i n root growth of treated stem cuttings are interpreted as mainly due to a r e l a t i v e l y e arly root i n i t i a t i o n compared with the control cuttings. The lack of s i g n i f i c a n t difference between the e f f e c t s of 100 pprn KNap and 10 ppm IBA treatments on bean cuttings indicates that the r o o t - i n i t i a t i o n e f f e c t of IBA i s s i m i l a r to that of KNap. IBA i s an established rooting hormone, and proved to be superior to IAA (e.g., Audus, 1959). As i s t y p i c a l of the e f f e c t of auxin, there i s a stimulation of adventitious root i n i t i a t i o n by KNap. 39 i c ) Elongation of pea stem segments. LITERATURE REVIEW The behaviour of stem segments of Pisum under the influence of synthetic auxin i n various experimental conditions has been studied by Galston and Hand (1949); Nitsch and Nitsch (1956\ and others. The response of such segments has been" employed to detect and characterise n a t u r a l l y -occurring auxin (Larsen, 1961). Galston and Hand (1949) who incubated 8-day-old dark-grown Alaska pea stem segments i n various concentrations of IAA at 25° C for 24 hours, found that h a l f maximal growth was generally produced by 0.01 mg/1, and maximal growth by 0.1 and 1.0 mg/1. The segments immersed i n 10 mg/1 IAA had a "water soaked" appearance. Maximum response i n the s t r a i g h t growth t e s t was obtained by the use of 0.1 mg/1 IAA, pH 6.1 (K^PO^ -Na^HPO^) i n 2% sucrose, and at 30° C i n darkness (Galston and Hand, 1949). 40 W h i te l i g h t r e t a r d s t h e e l o n g a t i o n o f e t i o l a t e d pea stem segments i n v a r y i n g c o n c e n t r a t i o n s o f IAA, and t h i s l i g h t - i n d u c e d i n h i b i t i o n o f g r o w t h c o u l d n o t be e x p l a n i e d i n terms o f p h o t o i n a c t i v a t i o n o f IAA. A t low exogenous IAA l e v e l s , t h e r e i s r e l a t i v e l y g r e a t e r e f f i c i e n c y i n t h e u t i l i s a t i o n o f IAA b y t h e segments. T h i s e f f i c i e n c y i s g r e a t e r i n t h e d a r k t h a n i n l i g h t ( G a l s t o n and Hand, 1949). The e f f e c t o f s u g a r on e l o n g a t i o n o f stem segments i s d e b a t a b l e . C h r i s t i a n s e n and Thimann (1950) r e p o r t e d t h a t s u c r o s e has no e f f e c t on e l o n g a t i o n o f p e a stem segments, whereas G a l s t o n and Hand (1950) r e p o r t e d an i n c r e a s e i n e l o n g a t i o n v / i t h 2% s u c r o s e . However, more i n v e s t i g a t o r s i n c l u d e 2% s u c r o s e i n t h e i r t r i a l s t h a n o t h e r w i s e . The r e s p o n s e o f segments t o t e s t m a t e r i a l s depends g r e a t l y on t h e p o r t i o n o f t h e stem from w h i c h t h e segments a r e - t a k e n . P u r v e s and H i l l m a n (1958) s u g g e s t e d t h a t segments t a k e n f u r t h e s t from t h e apex o f t h e i n t e r n o d e s r e s p o n d w e l l t o exogenous IAA. C h r i s t i a n s e n and Thimann (1950) s u g g e s t e d t h e use o f segments t a k e n from t h i r d i n t e r n o d e s . N i t s c h and N i t s c h (1956) u t i l i s e d segments t a k e n from t h e f i r s t i n t e r n o d e s . -9 The l o w e r l i m i t o f t h i s t e s t i s 10 mg/1 IAA ( e . g . , B e n t l e y , 1958). To d a t e , no l i t e r a t u r e i s a v a i l a b l e on t h e e f f e c t o f HNap on t h e growth o f stem segments. 42 MATERIALS AND METHODS The procedure adopted i n t h i s i n v e s t i g a t i o n was modified from Christiansen and Thimann (1968). Uniform seeds of Pisum sativum L c u l t i v a r Alaska were soaked f o r s i x hours i n d i s t i l l e d water and sown i n verm i c u l i t e i n wooden f l a t s . The plants were grown i n darkness at a i r temperatures of 23° + 0.5° C, and a r e l a t i v e humidity of 95% + 2%. A f t e r seven days, uniform plants were selected and 19.5 mm segments were cut from the subapical portion of the t h i r d internodes with razor blades mounted 19.5 mm apart on a p l a s t i c block. This region has been shown to respond w e l l to auxin (Purves and Hillman, 1958). Immediately following e x c i s i o n the segments were washed i n d i s t i l l e d water, b l o t t e d dry with paper towels, and divided into groups of f i v e . Each group was weighed and placed i n p e t r i dishes containing one of the following s o l u t i o n s : 1) IAA (K s a l t ) 0.1 ppm 2) IAA (K sa l t ) 1.0 ppm 3) CHCA (K sa l t ) 0.1 ppm 43 4) CHCA (K s a l t ) 1.0 ppm 5) CHCA (K s a l t ) 10.0 ppm 6) KNap 0.1 ppm 7) KNap 1.0 ppm 8) KNap 10.0 ppm 9) D i s t i l l e d w a t e r s e r v e d as c o n t r o l . W i t h t h e e x c e p t i o n o f t h e c o n t r o l , t h e pl-l o f t h e above s o l u t i o n s was a d j u s t e d t o 10 w i t h I N KOH. The stem segments were i n c u b a t e d f o r 24 h o u r s i n d a r k n e s s a t an a i r t e m p e r a t u r e o f 29° C. The e x p e r i m e n t was c o n d u c t e d i n a ran d o m i s e d c o m p l e t e b i o k d e s i g n , a r r a n g e d i n t h r e e r e p l i c a t i o n s , w i t h f i v e segments i n each r e p l i c a t i o n . A t t h e c o m p l e t i o n o f t h e s p e c i f i e d i n c u b a t i o n p e r i o d , t h e l e n g t h s o f t h e segments were measured w i t h a m i c r o m e t e r . A l l o b s e r v a t i o n s and m a n i p u l a t i o n s were p e r f o r m e d under two 40-watt r e d S y l v a n i a lamps. The r e s u l t s were s u b j e c t e d t o a n a l y s i s o f v a r i a n c e and Duncan's New M u l t i p l e Range T e s t (Duncan, 1955) f o r m u l t i p l e c o m p a r i s o n o f t r e a t m e n t means. 44 RESULTS The e f f e c t of treatment on elongation of stem segments was assessed using t h i s formula: Increase of treated over c o n t r o l — x 100 Increase i n Control The stimulatory e f f e c t of the treatments on segment elongation, and on increases i n weight of segments was s i g n i f i c a n t at the 0.01 l e v e l . A r i s e i n concentrations of the IAA, CHCA, and KNap . treatments resulted i n an increase i n elongation of the segments, and a corresponding increase i n weight of the segments (Table i v ) . The elongation of segments i n 0.1 ppm IAA (ca 339% increase over c o n t r o l segments) did not d i f f e r s i g n i f i c a n t l y from that of 1.0 ppm KNap ( ca 279% over c o n t r o l segments), and also from the induced elongation by 1.0 and 10 ppm CHCA (ca 327 and 360% increase over control segments). This suggests that the stimulatory e f f e c t s of CHCA and KNap are s i m i l a r to that of IAA. In low KNap concentration (0.1 ppm), there was no s i g n i f i c a n t difference of induced elongation over the con t r o l segments. 45 KNap at 10 ppm had greater stimulatory e f f e c t on elongation (ca 473% over c o n t r o l segments) than that of IAA (ca 339% over c o n t r o l segments). The increases i n weights of segments receiving 0.1 ppm IAA (ca 70%), 10 ppm KNap (ca 67%), and 1.0 ppm KNap d i d not d i f f e r s i g n i f i c a n t l y from one another, according to Duncan's New Multi p l e Range Test (Duncan, 1955). 46 Table IV. Response of dark-grown Alaska pea stem segments to exogenously applied IAA, CHCA, and KNap. Treatment Average Average Increase Average Increase (ppm) length increase i n increase i n of 15 i n length i n t o t a l weight segments length over weight of over (mm) (mm) con t r o l 5 segments co n t r o l (°/o) + (mg) <<&) 1 Control 19.83* 0.33 21.00 2 IAA 0.1 20.95 1.45 339.39 35.67 69.85 3 IAA 1.0 22.10 2.60 687.87 74.67 255.57 4 CHCA 0.1 20.24 0.74 124.24 21.67 3.19 5 CHCA 1.0 20.91 1.41 327.27 27.00 28.57 6 CHCA 10 21.02 1.52 360.60 29.67 41.28 7 KNap 0.1 20.16 0.66 100.00 25.33 20.62 8 KNap 1.0 20.75 1.25 278.78 31.00 47.61 9 KNap 10 21.39 1.89 472.72 35.00 66.66 Segment elongation ATreatment number: 3 9 6 2 5 8 4 7 1 Increase i n weight ATreatment number: 3 2 9 8 6 5 7 4 1 A Corresponds to the treatment i n Table V. Treatments underscored by the same l i n e do not d i f f e r s i g n i f i c a n t l y , according to Duncan's New Multiple Range Test. + Value from three r e p l i c a t i o n s . * I n i t i a l length was 19.5 mm for a l l segments. DISCUSSION The widely held concept of IAA-induced c e l l elongation i s that the primary e f f e c t of IAA i s on the c e l l wall, and that IAA regulates c e l l expansion by loosening the c e l l walls (Heyn, 1931, 1940; Cleland and Bonner, 1956; Burstrom, 1961; Preston and Hepton, 1960). Burstrom (1942) suggested, i n the case of wheat roots, the influence of IAA on root growth was mainly due to i t s e f f e c t on c e l l elongation rather than c e l l m u l t i p l i c a t i o n . The same concept may apply to the e f f e c t of other auxin-like substances on stem segments. The lack of s i g n i f i c a n t difference i n the e f f e c t s of 0.1 ppm IAA, 1.0,and 10 ppm CHCA, and 1.0 ppm KNap on augmentation of elongation of pea stem segments provides evidence that the behaviour of CHCA and KNap at the said concentrations was analogous to that of IAA. The consequence of t h i s analogy leads to the suggestion that i n the promotion of c e l l elongation, CHCA and KNap f i r s t relax the c e l l walls, and, as a r e s u l t of t h i s relaxation, water i s taken i n osmotically. The absence of an active process i n v/ater uptake during IAA-induced c e l l expansion has been amply demonstrated (Cleland and Bonner, 1956; 48 O r d i n e t a l , 1956; L e v i t t , 1953; Thimann, 1954). That c e l l e nlargement i s l a r g e l y due t o i n c r e a s e d w a t e r u p t a k e i s s u b s t a n t i a t e d i n t h i s e x p e r i m e n t by marked i n c r e a s e s i n w e i g h t i n KNap- and CHCA-treated segments. From t h e r e p o r t s o f i n c r e a s e s i n de novo s y n t h e s i s and/or a c t i v i t y o f c e r t a i n enzymes i n b u s h beans r e s u l t i n g from KNap and CHCA t r e a t m e n t s ( F a t t a h and Wort, 1970; S e v e r s o n , 1972), and t h a t KNap a c t s a t t h e g e n e t i c l e v e l (Wort e t a l , 1971), i t i s t e m p t i n g t o s p e c u l a t e t h a t t h e KNap- and CHCA-augmented i n c r e a s e s i n c e l l e l o n g a t i o n may be m e d i a t e d t h r o u g h t h e s t i m u l a t i o n o f t h e s y n t h e s i s o f h y d r o x y p r o l i n e - r i c h p r o t e i n , and t h e subsequent i n c o r p o r a t i o n o f t h i s p r o t e i n i n t o t h e c e l l w a l l i n o r d e r f o r c e l l e l o n g a t i o n t o o c c u r . T h i s e x p l a n a t i o n i s i n agreement w i t h t h e s u g g e s t i o n b y C l e l a n d (1967) w i t h r e s p e c t t o I A A - i n d u c e d e l o n g a t i o n o f Avena c o l e o p t i l e s . 49 id) Abscission of debladed p e t i o l e s of bean plants. LITERATURE REVIEW There i s s t i l l uncertainty p r e v a i l i n g at the present time with respect to the physiology of abscission. Since 1933, v/hen Laibach found that the abscission of debladed p e t i o l e s was delayed by the a p p l i c a t i o n of auxin-rich orchid p o l l i n i a to the cut surfaces of the debladed p e t i o l e s , i t i s recognised that auxin plays a regulatory r o l e i n abscission. I n h i b i t i o n of abscission by the a p p l i c a t i o n of 5 mg/1 IAA to debladed p e t i o l e s of Coleus blumei Benth. by La Rue (1936) confirmed the c l a s s i c a l work of Laibach. E a r l y investigations showed that l e a f auxin decreased with age (Avery, 1935; Goodwin, 1937; Went and Thimann, 1937). Working with Coleus, Myers (1940) found p o s i t i v e c o r r e l a t i o n s between age of leaves, amount of d i f f u s i b l e auxin, and times required for abscission of i n t a c t or debladed leaves. Shoji et a l (1951) reported that free auxin i n the mature healthy l e a f l e t s of bean (Phaseolus v u l g a r i s L), i s about 50 three times that i n the l e a f stalk. Immediately before abscission, l e a f l e t auxin f e l l to a l e v e l approximately equal to that of the l e a f stalk, while stalk auxin remained unchanged. Similar changes were observed i n Coleus and cotton (Jacobs, 1955; Carns, 1957, r e s p e c t i v e l y ) . The process of abscission i s affected by a multitude of compounds and environmental factors: 1) Auxin Auxin retards or promotes abscission depending on the s i t e of application with respect to the abscission zone (e.g., Addicott et a l , 1955). The regulatory e f f e c t of auxin on abscission i s governed by the time of i t s application to p e t i o l e s a f t e r the removal of the l e a f blades (Rubinstein and Leopold, 1962, 1963), and by .the concentration of auxin (e.g., Gaur and Leopold, 1955). The role of auxin i n the regulation of abscission i s discussed. 2) Abscisic acid (Abscisin I I : Addicott et al_, 1964; Ohkuma et a l , 1963, 1965; Dormin: Cornforth et a l , 1965; Eagles and Wareing, 1963; Wareing et a l , 1964). 51 A b s c i s i c a c i d was thought to be an abscission-regulating hormone based on the f a c t that the amounts of t h i s hormone increase during periods of ageing, and that ageing i s c o r r e l a t e d with abscission.. The a p p l i c a t i o n of a b s c i s i c a c i d to explants accelerates abscission. 3) G i b b e r e l l i c acids G i b b e r e l l i c acids promote abscission (Bornman, 1965; Cams et at, 1951; Chatterjee and Leopold, 1964; Greenblatt, 1965). 4) Ethylene Ethylene i s a potent accelerant of abscission (e.g., Zimmerman et a l , 1931) . Its e f f e c t on the abscission process has been studied by Gawadi and Avery (1950); H a l l (1952). Ethylene accelerates abscission through i t s i n h i b i t o r y influence on the b a s i p e t a l transport of auxin and on the i n i t i a t i o n of s p e c i f i c RNA, and protein synthesis (Beyer and Morgan, 1971) . 5) Amino acids and carbohydrates Rubinstein and Leopold (1962) reported that alanine and glutamic acid promoted abscission of bean p e t i o l e s . 52 Yager and Muir (1958) found that methionine was a potent promoter of abscission. . Carbohydrates have been shown to retard or promote abscission under a wide range of experimental conditions (Biggs and Leopold, 1957; Brown and Addicott, 1950). 6) Environmental factors An extensive review of the influence of environmental factors on abscission i s described i n a paper by Addicott * (1968).With bean l e a f l e t explants, the rate of abscission i s very low below 5° C, r i s e s with temperature to a maximum o o between 25 and 30 C, and f a l l s at higher temperatures (Yamaguchi, 1954). This phenomenon may be associated with enzyme a c t i v i t y as cellulasehas been found to break down c e l l walls (e.g., Abeles and Leather, 1971). Exposure to r e l a t i v e l y extreme temperatures i s often followed by increased abscission of leaves, flowers, and f r u i t s (Chandler, 1925). The autumn l e a f abscission of deciduous trees i s corr e l a t e d with shortening of photoperiod, e.g., sugar maple (Acer saccharum) (Wiesner, 1904). Under reduced oxygen tensions, abscission i s retarded (Sampson, 1918). * An explant i s a small segment of a l e a f containing an abscission zone. 53 Oxygen acceleration of abscission may be the r e s u l t of i n a c t i v a t i o n of auxin by IAA. oxidase system (e.g., Morgan and H a l l , 1963). The auxin acceleration of abscission was f i r s t observed following applications of 10, 105, and 525 mg/1 IAA to the proximal side of the abscission zones of excised l e a f l e t s of bean (Addicott and Lynch, 1951). However, applications of a range of IAA concentrations 10-1000 mg/1 d i s t a l to the abscission zones were shown to retard abscission. They obtained s i m i l a r r e s u l t s with 2,4-D and 2,4,5-T. Retardation of abscission was found to be i n d i r e c t pro-portion to the concentration of IAA applied. The work of.Addicott and Lynch (1951) has been confirmed and extended by several workers. Jacobs (1955) working with Coleus, indicated that auxin moving from the a p i c a l bud and from i n t a c t leaves accelerated the abscission of the lower debladed p e t i o l e s . With f i e l d cotton, Louie (1960) found that removal of the a p i c a l bud retarded abscission , and a p p l i c a t i o n of IAA to the stem stump accelerated abscission of debladed p e t i o l e s of the upper leaves. The employment of p e t i o l e a b s c i s s i o n serves a s u i t a b l b i o a s s a y t o evaluate the a u x i n - l i k e p r o p e r t i e s o f t e s t compounds. No l e s s than s i x t h e o r i e s have been proposed i n an attempt t o e l u c i d a t e the p h y s i o l o g y o f a b s c i s s i o n . The more recent t h e o r i e s are: 1) Auxin g r a d i e n t ( A d d i c o t t , Lynch, and Cams, 1955) This theory i s formulated on the f o l l o w i n g p r i n c i p l e s a) A g r a d i e n t o f auxin e x i s t s across the a b s c i s s i o n zone. b) T y p i c a l l y , the g r a d i e n t appears t o be from r e l a t i v e l y h i g h auxin d i s t a l l y , t o r e l a t i v e l y low au x i n p r o x i m a l l y . c) A lowering o f the g r a d i e n t i n i t i a t e s a b s c i s s i o n . d) The degree o f lowering o f the auxin g r a d i e n t determines the r a t e o f a b s c i s s i o n . This theory i s supported by T e r p s t r a (1956) and Louie (1960). 2) Two-stage theory (Rubinstein and Leopold, 1962, 1963) This theory apparently supersedes the auxin g r a d i e n t -4 theory of A d d i c o t t e t a l , (1955). U t i l i s i n g 5 x 10 M 55 radioactive NAA on bean p e t i o l e explants, Rubinstein and Leopold (1963) were able to demonstrate the lack of an auxin gradient across the abscission zone. Stage I i s the induction stage of about s i x hours or more, and i s i n h i b i t e d by auxin. Stage II i s accelerated by auxin of s i m i l a r concentration. Gaur and Leopold (1955) reported that low concentrations of NAA (1-10 ppm) promoted abscission and high concentrations (100-1000 ppm) retarded abscission whether the. auxin was applied d i s t a l l y or proximally with respect to the abscission zone. The "two-stage" response to NAA i s interpreted by Rubinstein and Leopold (1962) as a t t r i b u t a b l e to the "two-stage" e f f e c t . They proposed that the promotion of abscission by d i s t a l a p p l i c a t i o n s of low auxin concentrations i s "a consequence of an amount of auxin j u s t low enough to allow the induction stage to proceed to completion yet high enough to stimulate the second stage..." This "two-stage" e f f e c t of auxin on abscission i s i n corro-boration with the two-phase scheme of auxin action on growth as proposed by Thimann (1951), whereby low auxin concentration promotes growth, while high auxin concentration i s i n h i b i t o r y . 56 3) Aqeinq-ethylene theory (Abeles, 1968) Abeles (1968) noted t h a t ethylene i n i t i a t e s s p e c i f i c RNA and p r o t e i n syntheses necessary f o r c e l l s e p a r a t i o n . As the t i s s u e ages, i t becomes i n c r e a s i n g l y s e n s i t i v e t o ethylene. Ethylene p r o d u c t i o n by p l a n t t i s s u e s i n response t o wounding was rep o r t e d by Burg (1962), and suggested t h a t ethylene produced by f r e s h l y - c u t bean explants (ca 3 m i c r o l i t r e s per g per hour) i s s u f f i c i e n t t o s t i m u l a t e a b s c i s s i o n (Burg, 1968). Beyer and Morgan (1971) i n d i c a t e d t h a t endogenously produced ethylene may f u n c t i o n i n p a r t t o r e g u l a t e l e a f a b s c i s s i o n through i t s i n h i b i t o r y e f f e c t on auxin t r a n s p o r t and the i n d u c t i o n of the s y n t h e s i s o f the c e l l w a l l - d e g r a d i n g enzyme c e l l u l a s e . Horton and Osborne (•1967) reported t h a t c e l l u l a s e was l o c a l i s e d i n the s e p a r a t i o n l a y e r , and t h a t ethylene i n c r e a s e d c e l l u l a s e a c t i v i t y , w h i l e 2,4,5-T decreased i t . The l a t t e r two t h e o r i e s have s u b s t a n t i a l bases, and o f f e r s a t i s f a c t o r y e x p l a n t i o n s o f the process o f a b s c i s s i o n . The d i s c r e p a n c i e s i n the f i n d i n g s o f v a r i o u s researchers may be due t o the use of d i f f e r e n t s p e c i e s o f p l a n t s . 57 P e t i b l a r explant technique i s popular with inv e s t i g a t o r s i n t h i s f i e l d . Since t h i s technique does not account for the c o r r e l a t i v e influence of other plant parts on the abscission zone i n question, the r e s u l t s obtained from such studies may not r e f l e c t the true or close-to-true s i t u a t i o n i n i n t a c t plants. In t h i s respect, the employment of i n t a c t plants to t e s t the a c t i v i t y of various compounds i n an e f f o r t to gain i n s i g h t into the process of abscission may prove to be superior. No information i n the l i t e r a t u r e has been encountered with regard to the influence of HNap on abscission. 58 MATERIALS AND METHODS Four seeds of bush bean (Phaseolus vulgaris L. c u l t i v a r Top Crop), were sown i n composted s o i l i n 15-cm p l a s t i c pots. Uniformity of plants was obtained by reducing to two seedlings i n each pot seven days a f t e r sowing, and f i n a l l y to one plant,two days before treatment. The plants were arranged i n rows of four, and grown i n a greenhouse under natural l i g h t conditions. The number of sunshine hours for the months of September and October, 1971 were: September: t o t a l 162.8 hours, average 5.43 hours, range 0.00-11.7 hours. October: t o t a l 121.2 hours, average 3.90 hours, range 0.00-9.7 hours. The l i g h t i n t e n s i t y varied from 12,000 to 20,000 lux. The procedure used i n t h i s investigation was modified from M i t c h e l l et a l , (1968). Ten days from sowing, the blade of a primary l e a f of each plant was severed just below the pulvinus. The debladed petioles were treated by applying, with a wooden toothpick, l a n o l i n -Tween 20 containing one of the t e s t compounds namely: KNap, CHCA (K s a l t ) , NAA, and lanolin-Tween 20. The con-centrations employed i n each case, except the lanoline-Tween 20, were 1.0, 10, 100, and 1000 ppm. 59 The treatments were applied around the debladed p e t i o l e as a band of about f i v e mm wide, and at a distanca of about f i v e mm from the stem. The determination of abscission was made by ap p l i c a t i o n of a constant f i v e g pressure once each day i n a downward d i r e c t i o n against the upper surface of each debladed p e t i o l e , at a distance of 8-10 mm from the stem. This was accomplished by pressing the metal rod of a pressure applicator against the p e t i o l e as indicated i n Plate 3. The number of p e t i o l e s abscised each day upon a p p l i -cation of the f i v e g pressure was recorded. The number of days required for 50% of the pe t i o l e s i n each treatment to abscise was taken for comparison of the treated and control plants. This i n v e s t i g a t i o n was set up i n a randomised complete block design involving three blocks. Four plants of each treatment were arranged i n a row i n each block. Analysis of variance and Duncan's New Multiple Range Test (Duncan, 1955) for multiple treatment comparisons were performed on the r e s u l t s obtained. P l a t e 3. Determination of a b s c i s s i o n of a p e t i o l e by the use of a f i v e gramme pressure a p p l i c a t o r . RESULTS The a p p l i c a t i o n o f l a n o l i n c o n t a i n i n g 1.0 ppm KNap t o the d i s t a l end o f debladed p e t i o l e s o f ten-day-old bean p l a n t s r e s u l t e d i n a s i g n i f i c a n t a c c e l e r a t i o n o f a b s c i s s i o n (9 days) compared w i t h about 11 days i n c o n t r o l p l a n t s (Table V ). The times r e q u i r e d f o r 50% a b s c i s s i o n i n p l a n t s which r e c e i v e d h i g h KNap c o n c e n t r a t i o n s (10, 100, and 1000 ppm) were not s t a t i s t i c a l l y d i f f e r e n t from t h a t o f the c o n t r o l p l a n t s , or from each other. The times r e q u i r e d f o r 50% a b s c i s s i o n i n p l a n t s t r e a t e d w i t h 10 and 1000 ppm CHCA (13 and 12.67 days, r e s p e c t i v e l y ) were s i g n i f i c a n t l y d i f f e r e n t from t h a t o f c o n t r o l p l a n t s , but not from p l a n t s t r e a t e d w i t h 100 ppm NAA. In p l a n t s t r e a t e d w i t h 1000 ppm NAA, the p e r i o d r e q u i r e d f o r 50% a b s c i s s i o n was 25 days which was s i g n i f i c a n t l y d i f f e r e n t from t h a t o f c o n t r o l p l a n t s a t the 0.01 l e v e l . 62 Table V . A b s c i s s i o n o f debladed p e t i o l e s o f bean (Phaseolus  v u l g a r i s L) i n response t o KNap, CHCA, and NAA treatments. Treatment (ppm) +Average time to 50% a b s c i s s i o n (days) L a n o l i n alone (c o n t r o l ) 10.67 2 KNap 1.0 9.00 3 KNap 10 12.00 4 KNap 100 11.67 5 KNap 1000 12.00 6 CHCA (K s a l t ) 1.0 12.00 7 CHCA (K s a l t ) 10 13.00 * 8 CHCA (K s a l t ) 100 12.33 9 CHCA (K s a l t ) 1000 12.67 * 10 NAA . 1.0 12.00 11 NAA ; 10 12.33 12 NAA ; 100 13.00 * 13 NAA : 1000 25.00 ** A Treatment number: 13 12 7 9 8 11 3 5 6 10 4 1 2 +Value from 12 p l a n t s . ATreatment number corresponds t o treatment i n Table VI. The treatments underscored by the same l i n e do not d i f f e r s i g n i f i c a n t l y from one another. * Value 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 v a l u e . ** Value d i f f e r s from the c o n t r o l value a t the 0.01 l e v e l . DISCUSSION It has been recognised f o r a long time that auxin regulates abscission. The two-phase theory of abscission proposed by Rubinstein and Leopold (1962) gives a s a t i s f a c -tory e l u c i d a t i o n of the process of abscission. According to these two workers, phase I of the abscission process i s i n h i b i t e d by auxin,and phase II i s accelerated by auxin of s i m i l a r concentration. In recent years, the involvement of ethylene i n the abscission process has received considerable i n t e r e s t by researchers. That ethylene i s a potent accelerant of abscission has been suggested by, for example, Burg (1962); Pratt and Goeschl (1969). The action of ethylene to promote abscission i s i n d i r e c t . It may act through i t s e f f e c t s on auxin l e v e l . Ethylene has been shown to i n h i b i t polar transport of auxin (Beyer and Morgan, 1971), to increase auxin destruction (Hall and Morgan, 1964), and to i n i t i a t e the synthesis of s p e c i f i c RNA and protein for c e l l separation (Abeles, 1968). 64 The r e t a r d a t i o n e f f e c t o f 10 and 1000 ppm CHCA on a b s c i s s i o n may be a t t r i b u t e d t o t h e enhancement o f a u x i n a c t i o n and/or IAA b i o s y n t h e s i s . I t was d e m o n s t r a t e d i n t h e p r e s e n t i n v e s t i g a t i o n s , t h a t 100 ppm KNap s t i m u l a t e d IAA b i o s y n t h e s i s i n bean e p i c o t y l s . The l a c k o f s i g n i f i c a n t d i f f e r e n c e i n d e l a y i n g a b s c i s s i o n b y 10 and 1000 ppm CHCA, and 100 ppm NAA s u g g e s t s t h a t t h e r e t a r d a t i o n e f f e c t o f CHCA i s s i m i l a r t o t h a t o f a u x i n . I t i s p o s s i b l e t h a t CHCA a c t e d i n t h e f i r s t phase o f t h e two-phase t h e o r y o f a b s c i s s i o n o f R u b i n s t e i n and L e o p o l d (1962). A n o t h e r p o s s i b i l i t y i s t h a t CHCA i n h i b i t e d e t h y l e n e s y n t h e s i s o r a c t i o n . The a c c e l e r a t i o n o f a b s c i s s i o n b y t h e low KNap c o n c e n t r a t i o n (1.0 ppm) may be i n t e r p r e t e d as t h e enhancement e f f e c t o f KNap on t h e b i o s y n t h e s i s and/or a c t i o n o f e t h y l e n e . CHAPTER II EFFECT OF POTASSIUM NAPHTHENATES ON IAA BIOSYNTHESIS AND DEGRADATION. 66 i i a ) E f f e c t o f KNap on IAA b i o s y n t h e s i s . LITERATURE REVIEW IAA i s w e l l e s t a b l i s h e d as t h e p r i n c i p a l hormone o f h i g h e r p l a n t s . Y e t t h e pathways o f IAA b i o s y n t h e s i s i n h i g h e r p l a n t s a r e o b s c u r e , and a r e s t i l l t h e s u b j e c t o f much i n v e s t i g a t i o n and c o n t r o v e r s y . I t appears t h a t t h e pathways o f IAA b i o s y n t h e s i s d i f f e r i n d i f f e r e n t p l a n t s p e c i e s . There i s ample e v i d e n c e t o e s t a b l i s h t h a t t r y p t o p h a n i s t h e p r e d o m i n a n t n a t u r a l p r e c u r s o r o f IAA i n h i g h e r p l a n t s as w e l l as i n numerous m i c r o o r g a n i s m s (Moore and Shaner, 1967, 1968; Zenk and S c h e r f , 1963; S h e r w i n and P u r v e s , 1969; M i u r a and M i l l s , 1971; P h e l p s and S e q u i r a , 1967; Wightman, 1963). The c o n v e r s i o n o f t h e D-enantiomer o f t r y p t o p h a n t o t h e L - enantiomer i n c e l l c u l t u r e s o f t o b a c c o was d e m o n s t r a t e d by M i u r a and M i l l s (1971). Gordon (1961) n o t e d t h a t w i t h s e v e r a l p l a n t p r e p a r a t i o n s , D - t r y p t o p h a n was e q u a l t o o r more e f f e c t i v e t h a n L - t r y p t o p h a n as a p r e c u r s o r o f IAA. S i m i l a r l y , Kim "67. 14 and Rohringer (1969) reported that D-tryptophan-methylene-C 14 was incorporated more e f f i c i e n t l y i n t o IAA-C than L-tryptophan-14 methylene-C by excised wheat leaves. Several researchers question the v a l i d i t y of the claim that tryptophan i s the natural precursor of IAA (Libbert et a l , 1966; Winter, 1966; Thimann and Grochowska, 1968; Black and Hamilton, 1971). The conversion of tryptophan to IAA i n various plant species has been worked out, and, according to workers i n t h i s area, t h i s conversion follows two major proposed pathways: indolepyruvic acid and indoleacetaldehyde, or tryptamine and indoleacetaldehyde (Larsen, 1951; Gordon, 1956, 1961; Fawcett, 1961; Wightman, 1962, 1968; Moore and Shaner, 1968; Kim and Rohringer, 1969; Srivastava and Shaw, 1962; Erdmann and Schiewer, 1971). The following pathway of IAA biosynthesis has been demonstrated i n the t i p s of green pea seedlings (Pisum sativum) (Moore and Shaner, 1968; Erdmann and Schiewer, 1971); i n oat c o l e o p t i l e s (Erdmann and Schiewer, 1971); 68 i n r u s t fungus, Melampsora l i n i (Pers.) Lev. ( S r i v a s t a v a and Shaw, 1962); and i n mung bean (Phaseolus aureus) (Wightraan, 1968).: tryptophan —*»indolepyruvic a c i d indoleacetaldehyde — I A A Another pathway: tryptophan-*tryptamine-•indoleacetaldehyde-*IAA was found t o occur i n tomato p l a n t s (Lycopersicum esculentum) (Wightman, 1963). The e x i s t e n c e o f y et another pathway: t r y p t o p h a n - ^ i n d o l e a c e t o n i t r i l e - * - indoleacetaldehyde -••IAA was detected i n cabbage by Wightman (1962). Another h y p o t h e t i c a l route f o r IAA b i o s y n t h e s i s i n Avena c o l e o p t i l e s i s given by Winter (1966): a n t h r a n i l o n i t r i l e - * a n t h r a n i l i c acid-*» i n d o l e - * tryptamine-* IAA Tryptamine r a t h e r than tryptophan i s the p r e c u r s o r o f IAA i n Avena c o l e o p t i l e s was the c o n c l u s i o n reached by Thimann and Grochowska (1968). These two workers emphasised t h a t the conversion of tryptophan to tryptamine d i d not occur, and suggested t h a t the conversion o f t r y p t o -phan t o IAA was due t o b a c t e r i a l contamination. This was s u b s t a n t i a t e d by Winter (1966) and Bl a c k and Hamilton (1971). 69 The f a i l u r e of exogenously supplied tryptophan to promote elongation of Avena c o l e o p t i l e s was the r e s u l t of i t s incorporation into protein and consequent u n a v a i l a b l i t y f o r conversion to IAA under s t e r i l e conditions, according to Black and Hamilton (1971). The conversion of tryptophan to IAA i n bean (Phaseolus v u l g a r i s L) shoot experiments with 1 4 tryptophan-C was reported by Black and Hamilton (1971). The findings of Perley and Stowe (1966) with b a c t e r i a l cultures of B a c i l l u s cereus s t r a i n KVT, showed that d i r e c t decarboxylation of tryptophan to tryptamine i s a very rare reaction. To the best of my knowledge, there i s only one pub l i c a t i o n on the e f f e c t of HNap on the content of auxin i n plants. Bazanova (1970) found that Sh-8 (or HNap) at 0.005% caused r e d i s t r i b u t i o n of endogenous auxin and i n h i b i t o r s i n various organs of f i n e - f i b r e d cotton, increased the t r a n s l o c a t i o n of growth-regulating substances from vegetative to reproductive organs, and enhanced the a c t i v i t y of natural growth-regulating substances and i n h i b i t o r s i n reproductive organs. 70 MATERIALS AND METHODS Bush bean seeds (Phaseolus v u l g a r i s L, c u l t i v a r Top Crop) were s u r f a c e s t e r i l i s e d by washing w i t h 95% ethanol f o r ten minutes, then w i t h s t e r i l e water. The seeds were soaked f o r 12 hours i n 100 ppm (0.01%) s t e r i l i s e d KNap (Wort and Patel,'1970; Naghibin, 1966; Ejubov, 1966). The seeds used as c o n t r o l were soaked i n s t e r i l e water f o r the same l e n g t h o f time. The seeds were sown i n rows i n s t e r i l i s e d vermi-c u l i t e ( " T e r r a - l i t e " , Grace C o n s t r u c t i o n M a t e r i a l s L t d . , Vancouver), s a t u r a t e d w i t h 1.0% Captan f u n g i c i d e . S t e r i l i z a -o txon was c a r r i e d out a t 248 C f o r 15 minutes a t a pressure o f 15 l b per square i n c h . The p l a n t s were allowed t o grow f o r 14 days i n the dark, a t a i r temperatures o f 22.5 + 1° C, and 95 + 2% r e l a t i v e humidity. A p i c a l regions (5-8 cm) i n c l u d i n g leaves were e x c i s e d and washed v/ith c o l d d i s t i l l e d water. Immediately a f t e r e x c i s i o n , 20 g f r e s h weight o f p l a n t t i s s u e s was homogenised i n a Waring blendor a t f u l l speed f o r ten minutes i n 40 ml o of KH 9P0 A-Na_HP0 A b u f f e r i n a c o l d room at 4 C (Moore and 71 Shaner, 1968). The concentration of the phosphate b u f f e r was 0.1 at pH 7.4 (Moore and Shaner, 1968) containing 0.1 M sucrose (Wightman, 1968), ten units per ml p e n i c i l l i n G, and 100 units per ml streptomycin sulphate (Valdovinos and Perley, 1966). The crude homogenates were f i l t e r e d through four layers of cheesecloth and kept i n an ice bath throughout the prepara-t i v e procedures. The f i l t r a t e thus obtained, was c e n t r i -fuged at 10,000 g_ for 20 minutes at 4° C (Moore and Shaner, 1968) and the r e s u l t i n g supernatant was used as the enzyme extract. Enzyme extracts were dialysed against phosphate o bu f f e r for 24 hours at 4 C with constant a g i t a t i o n and one change of external b u f f e r . Reaction mixtures, modified from Moore and Shaner (1968), contained three ml enzyme extract and three ml of 0.1 M KH 2P0 4-Na 2HP0 4 buffer, pH 7.4, containing: 30 micromoles alpha-ketoglutaric acid 0.6 micromoles pyridoxal phosphate 0.6 micromoles thiamine pyrophosphate 0.6 micromoles nicotinamide adenine dinucleotide 0.03 M D-tryptophan 0.0005 M diethyldithiocarbamic a c i d 72 The above chemicals were purchased from Sigma Chemical Company, St. Louis, Missouri. The D-enantiomer of tryptophan was found to be the best precursor of IAA (Kim and Rohringer, 1969). The concentra-t i o n of D-tryptophan (0.03 M) was adopted from Wightman and Cohen (1968), while the i n c l u s i o n of 0.0005 M d i e t h y l d i t h i o -carbamic acid which i s a potent i n h i b i t o r of IAA oxidase, was adopted from Wagenknecht and Burris (1950). The reaction mixtures, held i n t e s t tubes, were incubated i n darkness for s i x hours at 35° C (Wightman and Cohen, 1968) i n a water bath with thermostatic controls (National Appliance Company, Portland, Oregon). Enzymatic reactions were stopped by lowering the pH to 3.0 with two drops of 85% orthophos-phoric acid. • The estimations of protein i n enzyme extracts were made with a view of assessing the s p e c i f i c a c t i v i t y of the enzymes involved i n IAA biosynthesis. The method of Lowry e_t a l (1951) as modified by Eggstein and Kreutz (1955) was used. To one ml of enzyme extract was added f i v e ml of a l k a l i n e copper solution (50 ml of 2% Na 2C0 3 i n 0.1 N NaOH plus 1.0 ml of 0.5% CuSO^.SB^O i n 1.0% sodium c i t r a t e ) . The mixture was thoroughly mixed and allowed to stand for ten minutes at room temperature (23-24° C). Thereafter, 0.5 ml of 1.0 N Folin-Ciocalteau reagent (commercial reagent 2 N d i l u t e d with 73 water to give a solut i o n of 1.0 N i n acid) was pipetted r a p i d l y into the mixture with thorough mixing. The o p t i c a l density was measured at 650 nm af t e r 30 minutes. The amount of protein i n the enzyme extracts was ca l i b r a t e d by reference to a standard curve developed using c r y s t a l l i n e bovine albumin. Extraction and quantitative determination of IAA. A l l manipulations were performed i n red l i g h t , at room temperature (23-24° C). Prior to extraction, ten microgrammes of authentic IAA were added to each reaction mixture, and each reaction mixture was extracted twice with s i x ml of methylene chloride by the method of Moore and Shaner (1968). The combined extracts of each reaction mixture were evaporated to dryness i n a r o t a r y - f i l m vacuum evaporator at 30° C. The •residue was dissolved i n two ml of 95% ethanol and a 0.5 ml aliquot of each extract was spotted on Whatman No. 1 chromatographic paper (26 x 36 cm) and co-chromatographed with ten microgrammes of authentic IAA. The chromatograms were developed i n 10:1:1 (v/v) 95% isopropanol:28% ammonium hy d r o x i d e : d i s t i l l e d water (Kuraishi and Muira, 1963; Artemenko and Chkanikov, 1970) i n an ascending manner (Wightman, 1963) for 16 hours, at 4° C i n the dark. The spots developed on the chromatograms were located under u l t r a v i o l e t l i g h t , and immediately sprayed with Ehrlich's reagent. E h r l i c h ' s reagent was prepared by mixing 2% p-dimethylaminobenzaldehyde i n 10 N 74 HC1 with acetone i n the proportion of 1:1 (v/v) immediately before use (Wightman, 1963). Quantitative determinations of IAA were made 15 minutes following treatment with E h r l i c h ' s reagent. The o p t i c a l density of the spots was determined by means of a densitometer (Photovolt Densitometer, Photovolt Corporation, New York City, Model 501 A). Maximum transmission values of the sopts obtained with the densitometer were used to compute the amount of IAA present i n plant tissues by c a l i b r a t i o n with values of a standard curve. Transmission values were employed c h i e f l y because of t h e i r l i n e a r r e l a t i o n -ship to the logarithm of IAA concentration (Vlitos and Meudt, 1953). A standard curve was developed by d e l i v e r i n g 1.0, 2, 5, and 20 microgrammes of authentic IAA to a chromatogram with a m i c r o l i t r e syringe. The chromatogram was developed and treated i n the same manner as described i n the preceding paragraph. The concentrations were adopted from V l i t o s and Meudt (1953). A l l solvents used i n chromatography were r e d i s t i l l e d . A randomised complete block design was used i n t h i s investigation involving three blocks with two determinations per treatment i n each block. The r e s u l t s obtained were subjected to analysis of variance. 75 RESULTS Soaking the bean seeds i n a 100 ppm s o l u t i o n o f KNap f o r 24 hours p r i o r t o sowing r e s u l t e d i n a 140.5% i n c r e a s e i n the content of IAA i n the e p i c o t y l s o f dark-grown bean p l a n t s , determined 14 days a f t e r treatment. The i n c r e a s e was s i g n i f i c a n t a t the 0.01 l e v e l (Table VI..:). The amount o f p r o t e i n (1.26 mg per g f r e s h weight) i n the e p i c o t y l s o f KNap-treated bean p l a n t s was l e s s than t h a t i n the c o n t r o l p l a n t s (1.96 mg per g f r e s h weight).. The s p e c i f i c a c t i v i t y o f the enzymes r e s p o n s i b l e f o r the conversion of tryptophan to IAA, expressed i n terms of microgrammes of IAA s y n t h e s i s e d per mg p r o t e i n per hour, was 1.34 i n the c o n t r o l p l a n t s and 5.03 i n the KNap-treated p l a n t s (Table V I I ). 76 T a b l e VI-'. IAA c o n t e n t o f t h e 5-8 cm t i p s o f e p i c o t y l s of 1 4 - d a y - o l d dark-grown P h a s e o l u s v u l g a r i s L s e e d l i n g s f o l l o w i n g t r e a t m e n t w i t h 100 ppm KNap. Treatment +Microgrammes o f IAA I n c r e a s e o v e r p e r g f r e s h w e i g h t c o n t r o l (%) D i s t i l l e d H 20 ( c o n t r o l ) 5.28 KNap 100 ppm 12.70** 140.5 + Mean o f s i x d e t e r m i n a t i o n s . ** V a l u e d i f f e r s s i g n i f i c a n t l y from t h e c o n t r o l v a l u e a t t h e 0.01 l e v e l . T a b l e V I I . S p e c i f i c a c t i v i t y o f t h e enzymes i n t h e c o n v e r s i o n o f t r y p t o p h a n t o IAA i n t h e e p i c o t y l s o f dark-grown bean p l a n t s . Treatment IAA c o n t e n t + P r o t e i n c o n t e n t microgrammes microgrammes p e r g f r e s h p e r g f r e s h w e i g h t w e i g h t S p e c i f i c a c t i v i t y o f enzymes microgrammes IAA p e r mg p r o t e i n p e r h o u r D i s t i l l e d H 20 ( c o n t r o l ) 5.28 KNap 100 ppm 12.70 1.96 1.26 1.34 5.03 + Mean o f s i x d e t e r m i n a t i o n s . DISCUSSION In view of the absence of reported pathways on the biosynthesis of IAA from tryptophan i n Phaseolus v u l g a r i s L plants, i t i s tempting to speculate that the pathway operating i n P. vu l g a r i s i s s i m i l a r to that of Phaseolus  aureus L (mung bean). It was reported that IAA i s synthe-sised i n excised E\_ v u l g a r i s shoots v i a tryptophan (Black and Hamilton, 1971). The pathway of.enzymatic conversion of tryptophan to IAA i n P. aureus has been worked out by Wightman and Cohen (1968), and, according to them, the pathway i s i n the following sequence: Tryptophan Indolepyruvic ac i d Indoleacetaldehyde —> IAA I II I I I The enzymes responsible for the conversion of tryptophan to IAA have been i d e n t i f i e d , and the properties of the i n d i v i d u a l enzymes involved i n each step of t h i s b i o s y n t h e t i pathway were determined by Wightman and Cohen (1968). According to these two investigators, the enzyme ca t a l y s i n g the p a r t i c u l a r step i n the pathway i s ind i c a t e d as follows: 78 Tryptophan transaminase i n step I; Indolepyruvic a c i d decarboxylase i n step II; and Aldehyde dehydrogenase i n step I I I . The increase i n the content of IAA i n the KNap-treated plants (140.5%) compared with c o n t r o l plants suggests KNap stimulation of IAA biosynthesis i n v i t r o . This KNap-stimulated IAA biosynthesis i n P. vu l g a r i s e p i c o t y l s may be mediated by e i t h e r the induction of de_ novo synthesis of the enzymes involved i n the conversion of tryptophan to IAA (mentioned i n the preceding paragraph), or by the stimulation of the a c t i v i t y of these enzymes, or i n combination. This suggestion i s made i n the l i g h t of reports on bush bean plants following KNap treatments, on the stimulation of protein synthesis by Severson (1972) and Wort et a l (1971); on the augmentation of s p e c i f i c a c t i v i t i e s of numerous enzymes i n crude extracts (Chu, 1969; Fattah, 1969; Fattah and Wort, 1970); and on increases i n RNA and DNA content (Wort et a l , 1971). The increases i n plant growth and crop y i e l d following applications of KNap reported i n the l i t e r a t u r e can be interpreted as due to KNap-induced IAA biosynthesis i n these plants. 79 i i b ) E f f e c t of KNap on IAA degradation. LITERATURE REVIEW The metabolism of IAA i n plants has been intensively-discussed but not well understood, even t i l l today. The s t a r t i n g point of discussion on t h i s subject dates back to 1934 when Thimann found that l e a f extracts of V i c i a  faba and Helianthus could i n a c t i v a t e IAA. But, he, however, did not demonstrate that the disappearance of IAA was enzymatic. Later, i n 1936, Larsen discovered that the i n a c t i v a t i o n of IAA was catalysed by an o x i d i s i n g enzyme. The c l a s s i c a l p u b l i c a t i o n of Tang and Bonner i n 1947 on some c h a r a c t e r i s t i c s of the IAA-degrading enzyme, IAA oxidase, i n e t i o l a t e d pea e p i c o t y l s was quoted repeatedly by investigators which led readers to b e l i e v e that IAA oxidase i s the only enzyme responsible for the destruction of IAA. The enzyme i s designated as IAA oxidase c h i e f l y because i n a c t i v a t i o n of IAA takes place only i n the presence of oxygen, and about one molecule of oxygen i s consumed per molecule of IAA inactivated, and one molecule of carbon 80 d i o x i d e i s l i b e r a t e d (Tang and Bonner, 1947). E v i d e n c e s u p p o r t i n g t h e c l a i m t h a t IAA o x i d a s e i s n o t a s i n g l e enzymed b u t a m i x t u r e o f enzymes i s f o u n d i n t h e work o f S e q u i r a and Mineo (1966); Lee (1971); Meudt (1967); Ray (1960); Hinman and Lang (1965). That IAA o x i d a s e may c o n s i s t o f a m i x t u r e o f p l a n t p e r o x i d a s e s , and t h a t IAA o x i d a s e t o g e t h e r w i t h p e r o x i d a s e s may e x i s t as a g g r e g a t e s was s u g g e s t e d b y S e q u i r a and Mineo (1966). Ray (1960) c o n s i d e r e d b o t h p e r o x i d a s e and IAA o x i d a s e a c t i v i t i e s were due t o one enzyme. IAA o x i d a s e has been i d e n t i f i e d as a heme p r o t e i n p e r o x i d a s e b y G o l d a c r e (1951). I t may be i n f e r r e d from t h e s e f i n d i n g s t h a t IAA o x i d a s e i s an isoenzyme o f p e r o x i d a s e . The a b i l i t y o f p e r o x i d a s e s t o o x i d i s e IAA has been r e p o r t e d b y Meudt (1967), u s i n g c r y s t a l l i n e h o r s e -r a d i s h p e r o x i d a s e ; Hinman and Lang (1965); Ray (1960), u s i n g p e r o x i d a s e from t h e fungus Omphalia f l a v i d a . Seven p e r o x i d a s e s isoenzymes were d e t e c t e d i n h o r s e r a d i s h r o o t s , w i t h no i n t e r c o n v e r s i o n s among t h e isoenzymes (Shannon e t aJL, 1966) . Jermy and Thomas (1954) r e p o r t e d t h a t t h e isoenzymes o f h o r s e r a d i s h p e r o x i d a s e have s u b s t r a t e s p e c i f i c i t i e s . Enzymes of the polyphenol oxidase systems are also known to oxidise IAA (Leopold and Plummer, 1961; Skoog, 1944) That the IAA oxidase system i n e t i o l a t e d pea e p i c o t y l s possessed a considerable degree of substrate s p e c i f i c i t y , was one of the conclusions reached by Tang and Bonner (1947). They reported that the IAA oxidase system d i d not attack indoleacetamide, indolebutyric acid, indolepyruvic acid, indolecarboxylic a c i d or tryptophan. The products of IAA oxidation have been studied, but no c l e a r - c u t conclusions are a v a i l a b l e . Hinman and Lang (1965) suggested 3-methylene oxindole, and a neutral indole, and emphasised that the product composition i s h i g h l y depend-ent on IAA concentrations. That the end product i s ihdole-aidehyde, and the intermediates, i n d o l e - 3 - g l y c o l l i c and indole-3-glyoxylic acids were suggested by Goldacre (1951). A general agreement among inves t i g a t o r s i s that the indole r i n g remains i n t a c t (Tang and Bonner, 1947; Hinman and Lang, 1965; Meudt, 1967). The oxidation products are i n a c t i v e i n the Avena t e s t or i n chemical methods of IAA determination 82 Meudt (1967) indicated that the oxidative transform-a t i o n of IAA leads to the formation of b i o l o g i c a l l y a c t i v e products, provided p r e v a i l i n g conditions are such to prevent the formation of secondary oxidation products which i n a c t i v a t e IAA. Meudt and Galston (1962) suggested a mechanism by which IAA a t t a i n s b i o l o g i c a l a c t i v i t y i n plants. The primary product of IAA oxidation i s oxindole (Hinman and Lang, 1965), and i s spared from destruction by binding to RNA. The-oxindole-RNA complexes then stimulate plant growth. These complexes gave p o s i t i v e reactions i n Salkowski and E h r l i c h reagents (Meudt and Galston, 1962). A support f o r t h i s hypothesis comes from £he study of the d i s t r i b u t i o n pattern of peroxidase a c t i v i t y i n plants. Meudt (1967) found that the d i s t r i b u t i o n pattern of peroxidase a c t i v i t y c o r r e l a t e s with the growth centres of the plant. Peroxidase a c t i v i t y i s higher i n actively-growing tissues than i n mature and dormant t i s s u e s . In the l i g h t of evidence presented, IAA oxidase should not be thought of as a sing l e component, but rather as a mixture o f peroxidase isoenzymes. I t i s a p p r o p r i a t e t o co n s i d e r IAA oxidase as a system o f enzymes, hence the term "IAA oxidase system" should be used. The l a t t e r term has been used by i n v e s t i g a t o r s f o r example, Goldschmidt, Goren, and Monselise (1967); Lee (1971). Babaev (1966) r e p o r t e d t h a t soaking c o t t o n seeds i n s o l u t i o n s o f HNap (10 mg/1), and the a p p l i c a t i o n o f the a c i d t o the s o i l (20 mg/kg o f dry s o i l ) a c t i v a t e d peroxidase a c t i v i t y i n the r o o t s . 84 MATERIALS AND METHODS Uniform seeds of Phaseolus v u l g a r i s c u l t i v a r Top Crop were surface s t e r i l i s e d by washing i n 95% ethanol for ten minutes. The seeds were sown i n s t e r i l i s e d vermiculite, saturated v/ith 1.0% Captan fungicide, contained i n wooden f l a t s , and allowed to grow i n darkness at an a i r temperature of 22.5 + 0.5° C and a r e l a t i v e humidity of 95 + 2%. Thirteen-day-old dark-grown seedlings were c a r e f u l l y removed from the vermiculite and washed v/ith tap water Treatment was ef f e c t e d by immersing the root systems i n 100 ppm (0.01%) KNap s o l u t i o n for 24 hours i n darkness at o room temperature (23-24 C). The root systems of c o n t r o l plants were soaked i n d i s t i l l e d water under s i m i l a r conditions. Subsequent handling of plant materials was performed i n red l i g h t at room temperature. E p i c o t y l s (5-8 cm) including leaves, were harvested, washed v/ith i c e c o l d d i s t i l l e d water, and immediately frozen with l i q u i d nitrogen. The following procedure was modified from Sequeira and Mineo (1966). The frozen plant t i s s u e s were ground to a f i n e 85 powder i n a motar with p e s t l e . The powder was suspended i n c o l d 0.02 M K^PO^-Na^PC^ buffer, pH 6.1, i n the proportion of ten g of frozen t i s s u e to 50 ml buffer, and s t i r r e d for 1.5 hours i n the col d room at 4° C. The mixture was centrifuged at 18,000 £ f o r ten minutes at 4° C. The supernatant was decanted into an 150-ml Erlenmeyer f l a s k and s o l i d (NH.).SO, was added gently to 35% saturation 4 2 4 (Green et a l , 1955). The p r e c i p i t a t e which was formed at 4 ° C i n 24 hours, was removed by c e n t r i f u g a t i o n at 18,000 £ fo r ten minutes. The supernatant was then brought to 70% saturation with s o l i d (NH^^SO^, and the p r e c i p i t a t e which was formed at 4° C i n . 24 hours, was recovered by c e n t r i f u g a t i o n at 18,000 2. f ° r t e n minutes, and resuspended i n 12.5 ml b u f f e r . The s o l u t i o n was dialysed for 24 hours i n the col d room (4° C) against 3000 ml bu f f e r v/ith one change o f external b u f f e r . This dialysed solution, which had both IAA oxidase and peroxidase a c t i v i t i e s , was re f e r r e d to as crude enzyme. No attempts were made to separate these two enzymes as both were found to degrade IAA (Ray, I960; Sequeira and Mineo, 1966; Meudt, 1970). 86 Assay for enzyme a c t i v i t y The a c t i v i t y of the IAA-destroying enzymes was determined by the Salkowski reaction (Tang and Bonner, 1947; Gordon and Weber, 1951; Gordon and Paleg, 1957). The reaction mixture (five ml) was prepared by the method of Sequeira and Mineo (1966), and consisted of the following: 0.5 ml enzyme extract 0.25 ml 1.0 mM Na s a l t of 2,4-dichlorophenol 1.00 ml 1.0 mM IAA i n 0.5 mM MnCl 2 (1:1, v/v) 3.25 0.02 M KH 2P0 4-Na 2HP0 4, pH 6.1 The reaction mixture was shaken i n a water bath (Dubnoff Metabolic Shaking Incubator, P r e c i s i o n S c i e n t i f i c , Chicago) for two hours at 30° C i n darkness, and then 1.0 ml of Salkowski reagent was added. The mixture was shaken for an ad d i t i o n a l three hours and absorbance at 525 nm was determined with a Bausch and Lomb Spectronic 20 colorimeter using a blue-sensing phototube , type CEA-59RX. The Salkowski reagent was prepared according to Gordon and Weber (1951), by mixing 1.0 ml of 0.5 M F e C l 3 to 50 ml of 35% HC10.. 4 87 The r e s i d u a l IAA i n the reaction mixtures was determined by reference to a standard curve. The standard curve was developed by mixing a ser i e s of two ml known quantities of authentic IAA to four ml Salkowski reagent for 35 minutes. The absorbance was read at 525 nm w i t h a S p e c t r o n i c 20 spectrophotometer. The experimental setup used i n t h i s i n v e s t i g a t i o n was the randomised complete block design, comprising two r e p l i c a t i o n s with f i v e determinations per r e p l i c a t i o n i n each treatment. The r e s u l t s were subjected to analysis of variance. RESULTS When the root systems of 13-day-old dark-grown bean plants were immersed i n a 100 ppm solution of KNap for 24 hours, there was a 4% increase i n the a c t i v i t y of the IAA oxidase system compared with that of control plants. This stimulatory e f f e c t of KNap was s i g n i f i c a n t at the 0.01 l e v e l (Table VIII). The a c t i v i t y of the IAA oxidase system was measured i n terms of microgrammes of IAA destroyed per g fresh weight of e p i c o t y l t i s s u e . 89 Table v i l l . T h e a c t i v i t y o f the IAA oxidase system i n the 14-day-old dark-grown bean e p i c o t y l s f o l l o w i n g treatment w i t h 100 ppm KNap. Treatment I n i t i a l IAA +Residual IAA Amount of Amount of concentration concentration IAA IAA i n reaction i n reaction destroyed destroye'd mixture mixture (% increase (ugm/ml) (ugm/ml) (ugm/g f r wt) over control) C o n t r o l 175 26.27 371.82 KNap 100 ppm 175 20.20 386.00** 3.81 + Mean o f 15 determinations. ** Value d i f f e r s s i g n i f i c a n t l y from the c o n t r o l value a t the 0.01 l e v e l . 90 DISCUSSION It i s recognised that the IAA oxidase system i s involved i n the degradation of IAA. The increase (ca 4% over co n t r o l plants) i n the a c t i v i t y of the IAA oxidase system i n bean plants following treatment with 100 ppm KNap supports the report of increases i n peroxidase a c t i v i t y i n the roots of cotton plants resulted from seed soak with 10 ppm HNap by Babaev (1966). That peroxidase i s a component of the IAA oxidase system was suggested by, for example, Sequeira and Mineo (1966). The stimulation of IAA biosynthesis i n bean e p i c o t y l s by 100 ppm KNap reported by the author i n the present in v e s t i g a t i o n , and the a c t i v a t i o n of the IAA oxidase system by s i m i l a r KNap concentration, suggests that the a c t i v i t y of the IAA oxidase system i s induced by the substrate, IAA. It i s d i f f i c u l t to resolve the p.aradox that IAA oxidase system i s capable of i n a c t i v a t i n g IAA; and that the a c t i v i t y of t h i s system i s induced by the substrate, IAA, and correl a t e s v/ith the active growth centres of plants i . e . 91 greater a c t i v i t y i n a c t i v e l y grov/ing tissues than i n mature and dormant tissues (Meudt, 1967). This poses a fundamental question: how can there be active growth i f IAA i s c o n t i n u a l l y destroyed ? It i s possible that i n the a c t i v e l y growing plant t i s s u e s , IAA i s i n excess and able to sustain destruction, and, at the same time, maintains ac t i v e growth. A s o l u t i o n to t h i s question must await further research. 92 CONCLUSIONS 1) Potassium naphthenates (KNap) exhibited the following auxin-like properties: a) I n h i b i t i o n of root growth KNap at 1000 ppm i n h i b i t e d the growth of i n t a c t roots of cucumber. b) Stimulation of root i n i t i a t i o n The treatment of bean stem cuttings with 10 and 100 ppm KNap resulted i n the stimulation of root i n i t i a t i o n compared with control cuttings. The treatment of azalea stem cuttings with 100 and 1000 ppm KNap s i g n i f i c a n t l y increased root i n i t i a t i o n compared with control cuttings. c) Stimulation of elongation of pea stem segments KNap at 10 ppm had a greater stimulatory e f f e c t on elongation of pea stem segments than 0.1 ppm IAA. d) Retardation of pe t i o l e abscission The applications of 10 and 1000 ppm cyclohexanecarboxylic acid to the d i s t a l end of debladed pe t i o l e s of bean, resulted i n retardation of abscission. 2) When applied to the seeds for 12 hours p r i o r to sowing, 100 ppm KNap augmented the biosynthesis of IAA from tryptophan i n the 5-8 cm t i p s of dark-grown epic o t y l s of bean. 3) When applied to the roots of 13-day-old dark-grown bean plants for 24 hours, 100 ppm KNap stimulated IAA degradation i n the 5-8 cm t i p s of e p i c o t y l s . 93 BIBLIOGRAPHY Abeles, F.B., 1968 Role of RNA and protein synthesis i n abscission. 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