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Studies on the induction and release of seed dormancy in wild oats (Avena fatua L.) Tilsner, Heidy Rose 1986

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STUDIES ON THE INDUCTION AND RELEASE OF SEED DORMANCY IN WILD OATS (AVENA FATUA L.) By HEIDY ROSE TILSNER B.Sc. (Agr.)f The Uni v e r s i t y of B r i t i s h Columbia, 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES The Department of Plant Science We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 1986 © Heidy Rose T i l s n e r , 1986 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f P l a n t Science The U n i v e r s i t y o f B r i t i s h C o l u m b i a 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3 D a t e J u l y 8, 1986 j-6 n/Rn i i ABSTRACT The induction and release of secondary dormancy by anaerobiosis i n g e n e t i c a l l y pure dormant (AN-51, Mont 73) and nondormant (CS-40, SH-430) l i n e s of Avena fatua L. and the r o l e of a l t e r n a t i v e r e s p i r a t i o n i n the regulation of i t s primary and secondary dormancy were studied. These l i n e s d i f f e r e d with regard to the optimal period of anaerobiosis necessary f or induction of dormancy and/or the degree (% of seeds acquiring dormancy) and duration of dormancy induced. Secondary dormancy could be induced more e f f e c t i v e l y i n after-ripened seeds of dormant l i n e s than i n nondormant l i n e s where only a short-term dormancy could be induced ( i n 5-7 week old seeds). As with primary dormancy, wild oat biotypes e x h i b i t genetic v a r i a b i l i t y i n t h e i r secondary dormancy behaviour and factors such as temperature can modify the expression of t h i s t r a i t . The germination stimulants k i n e t i n , isopentenyl adenine, sodium azide, potassium n i t r a t e and ethanol, which break primary dormancy i n wild oats, stimulated germination of secondarily dormant seeds ( l i n e AN-51). Since these chemicals are s t r u c t u r a l l y diverse, primary and secondary dormancies appear to be regulated by s i m i l a r mechanism(s). Salicylhydroxamate (SHAM), an i n h i b i t o r of a l t e r n a t i v e r e s p i r a t i o n , did not i n h i b i t : 1. the r e s p i r a t i o n of embryos excised from after-ripened or secondarily dormant seeds, 2. the spontaneous release of secondary dormany i n nondormant l i n e s or 3. the release of secondary dormancy by a var i e t y of chemicals (except azide), suggesting that a l t e r n a t i v e r e s p i r a t i o n i s not involved i n the induction or release of secondary dormancy. Azide and cyanide released seed dormancy at s i m i l a r concentrations i i i and treatment durations. While cyanide released primary dormancy in seeds with l i t t l e after-ripening, azide was effective only in secondarily dormant seeds or seeds with more extensive after-ripening. Both inhibitors stimulated seed respiration to a similar extent. The release of dormancy by cyanide was always preceded by respiratory stimulation, but the latter appeared to be independent of germination. SHAM inhibited both the release of seed dormancy and the stimulation of seed respiration by azide but not by cyanide. Respiration was inhibited only when SHAM was applied concurrently with azide. When applied subsequent to azide treatment, SHAM had no effect. The respiration of seed pre-treated with azide and cyanide was insensitive to SHAM and therefore cannot be alternative. Studies were performed to determine the effect of pH on the stimulation of germination and respiration by cytochrome oxidase inhibitors. Although pH had l i t t l e effect on seed respiration and germination in controls and in the presence of cyanide, i t strongly affected the activity of azide. At pH 5, 1 mM azide inhibited both seed respiration and germination whereas at pH 7 i t stimulated both. SHAM at pH 7 did not affect the stimulation of respiration by azide, but inhibited i t in the unbuffered system and at pH 5. Thus, SHAM appears to alter azide activity by lowering pH, increasing the concentration of undissociated (active) azide, which then completely inhibits cytochrome oxidase and consequently, seed respiration and germination. The release of dormancy and the stimulation of respiration by cyanide and azide do not appear to be related to the inhibition of cytochrome-mediated respiration or the stimulation of alternative respiration. iv. TABLE OF CONTENTS PAGE Abstract i i Table of contents i v L i s t of tables v i L i s t of fig u r e s v i i i Acknowledgement x I. Introduction and l i t e r a t u r e review. A. Introduction 1 B. Seed dormancy i n wild oat 2 C. Stimulation of wild oat seed germination 6 by chemicals D. Seed dormancy and a l t e r n a t i v e r e s p i r a t i o n 1. A biochemical overview of the a l t e r n a t i v e r e s p i r a t o r y pathway 8 2. The phy s i o l o g i c a l s i g n i f i c a n c e of a l t e r n a t i v e r e s p i r a t i o n 10 3. A l t e r n a t i v e r e s p i r a t i o n and the regulation of seed dormancy ..12 E. Secondary seed dormancy 14 F. Objectives 16 I I . Materials and methods. A. Seed source 17 B. The induction of secondary dormancy 18 C. Germination studies 18 D. Oxygen-uptake measurements 19 1. Intact seeds. 2. Excised embryos. E. S t a t i s t i c a l a n alysis 22 I I I . Results. A. The induction and release of secondary dormancy 1. Characterization of the induction of secondary dormancy i n pure l i n e s 23 2. The e f f e c t of temperature and seed age on the induction of secondary dormancy 26 3. Stimulation of germination of secondarily dormant seeds by chemicals 31 4. Determination of re s p i r a t o r y components 31 5. E f f e c t of the induction of secondary dormancy on resp i r a t o r y components 36 B. E f f e c t s of resp i r a t o r y i n h i b i t o r s on germination. 1. Release of primary and secondary dormancy 39 PAGE 2. E f f e c t of SHAM on the release of dormancy by azide and cyanide 44 C. E f f e c t s of i n h i b i t o r s on seed r e s p i r a t i o n 1. Stimulation of seed r e s p i r a t i o n by azide and cyanide 47 2. The nature of azide and cyanide induced respiration....47 3. The resp i r a t o r y partioning of azide and cyanide induced r e s p i r a t i o n 53 D. The e f f e c t of pH on the action of resp i r a t o r y i n h i b i t o r s 1. Respiratory studies 57 2. Germination studies 60 IV. Discussion A. The induction and release of secondary dormancy 66 B. A l t e r n a t i v e r e s p i r a t i o n and the regulation of secondary dormancy 68 C. The act i o n of azide and cyanide on the release of primary and secondary dormancy 71 D. The stimulation of seed r e s p i r a t i o n by azide and cyanide 73 E. The e f f e c t of SHAM on the release of dormancy by azide and cyanide 75 F. The nature of azide and cyanide induced r e s p i r a t i o n . 1. The e f f e c t of SHAM on the induction of r e s p i r a t i o n by azide and cyanide 76 2. The e f f e c t of azide and cyanide on resp i r a t o r y components. a. A l t e r n a t i v e r e s p i r a t i o n 77 b. Residual r e s p i r a t i o n 79 c. Cytochrome mediated r e s p i r a t i o n 81 G. The e f f e c t of pH on the action of azide cyanide on seeds 81 1. The e f f e c t of pH on the stimulation of r e s p i r a t i o n by azide and cyanide 82 2. Comparison of the e f f e c t of SHAM and pH on the action of azide and cyanide 84 3. The e f f e c t of pH on the release of seed dormancy by azide and cyanide 87 H. General discussion 89 V. Conclusions 93 VI. Bibliography 95 v i LIST OF TABLES Page Table 1. The effect of SHAM on the release of secondary dormancy in line SH-430 28 Table 2. The effect of anaerobiosis temperature on the induction of secondary dormancy in seeds of nondormant (SH-430, CS-40) and after-ripened seeds of dormant (AN-51, Mont 73) lines . 30 Table 3. The effect of seed age on the induction of secondary dormancy in line SH-430 32 Table 4. Stimulation of germination of secondarily dormant seeds of line AN-51 by chemicals known to break primary dormancy 33 Table 5. Inhibition of embryo and seed respiration by 1 mM cyanide in the presence or absence of SHAM 37 Table 6. Effect of respiratory inhibitors on the oxygen-uptake of embryos excised from after-ripened, primarily and secondarily dormant seeds of line AN-51 38 Table 7. The effect of cyanide on percentage and rate of germination in primarily dormant seeds of line AN-51 40 Table 8. Time course of azide stimulated germination in secondarily dormant seeds of line AN-51 42 Table 9. Effect of after-ripening on the release of primary dormancy by azide and cyanide in line AN-51 45 Table 10. The effect of SHAM on the release of secondary dormancy in line AN-51 by azide and cyanide 46 Table 11. The effect of SHAM on the induction of respiration by 24 h cyanide or azide treatment in primarily dormant seeds of line AN-51 50 v i i Page Table 12. The e f f e c t of short cyanide pulse treatments on r e s p i r a t i o n and germination i n pri m a r i l y dormant seeds of l i n e AN-51 51 Table 13. The nature of azide and cyanide-induced r e s p i r a t i o n i n dormant seeds of l i n e AN-51 52 Table 14. Dose response f o r the i n h i b i t i o n of seed r e s p i r a t i o n i n l i n e AN-51 by azide 54 Table 15. Dose response f o r the e f f e c t of SHAM on the i n h i b i t o n of seed r e s p i r a t i o n ( l i n e AN-51) by azide 55 Table 16. The e f f e c t of cyanide and azide treatment on the response of primarily dormant seeds ( l i n e AN-51) to respir a t o r y i n h i b i t o r s 56 Table 17. The e f f e c t of pH on the action of respi r a t o r y i n h i b i t o r s on oxygen consumption of primarily dormant seeds of l i n e AN-51 58 Table 18. The e f f e c t of pH on the release of secondary dormancy by azide i n seeds of l i n e AN-51... 64 Table 19. The e f f e c t of pH on the release of primary dormancy by cyanide i n seeds of l i n e AN-51 65 v i i i LIST OF FIGURES Page F i g . 1. The cytochrome mediated and a l t e r n a t i v e pathways 9 F i g . 2. The e f f e c t of anaerobiosis duration on the induction of secondary dormancy i n a f t e r -ripened seeds of dormant l i n e s 24 F i g . 3. Induction of secondary dormancy i n ae r o b i c a l l y pre-imbibed af t e r - r i p e n d seeds of l i n e AN-51 25 F i g . 4. The induction of secondary dormancy i n ge n e t i c a l l y nondormant seeds of l i n e s SH-430 and CS-40 27 F i g . 5. Afte r - r i p e n i n g of secondarily dormant seeds of l i n e AN-51 during dry storage 29 F i g . 6. The e f f e c t of cyanide on the r e s p i r a t i o n of excised embryos ( l i n e AN-51) 34 F i g . 7. The e f f e c t of azide on the r e s p i r a t i o n of excised embryos ( l i n e AN-51) 35 F i g . 8. E f f e c t of azide pulse duration on the release of primary dormancy i n seeds of l i n e AN-51 41 F i g . 9. E f f e c t of cyanide pulse duration on the release of primary dormancy i n seeds of l i n e AN-51 43 F i g . 10. E f f e c t of cyanide on r e s p i r a t i o n and germination of primarily dormant seeds of l i n e AN-51 48 F i g . 11. E f f e c t of cyanide pulse duration on the r e s p i r a t i o n and germination of primarily dormant seeds of l i n e AN-51 49 F i g . 12. The e f f e c t of pH on azide-induced r e s p i r a t i o n i n primarily dormant seeds of l i n e AN-51 59 F i g . 13. The e f f e c t of pH on cyanide-induced r e s p i r a t i o n i n primarily dormant seeds of AN-51 61 ix Page Fig. 14. The effect of SHAM and pH on azide-induced respiration in primarily dormant seeds of line AN-51 62 Fig. 15. The effect of SHAM on cyanide-induced respiration in dormant seeds of line AN-51 63 X ACKNOWLEDGMENTS My h e a r t - f e l t thanks go to Dr. Mahesh K. Upadhyaya. His continued guidence and encouragement helped maintain my i n t e r e s t and enthusiasm through-out the course of t h i s project. I e s p e c i a l l y appreciated h i s patient reviewing and e d i t i n g of t h i s manuscript. I also thank Dr. N.R. Knowles f or h i s s t a t i s t i c a l advice. I g r a t e f u l l y acknowledge the rece i p t of a NSERC postgraduate scholarship. I thank my fri e n d s i n the department for making my graduate study at U.B.C. much more than an academic experience. 1 INTRODUCTION AND LITERATURE REVIEW I. INTRODUCTION Seed dormancy , a widespread phenomenon i n the plant kingdom, i s e c o l o g i c a l l y advantageous f o r several reasons. F i r s t l y , germination can be co n t r o l l e d so as to l i m i t emergence to times most conducive to seedling establishment. Secondly, a dormancy mechanism can function to secure a sui t a b l e place f o r germination ( i . e . seeds re q u i r i n g l i g h t f o r dormancy release w i l l germinate only i n the top few millimeters of s o i l ) . L a s t l y , seed dormancy prevents premature germination ( i . e . while seeds are s t i l l on the maternal plant; Bewley and Black, 1985). Seed dormancy i s of great a g r i c u l t u r a l s i g n i f i c a n c e . Although most crop species produce nondormant seed (because of human s e l e c t i o n f o r prompt and uniform seed germination), weed species display a wide d i v e r s i t y i n types of dormancy. V a r i a b i l i t y i n weed seed germinability ensures germination over an extented period making co n t r o l d i f f i c u l t . For t h i s study the d e f i n i t i o n of seed dormancy given by Simpson (1978) w i l l be used: "dormancy i s that state i n which a seed f a i l s to resume growth when i t i s rehydrated i n an environment that w i l l support normal germination and seedling growth of apparently i d e n t i c a l but nondormant seeds of the same species". 2 Seed dormancy can be divided into two categories: 1. Embryo dormancy and 2. Coat-imposed dormancy. In embryo dormancy, the co n t r o l mechanism resides within the embryo which f a i l s to germinate when removed from the r e s t of the seed t i s s u e . Coat-imposed dormancy i s maintained by a hard, impervious seed coat which encloses the embryo and endosperm (Bewley and Black, 1985). The control of embryo dormancy can be a function of: 1. the presence of germination i n h i b i t o r s , 2. the existence of a biochemical block that prevents or i s necessary for germination or 3. p h y s i o l o g i c a l immaturity of the embryo. Coat-imposed dormancy may be manifested by: 1. mechanical r e s t r a i n t ( i . e . "hard seeds"), 2. interference with water or oxygen uptake or 3. the presence of germination i n h i b i t o r s within the seed coat (Bewley and Black, 1982). Both the acquistion and subsequent release of dormancy are c o n t r o l l e d by an i n t e r a c t i o n between genetic and environmental f a c t o r s . Environmental f a c t o r s regulating the acquistion of seed dormancy include l i g h t , temperature and oxygen a v a i l a b i l i t y . Time i s important i n the release of seed dormancy during dry storage ( a f t e r -ripening) (Bewley and Black, 1985). B. SEED DORMANCY IN WILD OAT. Wild oats (Avena fatua L.) i s an important weed of cereal and o i l s e e d crops around the world. Origin a t i n g i n Eurasia (Frankton and Mulligan, 1970), i t s range now includes several European countries, North A f r i c a , A u s t r a l i a , New Zealand and North America. 3 It s d i s t r i b u t i o n i n Canada i s predominantely western, being most abundant i n the three p r a i r i e provinces and the Peace River region of B.C. (Sharma and Vanden Born, 1978). In terms of crop losses and weed control costs, A_. fatua i s Canada's most detrimental weed species (Alex, 1982). In 1978, estimated losses due to wild oats i n the grain growing areas of Canada exceeded $280 m i l l i o n (Alex, 1982). The persistance of wild oats as a weed l a r g e l y depends on the a b i l i t y of i t s seed to remain dormant i n the s o i l f o r varying periods. D i f f e r e n t biotypes of wild oats produce seed with dormancy ranging from a few weeks up to 5 years (Naylor and Jana, 1976; Sharma and Vanden Born, 1978). In addition, nondormant (through after-ripening) seeds are able to acquire secondary dormancy under adverse environmental conditions (Hay and dimming, 1959). An understanding of the p h y s i o l o g i c a l basis of seed dormancy i s e s s e n t i a l for the development of more e f f e c t i v e c o n t r o l measures for t h i s weed. Wild oats provide an excellent system for the study of seed dormancy. Geneti c a l l y pure l i n e s e x h i b i t i n g d i f f e r e n t seed dormancy behavior have been i s o l a t e d from f i e l d populations. By r a i s i n g these l i n e s under c o n t r o l l e d growth conditions i t i s possible to compare g e n t i c a l l y nondormant and dormant seeds i n the absence of confounding environmental e f f e c t s on dormancy development. The l o s s of wild oats seed dormancy through a f t e r - r i p e n i n g and the a b i l i t y of the a f t e r -ripened seeds to acquire secondary dormancy enables comparison of d i f f e n t states of dormancy within a l i n e thus circumventing genetic differences that could hamper such a study. Dormancy i n wild 4 oats i s believed to be embryonic i n o r i g i n . Embryos excised from f r e s h l y harvested dormant wild oats seeds f a i l e d to germinate when imbibed on water or nutrient media (Naylor and Simpson, 1961; Simpson, 1965) while embryos from nondormant seeds germinated under these conditions. As dormant seeds after-ripened, the percentage germination of excised embryos increased. Addition of GA^ ( g i b b e r e l l i c acid-3) to the medium overcame embryo dormancy (Simpson, 1965). Genetic (Naylor and Jana, 1976; Jana et a l . , 1979) and environmental influences on seed dormancy i n wild oats have been well documented. Environmental conditions during seed maturation and seed germination a l t e r the expression of dormancy i n wild oats (Sawhney and Naylor, 1979, 1980; Peters, 1982). Sawhney and Naylor (1979) found that high temperature during seed maturation greatly reduced the duration of primary dormancy i n some biotypes of wild oats. The los s of primary dormancy i n imbibed seeds however, i s favored by low incubation temperatures (Naylor and Fedec, 1978). Mature seeds of g e n e t i c a l l y pure l i n e s d i f f e r i n t h e i r germination response to temperature during imbibition (Naylor and Fedec, 1978). Water stress during seed maturation also a f f e c t s dormancy expression i n wild oats. Peters (1982) found that water s t r e s s , imposed from the time of panicle emergence to the completion of ripening, increased the germination percentage of mature seeds, i n d i c a t i n g that the duration of dormancy was much shorter i n seeds from water stressed plants compared to those from nonstressed plants. The biochemical basis of seed dormancy i n wild oats i s not known. 5 Much of the work inv o l v i n g the physiology of dormancy release i s of an empirical nature and has contributed l i t t l e to a general understanding of the mechanism of dormancy i n t h i s species. GA^ i s a potent dormancy-breaking treatment for wild oats seed (Naylor and Simpson, 1961b). Consequently, a mechanism whereby dormancy i s regulated by t h i s compound has been sought. Naylor and Simpson (1961b) observed two d i s t i n c t concentration optima for GA^ i n the presence and absence of sucrose. Alone, 50ppm GA^ was required to release dormancy i n f r e s h l y harvested seed but when given i n combination with sucrose, 0.5 ppm GAg released dormancy. This bimodal concentration optima was explained by a dual mode of action of GA^; high GA^ concentrations are required to stimulate starch breakdown i n endosperm whereas when applied with sucrose, a lower GA^ concentration was adequate to enable sugar u t i l i z a t i o n and germination by the embryo. Thus, while GA^ was required f o r endosperm mobilization, the release of dormancy could be e l i c i t e d by GA^ i n the absence of endosperm breakdown. Metzger (1983a), however, found no d i f f e r e n c e i n endogenous GA^ l e v e l s i n g e n e t i c a l l y dormant and nondormant l i n e s of wild oats. Furthermore, no s i g n i f i c a n t change i n g i b b e r e l l i c a c i d l e v e l occurred during the l o s s of dormancy through a f t e r - r i p e n i n g (Taylor and Simpson, 1980). Thus, dormancy i n wild oats cannot be explained simply i n terms of endogenous GA„ l e v e l . C. STIMULATION OF WILD OAT SEED GERMINATION BY CHEMICALS Adkins and Ross (1981) found that the growth regulator 6 ethylene could stimulate germination i n p a r t i a l l y after-ripened seeds of wild oats. They did not however f i n d any r e l a t i o n s h i p between endogenous ethylene production and germinability. Roberts and Smith (1977) proposed that n i t r a t e and n i t r i t e ions, both of which promote wild oats seed germination (Johnson, 1935; Adkins et a l . , 1984b), act to promote the reoxidation of NADPH to NADP v i a the oxidative pentose phosphate pathway (PPP) which i n turn promotes germination. They suggested that any compound stimulating the PPP w i l l release seed dormancy. Azide, an i n h i b i t o r of cytochrome oxidase, i s known to release dormancy i n wild oats (Fay and Gorecki, 1978). Roberts and Smith (1977) suggested that azide promotes seed germination by stimulating the PPP. In l i g h t of more recent research t h i s hypothesis has however, been dismissed. No r e l a t i o n s h i p between the l e v e l s of PPP dehydrogenases (Upadhyaya et a l . , 1981) or PPP a c t i v i t y measured by the C^/C^ r a t i o technique (Fuerst et a l . , 1983) and dormancy status could be found. S i m i l a r l y the release of dormancy by GAg did not r e s u l t i n the concomitant r i s e i n the a c t i v i t y of the PPP as Robert's hypothesis would require. This hypothesis i s therefore not substantiated i n wild oats. Recently Adkins et a l . (1984c) have suggested that n i t r a t e and n i t r i t e may release seed dormancy i n wild oats by promoting g l y c o l y s i s and/or the Kreb's cycle by enhancing the rate of reoxidation of NADH. Evidence supporting t h i s hypothesis i s also la c k i n g . 7 Anesthetics are another c l a s s of compounds known to release seed dormancy i n several species (Taylorson and Hendricks, 1979) i n c l u d i n g wild oats (Upadhyaya, unpublished data; Adkins et a l . , 1984a,d). Hendricks and Taylorson (1980) suggested that ethanol and some other anesthetics release seed dormancy by increasing c e l l membrane permeability. The bases of t h e i r conclusion were: 1. several metabolically i n e r t anesthetics ( i . e . chloroform, e t h y l ether) released seed dormancy, suggesting that these compounds release dormancy v i a t h e i r action on c e l l membranes rather than as r e s p i r a t o r y substrates, and 2. a p p l i c a t i o n of hydrostatic pressure (known to decrease c e l l membrane permeability, Hendricks and Taylorson, 1980) to seeds reversed the e f f e c t of applied anestetics (Hendricks and Taylorson, 1980). Adkins et a l . (1984a) found that ethanol, acetaldehyde and to a l e s s e r extent, procaine, chloralhydrate and methanol stimulated germination of dormant wild oats seeds. Since the two most e f f e c t i v e anesthetics (ethanol and acetaldehyde) are metabolically a c t i v e , i t was suggested that the promotion of germination by these compounds may be due to t h e i r r o l e as r e s p i r a t o r y substrates rather than by the v i r t u e of t h e i r e f f e c t s on c e l l membranes (Adkins et a l . , 1984d). Accordingly, a p p l i c a t i o n of ethanol to seeds i s thought to enhance the f l u x of metabolites through the Kreb's cy c l e , which the authors propose regulates seed dormancy i n wild oats. Substituted phthalimides have also been shown to release dormancy i n several weed species including wild oats (Metzger, 1983b; Upadhyaya et a l . , 1986). I t i s believed that these compounds act l i k e GA„ i n t h i s regard. 8 D. SEED DORMANCY AND ALTERNATIVE RESPIRATION Recent studies on the mechanism of the release of seed dormancy using r e s p i r a t o r y i n h i b i t o r s have implicated a l t e r n a t i v e (cyanide-i n s e n s i t i v e ) r e s p i r a t i o n as being important i n i t s reg u l a t i o n . Before discussing the evidence f o r t h i s theory the biochemistry and physiology of t h i s pathway w i l l be b r i e f l y reviewed. 1. A biochemical overview of the a l t e r n a t i v e pathway. The occurrence of CN-resistant r e s p i r a t i o n i n plant t i s s u e has been observed since the early 1930's (Henry and Nyns, 1975). Although early c l a s s i c a l studies of a l t e r n a t i v e r e p i r a t i o n involved almost ex c l u s i v e l y members of the Araceae, the phenomenon of CN-resistance i s now known to be widespread i n the plant kingdom (Henry and Nyns, 1975). The c e l l u l a r l o c a t i o n of a l t e r n a t i v e r e s p i r a t i o n has been ascribed to the mitochondria. James and Beevers (1950) reported that oxygen uptake i n mitochondria i s o l a t e d from CN-resistant spadix t i s s u e of Arum maculatum was CN-resistant. Subsequently numerous studies have reported s i m i l a r r e s u l t s i n a v a r i e t y of ti s s u e s (Solomos, 1977). Recent research i n d i c a t e s that components of the a l t e r n a t i v e pathway are embedded i n the inner mitochondrial membrane (Troostembergh and Nyns, 1976). The discovery of s e l e c t i v e i n h i b i t o r s of the a l t e r n a t i v e pathway (hydroxamic acids, Schonbaum et a l . , 1971; d i s u l f i r a m , Grovers and L a t i e s , 1981) has greatly advanced our knowledge of i t s biochemical 9 NADH (endogenous) I Fp (+20 mV) NADH-DEHYDROGENASEn-* Fp (-70 mV) I % 11 N — * ATP ubiquinone (+70 mV) 11 Fp (+110 mV) oxygen cyt b's ADP + Pi i C B 1 ^ w ATP cyt c cyt a ^ ADP + Pi X ATP cyt a, 1 oxygen Fig. 1 The cytochrome (A) and alternative (B) pathways. X • alternative oxidase. Midpoint redox potentials are given in brackets, (from: Lambers, 1980). 10 and p h y s i o l o g i c a l nature. Using these i n h i b i t o r s i t has been demonstrated that when the cytochrome pathway i s blocked ( i . e . i n the presence of cyanide) the contribution of a l t e r n a t i v e r e s p i r a t i o n increases. This suggests that the two pathways share some common components with the a l t e r n a t i v e pathway providing a "detour" f o r electrons i n the presence of i n h i b i t o r s of cytochrome-mediated r e s p i r a t i o n (Lambers, 1980). Bendall and Bonner (1971) demonstrated that CN-resistant mitochondria possess two oxidases - one being CN-resistant ( a l t e r n a t i v e oxidase) and the other CN-sensitive (cytochrome oxidase). The a l t e r n a t i v e r e s p i r a t o r y pathway was determined to branch from the main re s p i r a t o r y pathway before the b-cytochromes. I t i s now generally accepted that the branch point occurs at ubiquinone (Lambers, 1980, F i g . 1). While the exact nature of the a l t e r n a t i v e oxidase i s not known, current evidence suggests that: 1. i t i s a quinoloxidoreductase ( L a t i e s , 1982), 2. i t s a f f i n i t y f o r oxygen i s about 10 times lower than that of cytochrome oxidase (Solomos, 1977), 3. i t i s non-phosphorylating (Akimenko et al., 1979) and 4. i t s f i n a l end-product i s water (Rich e_t a_l., 1978) 2. The p h y s i o l o g i c a l s i g n i f i c a n c e of a l t e r n a t i v e r e s p i r a t i o n . The wide-spread occurrence of CN-resistant r e s p i r a t i o n has enticed s c i e n t i s t s to search f o r the p h y s i o l o g i c a l s i g n i f i c a n c e of t h i s pathway. Because i t i s non-phosphorylating, oxidative energy can be released i n the form of heat. In Arum spadix t i s s u e , where the capacity of the a l t e r n a t i v e pathway i s 3-4 times that of the 11 cytochrome pathway, the temperature of the spadix t i s s u e may be 20 C higher than ambient temperature when the a l t e r n a t i v e pathway i s f u l l y operative (Lambers, 1980). Meeuse (1975) suggests that t h i s r i s e i n temperature v o l a t i l i z e s compounds which a t t r a c t i n s e c t p o l l i n a t o r s . S i m i l a r i l y , i n Symplocarpus, heat production v i a the a l t e r n a t i v e pathway enables f l o r a l development and p o l l i n a t i o n to occur at sub-freezing temperatures (Meeuse, 1975). Nevertheless a l l t i s s u e s e x h i b i t i n g CN-resistance are not thermogenic. Solomos (1977) suggested that a l t e r n a t i v e r e s p i r a t i o n may be involved i n the c l i m a c t e r i c and ripening process of c e r t a i n f r u i t s . Cyanide was found to be as e f f e c t i v e as ethylene i n i n i t i a t i n g the c l i m a c t e r i c of some f r u i t s . In the same vein, ethylene-induced r e s p i r a t i o n was found to be CN-resistant (Solomos and L a t i e s , 1974). However, upon further examination using s e l e c t i v e i n h i b i t o r s of the a l t e r n a t i v e pathway, i t was found that even at the c l i m a c t e r i c peak, no a l t e r n a t i v e r e s p i r a t i o n was present i n ripening avocados or bananas (Theologis and L a t i e s , 1978b). S i m i l a r i l y , i n f i l t r a t i n g pear f r u i t s with SHAM (salicylhydroxamic acid - an i n h i b i t o r of a l t e r n a t i v e r e s p i r a t i o n ; Schonbaum et a l . , 1971) did not prevent the c l i m a c t e r i c from occurring (Janes and Frenkel, 1978). L a t i e s (1982) concluded that the c l i m a c t e r i c cannot be explained i n terms of the a l t e r n a t i v e pathway and warned against dogmatically assuming that CN-resistance automatically implies the i n vivo functioning of the a l t e r n a t i v e pathway i n the absence of cytochrome i n h i b i t o r s . A l t e r n a t i v e r e s p i r a t i o n has been measured i n the roots of many species. I t has been implicated as having a r o l e i n the regulation 12 of anion uptake, flood tolerance and i n providing a path f o r oxidation of excess substrates when storage capacity i s exceeded ( L a t i e s , 1982). Conclusive evidence supporting these hypotheses has yet to be reported. The a l t e r n a t i v e pathway may have a r o l e i n protecting some species from c h i l l i n g i n j u r y (Yoshida and Tagawa, 1979). When c h i l l -s e n s i t i v e Cornus c a l l u s t i s s u e was subjected to c h i l l i n g s t r e s s , one or more of the components of the cytochrome pathway embedded i n the inner mitochondrial membrane was disrupted, r e s u l t i n g i n the d i v e r s i o n of electrons through the a l t e r n a t i v e pathway. This enabled ATP production to continue, although the r e s p i r a t o r y c o n t r o l r a t i o dropped sharply. 3. Regulation of seed dormancy and a l t e r n a t i v e r e s p i r a t i o n . A l t e r n a t i v e r e s p i r a t i o n has been implicated as being p h y s i o l o g i c a l l y s i g n i f i c a n t i n the regulation of seed dormancy and germination i n several species. Evidence suggesting t h i s r o l e has been c i r c u m s t a n t i a l . Since cyanide and other i n h i b i t o r s of cytochrome-mediated electron transport are i n e f f e c t i v e at completely i n h i b i t i n g seed r e s p i r a t i o n , i t has been suggested that oxygen-uptake i n the presence of these i n h i b i t o r s i s a l t e r n a t i v e (Yentur and Leopold, 1976). I t has been proposed that cyanide increases the f l u x of electrons through the a l t e r n a t i v e pathway i n seeds, promoting germination (Hendricks and Taylorson, 1972). Further evidence suggesting involvement of a l t e r n a t i v e r e s p i r a t i o n i n germination 1 3 and/or release of dormancy has involved the use of inhibitors of this pathway. Yentur and Leopold (1976) found that SHAM inhibited seed germination in several species. Similarily, Yu et a l . (1979) found that SHAM (10 mM) inhibited red light-induced, release of dormancy in lettuce seeds (var. Grand Rapids); KCN i s known to release dormancy in these seeds (Taylorson and Hendricks, 1973). In these studies the hypothesis that electron flux through the alternative pathway i s required for the release of seed dormancy was advanced. Although numerous studies have shown alternative respiration to occur in late or post-germination stages of nondormant seeds (James and Spencer, 1979; Morohashi and Matsushima, 1983; Burguillo and Nicolas, 1977) l i t t l e research has been performed which directly examines i t s occurrence during the release of seed dormancy. The role of cytochrome mediated and alternative respiration in the release of dormancy of cocklebur seeds has however, been examined. Ethylene (Esashi et a l . , 1979b), cyanide (Esashi el: a l . , 1981a) and fluctuating temperature (Esashi et a l . , 1983), a l l of which stimulate germination in dormant cocklebur seeds, evoked either a concomitant increase in alternative respiration or an increase in the alternative/cytochrome respiration ratio. The latter was suggested to regulate cocklebur germination (Esashi et a l . , 1981b). Furthermore, the stimulatory effect of ethylene (which also increases alternative respiration in other systems, Solomos and Laties, 1975) on seed germination in cocklebur was negated by a simultaneous application of SHAM (Esashi et a l . , 1979b). It was proposed that ethylene acted on the seeds to 14 induce the a l t e r n a t i v e pathway which i n turn promoted the release of dormancy. SHAM a p p l i c a t i o n i n h i b i t e d the induced a l t e r n a t i v e r e s p i r a t i o n and thus germination. Azide has been shown to release seed dormancy i n wild oats (Fay and Gorecki, 1978). Upadhyaya et a l . (1982a) found that t h i s release of dormancy could be prevented by a simultaneous a p p l i c a t i o n of SHAM. I t was subsequently found that azide stimulated seed r e s p i r a t i o n well i n advance of germination and concurrent treatment of seeds with SHAM resulted i n an almost complete i n h i b i t i o n of r e s p i r a t i o n (Upadhyaya e_t a l . , 1983). The authors suggested that SHAM-sensitive ( a l t e r n a t i v e ) r e s p i r a t i o n may be associated with and may be necessary f o r the stimulation of germination by azide. Conclusive evidence supporting such an involvement of a l t e r n a t i v e r e s p i r a t i o n has not yet been presented. E. SECONDARY SEED DORMANCY. Secondary dormancy can be induced i n seeds of several species by a var i e t y of methods. For example, secondary dormancy has been induced by far - r e d l i g h t i n le t t u c e (Vidavar and Hsiao, 1975), elevated temperatures i n common ragweed (Baskin and Baskin, 1980), darkness i n Lamium amplexicaule (Taylorson and Hendricks, 1976) and anaerobiosis i n cocklebur (Esashi et a l . , 1978) and wild oats (Hay and Cumming, 1959) seeds. Very l i t t l e i s known about the regulation of secondary seed dormancy or how i t compares p h y s i o l o g i c a l l y with primary dormancy. 15 Esashi e_t a l . (1981b) have proposed that secondary dormancy i n cocklebur seeds r e s u l t s from t h e i r i n a b i l i t y to perform a l t e r n a t i v e r e s p i r a t i o n . While secondarily dormant seeds i n cocklebur were not responsive to cyanide alone, i t s a p p l i c a t i o n following pretreatment with ethylene increased both germination and the alterative/cytochrome r e s p i r a t i o n r a t i o . I t was suggested that cyanide was i n e f f e c t i v e at rel e a s i n g dormancy since the seeds were unable to perform a l t e r n a t i v e r e s p i r a t i o n . When ethylene was applied p r i o r to cyanide, the a l t e r n a t i v e pathway was induced, which was then promoted by subsequent cyanide treatment r e s u l t i n g i n germination. In wild oats, compounds such as n i t r a t e , peroxide and GA^ are known to promote germination of secondarily dormant seeds (Hay and Cumming, 1959). Since the work of Hay and Cumming (1959), several g e n e t i c a l l y pure l i n e s of wild oats d i f f e r i n g i n t h e i r dormancy behavior have been characterized and used i n the i n v e s t i g a t i o n of primary dormancy (Fuerst et a l . , 1983; Sawhney and Naylor, 1980; Naylor and Fedec, 1978). Whether these l i n e s d i f f e r i n t h e i r a b i l i t y to acquire secondary dormancy i s not known. Furthermore, several a d d i t i o n a l chemicals have been i d e n t i f i e d which stimulate germination i n seed of several weeds including wild oats (Adkins et a l . , 1984a,b; Esashi et a l . , 1978; Fay and Gorecki, 1978; Roberts and Smith, 1977; Upadhyaya et^ j^L., 1982a). These stimulants and pure l i n e s can be useful t o o l s i n the i n v e s t i g a t i o n of the induction and release of secondary dormancy i n wild oats. 16 F. Objectives. The objectives of t h i s study were to determine: 1. i f g e n e t i c a l l y pure l i n e s of wild oats d i f f e r with regard to the induction of secondary dormancy. 2. how temperature a f f e c t s the induction of secondary dormancy. 3. whether primary and secondary dormancies d i f f e r i n response to a va r i e t y of unrelated germination stimulants. 4. the involvement of a l t e r n a t i v e (SHAM-sensitive) r e s p i r a t i o n i n the induction and release of secondary dormancy. 5. the s i m i l a r i t i e s and differences of azide and cyanide-induced r e s p i r a t i o n and germination. 6. the r e l a t i o n s h i p between cyanide-induced seed r e s p i r a t i o n and the release of dormancy by t h i s compound. 7. the possible involvement of a l t e r n a t i v e r e s p i r a t i o n i n the stimulation of r e s p i r a t i o n and germination by azide and cyanide. 8. the e f f e c t s of pH on the ac t i o n of re s p i r a t o r y i n h i b i t o r s on wild oats seeds. 17 MATERIALS AND METHODS A. Seed source. In order to minimize extraneous v a r i a t i o n i n seed dormancy behavior, g e n t i c a l l y pure l i n e s of wild oats were used i n a l l experiments. Two dormant (AN-51 and Mont 73) and two nondormant (SH-430 and CS-40) l i n e s were used. Under our conditions, AN-51 and Mont 73 remained dormant for 6-12 months a f t e r harvest while SH-430 and CS-40 germinated at a rate of 100% a f t e r 2-3 weeks of a f t e r -ripening. Environmental factors (temperature and moisture l e v e l s ) are known to e f f e c t the development of seed dormancy during seed maturation (Sawhney and Naylor, 1979, 1980). For t h i s reason a l l plants raised f o r seed production were grown under c o n t r o l l e d conditions i n growth cabinets. Plants were grown i n 2.7 L pots (3 plants/pot) i n a 4:1 s o i l : p e a t mix (with 20-20-20 f e r t i l i z e r ) at a _2 constant temperature of 20 C and a 16 h photoperiod with 200 W m l i g h t i n t e n s i t y (Sylvania, VHO, cool-white fluorescent tubes supplemented with incandescent bulbs). Plants were f e r t i l i z e d with 100 mL p o t - 1 of 3 g L - 1 Peters 20:20:20 (%NPK plus Mg, Fe, Mn, Zn, Cu, B and Mo) f e r t i l i z e r at the f l a g - l e a f stage. Seeds were harvested at maturity and used e i t h e r within 4-8 weeks or were allowed to a f t e r -ripen f o r 1-2 years i n the dark at about 25 C. Seeds were dehulled p r i o r to use i n a l l experiments. 18 B. The induction of secondary dormancy. Secondary dormancy was induced by immersing after-ripened seeds i n deaerated water (approximately 50 uM dissolved oxygen) i n sealed Erlenmeyer f l a s k s at 25 C i n the dark for various durations (as s p e c i f i e d ) . Deaeration was accomplished by b o i l i n g d i s t i l l e d water and allowing i t to cool to the desired temperature i n an a i r - s e a l e d system which permitted the intake of boiled water to f i l l the a i r space produced on cooling. In experiments inv o l v i n g the e f f e c t of anaerobiosis temperature on the induction of secondary dormancy, dormancy was induced at 15, 20, 25 and 30 C. Upon completion of anaerobiosis, seeds were removed from the f l a s k s and t h e i r germination monitored under aerobic conditions (25 C, i n darkness) i n 10 cm P e t r i dishes (10 seeds/dish) l i n e d with two Whatman No. 1 f i l t e r disks wetted with 5 mL of water. Seeds were treated with Captan (50% wettable powder) p r i o r to placement i n the dishes. Seeds used to investigate the e f f e c t of pre-imbibition on the induction of secondary dormancy were imbibed f o r the s p e c i f i e d periods under aerobic conditions (at 25 C) and then transfered to Erlenmeyer f l a s k s f o r anaerobiosis. C. Germination studies. Seeds were placed embryo side-up i n 10 cm P e t r i plates (10 seeds/plate) l i n e d with two Whatman No. 1 f i l t e r disks wetted with 19 5 mL water or the indicated treatment s o l u t i o n . Chemicals were dissolved i n d i s t i l l e d water or buffer (50 mM sodium phosphate or citrate-phosphate buffer) of s p e c i f i e d pH. Buffers were prepared according to Gomori (1955) and d i l u t e d four f o l d ; the buffering capacity was found to be adequate for a l l experiments. In experiments where KCN was used, P e t r i plates were sealed with a s t r i p of Parafilm to minimize the escape of cyanide as gaseous HCN. Plates were stored i n a saturated atmosphere at 25 C i n the dark. Germination (protrusion of the coleorhiza through the testa) was recorded at s p e c i f i e d i n t e r v a l s . Seed v i a b i l i t y was determined by percentage germination on addition of 1 mL of 1.4 mM GA^ per dish at the completion of the experiment. Seed v i a b i l i t y generally exceeded 90%. Experiments were performed with ei t h e r 3 or 4 r e p l i c a t e s . D. Oxygen uptake measurements. 1. Intact seeds: Oxygen-uptake was monitored polarographically using a Clark-type oxygen electrode and a YSI Model 30 oxygen meter (Yellow Springs Instruments Co., Inc., Yellow Springs, Ohio). Upon completion of the incubation treatment i n P e t r i dishes, seeds were rinsed three times with d i s t i l l e d water suspended i n 5 mL of oxygen-saturated phosphate buffer (50 mM, pH 6.9) and oxygen consumption was recorded for 7-10 minutes. Two or 3 r e p l i c a t e s (of 10 seeds each) were used i n each experiment. 20 2. Excised embryos i . Oxygen uptake: Embryo sections (embryo and adhering pericarp and testa) were excised from seed imbibed on water under a d i s s e c t i n g microscope. Excised embryos were gently vortexed to remove adhereing starch grains and then suspended i n 3 mL phosphate buffer (50 mM, pH 6.9) with or without 5 mM SHAM. To determine res p i r a t o r y components, 10 /iL of concentrated NaN^ or KCN s o l u t i o n was i n j e c t e d d i r e c t l y i n t o the oxygen uptake vessel (to obtain desired i n h i b i t o r concentration) a f t e r a l i n e a r rate of oxygen consumption had been established. A l l experiments were performed using 3 r e p l i c a t e s of 10 embryos each. i i . C a l c u l a t i o n of res p i r a t o r y components: Respiratory components were determined from oxygen uptake data by the method of Theologis and La t i e s (1978a) f o r i n t a c t t i s s u e . This method has been adapted from the analysis of res p i r a t o r y components for i s o l a t e d mitochondria (Bahr and Bonner, 1973). Here the r e s p i r a t i o n i s described by the equation: Eq. 1. V t= p.(gi) + V( cyt + V. res where: V t V _ cyt t o t a l r e s p i r a t i o n rate = cytochrome-mediated r e s p i r a t i o n 21 (gi) = maximal possible contribution of the a l t e r n a t i v e pathway p = the f r a c t i o n of the a l t e r n a t i v e pathway which i s operative at any given time and therefore p(gi) represents the actual rate of a l t e r n a t i v e r e s p i r a t i o n (^ a^ t) V = r e s i d u a l r e s p i r a t i o n (that portion of t o t a l r e s p i r a t i o n that i s i n s e n s i t i v e to both i n h i b i t o r s of cytochrome-mediated and a l t e r n a t i v e r e s p i r a t i o n ) . Respiratory components can be determined i n both the presence and the absence of cytochrome oxidase i n h i b i t o r s - i n both cases the in t e r p r e t a t i o n of the r e l a t i v e contribution of each resp i r a t o r y component i s d i f f e r e n t . In the absence of cytochrome i n h i b i t o r s : V = t o t a l oxygen consumption va ^ t = V - oxygen consumption i n the presence of SHAM V = oxygen consumption i n the presence of both res SHAM and cyanide/azide vc y t = oxygen consumption i n the presence of SHAM - ^ r e s In the absence of i n h i b i t o r s of cytochrome oxidase represents the actual contribution of the cytochrome pathway while Valt represents the actual (engaged) contribution of the a l t e r n a t i v e pathway. In the presence of cytochrome i n h i b i t o r s : V = t o t a l oxygen consumption V n = oxygen consumption i n the presence of KCN - V 22 In the presence of cytochrome oxidase i n h i b i t o r s , V ^ represents the po t e n t i a l capacity of the a l t e r n a t i v e pathway which may or may not be engaged when the cytochrome pathway i s not blocked. E. S t a t i s i c a l analysis and graphics. A l l experiments were repeated two or more times with s i m i l a r r e s u l t s . Analysis of variance was performed f o r each experiment (U.B.C. MFAV). Student-Newman-Keul's multiple range test (SNK MRT) and l e a s t s i g n i f i c a n t d i f f e r e n c e (LSD) values were computed (U.B.C. MFAV and Completely Randomized and Randomized Complete Block Analysis of Variance program by D.C. K o l l e r and N.R. Knowles, respectively) for convenient mean separation when the F-value for the main e f f e c t of treatment was s i g n i f i c a n t . 23 RESULTS A. The induction and release of secondary dormancy 1. Characterization of the induction of secondary dormancy i n pure l i n e s . Anaerobiosis induced secondary dormancy i n after-ripened seeds of dormant l i n e s (AN-51 and Mont 73, F i g . 2). The optimum duration of anaerobiosis ( i . e . days of anaerobiosis f or maximum induction of dormancy) varied from 1 to 4 days f or AN-51 and from 4 to 10 days f or Mont 73; shorter periods were l e s s e f f e c t i v e whereas longer periods released secondary dormancy ( F i g . 2). The maximum dormancy induced was c o n s i s t e n t l y greater than 85% i n l i n e AN-51, but varied s u b s t a n t i a l l y f o r Mont 73. In general, secondary dormancy could be induced more e f f e c t i v e l y i n l i n e AN-51 than i n Mont 73. Pre-imbibition of after-ripened seeds of l i n e AN-51 under aerobic conditions a f f e c t e d t h e i r response to subsequent anaerobic treatment. With up to 4 h of pre-imbibition, secondary dormancy could be induced i n 85% of the seeds ( F i g . 3). Dormancy was induced to a le s s e r extent with longer (4 to 12 h) pre-imbibition. When seeds were imbibed a e r o b i c a l l y f o r longer than 12 h, v i s i b l e germination occurred p r i o r to and during anaerobiosis. Anaerobic treatment of these seeds s i g n i f i c a n t l y affected t h e i r a b i l i t y to develop i n t o seedlings (>1 cm c o l e o p t i l e and r a d i c l e ) and reduced t h e i r v i a b i l i t y ( F i g . 3). In contrast to the response of dormant l i n e s , seeds of nondormant l i n e s (CS-40, SH-430) developed a short dormancy (2 to 3 weeks; F i g . 24 0 | i 1 1 1 1 1 1 0 2 4 6 8 10 12 14 D A Y S O F A N A E R O B I O S I S Fig. 2 The effect of anaerobiosis duration on the induction of secondary dormancy in after-ripened seeds of dormant lines AN-51 and Mont 73. Final germination was recorded 30 days after the completion of anaerobiosis. Values are the means of 4 replicates. F-value for interaction of line X time was significant at the p= 0.05 level. 25 0 5 10 15 20 HOURS OF PREIMBIBITION F i g . 3 Induction of secondary dormancy i n a e r o b i c a l l y pre-imbibed after-ripened seeds of l i n e AN-51. A . % germination immediately before anaerobiosis (LSD = 6%)ix. t % germination immediately a f t e r anaerobiosis (LSD = 2%); • , % germination 24 days a f t e r anaerobiosis; H , % seedling establishment (LSD = 29%). Seeds were imbibed under aerobic conditions for the s p e c i f i e d period and then subjected to 3 day anaerobiosis. Seedlings establishment r e f e r s to germinated seeds with a c o l e o p t i l e and r a d i c l e >1 cm. 26 4). Secondarily dormant seeds of dormant l i n e s did not germinate for up to 10 months i n an imbibed state (at 25 C, data not shown). A f t e r -ripened seeds of these l i n e s , not having received anaerobic treatment, gave germination rates of 100% i n l e s s than 3 days when imbibed under aerobic conditions. In contrast, secondarily dormant seeds of nondormant l i n e s showed a rapid increase i n germination 1 to 2 weeks a f t e r completion of anaerobiosis ( F i g . 4). This increase always occurred e a r l i e r i n l i n e CS-40 than i n l i n e SH-430 ( F i g . 4) and was not affected by the presence of 3 mM SHAM (Tab. 1). The optimum period of anaerobiosis for both nondormant l i n e s varied between 4 to 10 days (data not shown). Secondarily dormant seeds of l i n e AN-51 after-ripened r a p i d l y during dry storage; germination increased from zero to > 90% over 22 weeks of dry storage ( F i g . 5). 2. The e f f e c t of temperature and seed age on the induction of secondary dormancy. Temperature during anaerobiosis s i g n i f i c a n t l y affected the induction of secondary dormancy i n l i n e s Mont 73, SH-430 and CS-40; higher temperatures (25 and 30 C) being more e f f e c t i v e at inducing dormancy (Tab. 2). In Mont 73 t h i s e f f e c t was evident only with short (1 day) anaerobiosis (Tab. 2); with a 3 day anaerobiosis treatment temperature did not a f f e c t the induction of dormancy i n dormant l i n e s (data not shown). 27 Fig. 4 The induction of secondary dormancy in genetically nondormant seeds of lines SH-430 and CS-40. Seeds were subjected to 4 day anaerobiosis. Germination was then monitored under aerobic conditions. Values are the means of 4 replicates. Bars represent the LSD (0.05) at 9 and 13 days after anaerobiosis. 28 Table 1. E f f e c t of SHAM on the release of secondary dormancy i n l i n e SH-430. Treatment Percentage germination  7 days 14 days 21 days Water con t r o l 20a 40b 50c SHAM, 3 mM 23a 35b 45c Secondary dormancy was induced by a 3-day anaerobiosis treatment; percent germination was recorded at 7, 14 and 21 days thereafter. Each value i s the mean of four r e p l i c a t e s . Values within columns and rows that are 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 at the p= 0.05 l e v e l according the the Student Newman Keul's MRT. F-value f o r diff e r e n c e from c o n t r o l was not s i g n i f i c a n t at the p= 0.05 l e v e l . 29 WEEKS OF AFTER—RIPENING F i g . 5 A f t e r - r i p e n i n g of secondarily dormant seeds of l i n e AN-51 during dry storage. After-ripened seeds were subjected to four day anaerobiosis, a i r - d r i e d and stored at room temperature. Germination was tested- at the indicated i n t e r v a l s . Values represent the means of 4 r e p l i c a t e s . 30 Table 2. E f f e c t of anaerobiosis temperature on the induction of secondary dormancy i n seeds of nondormant (SH-430, CS-40) and after-ripened seeds of dormant (AN-51, Mont 73) l i n e s . Percentage germination Anaerobiosis Experiment I Experiment II temperature (°C) AN-51 Mont 73 SH-430 CS-40 15 18a 55b 85a 88a 20 10a 45b 50b 50b 25 8a 15a 10c 18c 30 5a 0a 13c 18c Line * ns Temperature * * Line X Temperature * ns F-value was s i g n i f i c a n t at the p= 0.05 l e v e l , ns - not s i g n i f i c a n t . Secondary dormancy was induced by 3-day anaerobiosis i n the nondormant and 1-day anaerobiosis i n the dormant l i n e s ; germination was recorded at 6 and 25 days a f t e r anaerobiosis i n nondormant and dormant l i n e s , r e s p e c t i v e l y . Each value i s the mean of four r e p l i c a t e s ; values within columns and rows that are 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 at the p= 0.05 l e v e l according to the SNK MRT. 31 Seed age had a pronounced e f f e c t on the induction of secondary dormancy i n a nondormant l i n e . Secondary dormancy i n SH-430 could be induced only i n seeds stored f o r l e s s than 2 months. Older seeds f a i l e d to acquire secondary dormancy under these conditions (Tab. 3). Seeds used i n t h i s experiment germinated 100% within 3 days when not subjected to anaerobiosis. On the other hand, seeds of dormant l i n e s were able to acquire secondary dormancy even a f t e r 2 years of a f t e r -ripening. 3. Stimulation of germination of secondarily dormant seeds. Ethanol (175 mM), k i n e t i n (1 mM), isopentenyl adenine (1 mM), potassium n i t r a t e (100 mM) and sodium azide (1 mM) stimulated germination (by >85%) i n secondarily dormant seeds of l i n e AN-51 (Tab. 4). With the exception of azide, SHAM did not i n h i b i t the promotion of germination by these stimulants (Tab. 4). Germination was increased with SHAM alone and reduced i n the presence of azide and SHAM. 4. Determination of resp i r a t o r y components. Azide and cyanide were compared f or t h e i r r e l a t i v e effectiveness as i n h i b i t o r s of cytochrome-mediated r e s p i r a t i o n i n excised embryos. Maximal i n h i b i t i o n of r e s p i r a t i o n was greater with cyanide (65%) than with azide (15%) ( F i g . 6 and 7). Cyanide was also e f f e c t i v e at lower concentrations than azide; maximal i n h i b i t i o n was achieved with 0.5 mM cyanide ( F i g 6.), whereas 1.5 mM azide ( F i g . 7) was required to e l i c i t 32 Table 3. E f f e c t of seed age on the induction of secondary dormancy i n nondormant l i n e SH-430. Seed age Percent germination 5 days 10 days 15 days 5 weeks Oa 23a 65a 7 weeks 20b 70b 100b 4 months 100c 100b 100b 7 months 98c 98b 100b 14 months 100c 100b 100b Secondary dormancy was induced by 5-day anaerobiosis. Each value i s the mean of four r e p l i c a t e s ; values within columns 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 at the p= 0.05 l e v e l according to the SNK MRT. The F-values f o r seed age and observation time were s i g n i f i c a n t at the p= 0.05 l e v e l ; the i n t e r a c t i o n was not s i g n i f i c a n t . 33 Table 4. Stimulation of germination of secondarily dormant seeds ( l i n e AN-51) by chemicals known to break primary dormancy. Percent germination Treatment - SHAM + SHAM (3 mM) Control 13a 30b Ki n e t i n (1 mM) lOOd lOOd Isopentenyl adenine (1 mM) lOOd lOOd Sodium azide (1 mM) 98d 50c Potassium n i t r a t e (100 mM) 93d 90d Ethanol (175 mM) 85d 98d Germination was recorded 12 days a f t e r the onset of treatment. Each value i s the mean of four r e p l i c a t e s . Values within columns and rows 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 at the p= 0.05 l e v e l according to the SNK MRT. F-values f o r the main e f f e c t s of germination stimulants, +/- SHAM and t h e i r i n t e r a c t i o n were s i g n i f i c a n t at the p= 0.05 l e v e l . 34 F i g . 6 The e f f e c t of cyanide on the r e s p i r a t i o n of excised embryos ( l i n e AN-51). Embryos were excised from seeds imbibed on water for two hours. KCN was injected d i r e c t l y into the oxygen-uptake vessel a f t e r a l i n e a r rate of oxygen consumption had been recorded. Values represent the means of 3 r e p l i c a t e s . 35 15 F i g . 7 The e f f e c t of azide on the r e s p i r a t i o n of excised embryos ( l i n e AN-51). Embryos were excised from seeds imbibed on water for two hours. Azide was inje c t e d d i r e c t l y into the oxygen-uptake vessel a f t e r a l i n e a r rate of oxygen consumption had been recorded. Values represent the means of 3 r e p l i c a t e s . 36 maximum re s p i r a t o r y i n h i b i t i o n . At low concentrations (0.25 - 1.25 mM) azide stimulated embryo r e s p i r a t i o n by 7 to 12 %. Consequently, cyanide was used to i n h i b i t cytochrome-mediated r e s p i r a t i o n i n subsequent experiments involving excised embryos. SHAM alone did not i n h i b i t oxygen uptake of excised embryos or whole seeds (Tab. 5). I t did, however, i n h i b i t the r e s p i r a t i o n of excised embryos (by 35 to 50%) i n the presence of cyanide (Tab. 5). SHAM (5 and 10 mM) was not e f f e c t i v e at blocking oxygen uptake i n the presence of 1 mM cyanide i n i n t a c t seeds (Tab. 5). 5. Respiratory components of embryos excised from after-ripened, primarily and secondarily dormant seeds. Relative contributions of CN-sensitive and SHAM-sensitive ( a l t e r n a t i v e ) pathways to t o t a l r e s p i r a t i o n of embryos excised from nongerminated seeds (after-ripened, primarily dormant and secondarily dormant) were determined. The induction of secondary dormancy had no e f f e c t on the t o t a l r e s p i r a t i o n of embryos excised from secondarily dormant seeds (Tab. 6). Primarily dormant seeds had higher t o t a l r e s p i r a t i o n , almost a l l of which was cytochrome-mediated (Tab. 6). Cyanide (1 mM) s u b s t a n t i a l l y i n h i b i t e d r e s p i r a t i o n (by 44 to 85%) i n a l l cases (Tab. 6), SHAM alone had no s i g n i f i c a n t e f f e c t on oxygen-uptake regardless of dormancy status (Tab. 6). Oxygen-uptake i n the presence of cyanide was greater i n secondarily dormant and a f t e r -ripened seeds than i n primarily dormant seeds (Tab. 6). Residual r e s p i r a t i o n was s i m i l a r i n a l l cases (12-16% of t o t a l r e s p i r a t i o n -Tab. 6). 37 Table 5. I n h i b i t i o n of seed and embryo r e s p i r a t i o n by 1 mM cyanide i n the presence or absence of SHAM. Oxygen uptake Oxygen uptake (nmol min ^  seed ^)  Seed Embryo i n i t i a l +KCN i n i t i a l +KCN Buffer c o n t r o l 0.53a 0 .26b (51) 1 0.32a 0.14b (56) 5 mM SHAM 0.54a 0 .24b (56) 0.28a 0.07c (75) 10 mM SHAM 0.53a 0 .23b (57) 0.30a 0.09c (70) SHAM ns ns KCN * * SHAM X KCN ns * F-value s i g n i f i c a n t at the p= 0.05 l e v e l , ns - not s i g n i f i c a n t . Values i n brackets represent % i n h i b i t i o n compared to i n i t i a l oxygen uptake. Seeds were imbibed on water for 24 h. Embryos were excised from seeds imbibed on water f o r 2 h. Oxygen uptake was determined i n 50 mM phosphate buffer (pH 6.9) with or without SHAM. After a l i n e a r rate of oxygen uptake had been established 10 uL of concentrated KCN sol u t i o n was in j e c t e d d i r e c t l y i n t o the oxygen uptake vessel to achieve 1 mM KCN concentration and subsequent oxygen uptake monitored. Values are the means of three r e p l i c a t e s . Means 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 at the p= 0.05 l e v e l according to the SNK MRT. 38 Table 6. E f f e c t of resp i r a t o r y i n h i b i t o r s on the oxygen uptake of embryos excised from after-ripened, primarily and secondarily dormant seeds of l i n e AN-51. Oxygen uptake (nmol embryo ^ min ^ )  Dormancy Buffer KCN SHAM KCN + SHAM V _ V , 1 V , 2 J cyt a l t a l t status only (V ) (1) (2) (3) (4) (3 - 4) (1 - 3) (2 - 4) After-ripened 0.18a 0.07c 0.18a 0.02d 0.16 0 0.05 Secondarily dormant 0.18a 0.10c 0.16a 0.03d 0.13 0 0.07 Primarily dormant 0.27b 0.04d 0.28b 0.03d 0.25 0 0.01 Engaged ( i n the absence of KCN); p o t e n t i a l ( i n the presence of KCN). Inh i b i t o r concentrations were 1 mM KCN and 5 mM SHAM. Intact seeds were imbibed f or 2 h on water p r i o r to embryo exc i s i o n . Oxygen uptake was determined i n 50 mM phosphate buffer with or without SHAM. KCN was in j e c t e d d i r e c t l y into the oxygen uptake vessel and subsequent oxygen uptake recorded. Values are the means of 3 r e p l i c a t e s . Means within columns and rows 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 at the p= 0.05 l e v e l according to the SNK MRT. F-values for the main e f f e c t s of dormancy status, i n h i b i t o r treatment and t h e i r i n t e r a c t i o n were s i g n i f i c a n t at the p= 0.05 l e v e l . 39 B. E f f e c t s of re s p i r a t o r y i n h i b i t o r s on seed germination 1. The release of primary and secondary dormancy by azide and cyanide Experiments comparing the release of primary and secondary dormancy by the cytochrome oxidase i n h i b i t o r s cyanide and azide were performed. Cyanide e f f e c t i v e l y promoted the release of primary dormancy at concentrations ranging from 0.3 to 3 mM, with 0.5 and 1.0 mM giving the highest germination percentages (Tab. 7). Germination stimulated by 1 and 3 mM cyanide was delayed several days ( c f . germination stimulated by 0.5 mM cyanide) (Tab. 7). I t was necessary to s e a l P e t r i plates containing cyanide with Parafilm to retard l o s s of HCN gas. No stimulation of germination occurred when cyanide (0.5-10.0 mM) was applied i n unsealed dishes (data not shown). To determine the optimal duration of azide and cyanide treatment for breaking dormancy, i n h i b i t o r treatments were given for d i f f e r e n t durations and germination was monitored. The r e s u l t i n g curve f o r azide was quadratic ( F i g . 8). In t h i s experiment maximum release of dormancy occurred a f t e r 4-12 h azide treatment ( F i g . 8). Although longer ( i . e . 16-24 h) treatments were l e s s e f f e c t i v e at releasing dormancy, continuous azide treatment broke dormancy to a s i m i l a r extent as the treatment for optimum duration (4 to 12 h, Fig.8) but germination was delayed up to 8 days (Tab. 8). Cyanide treatments of d i f f e r e n t duration gave a s i m i l a r dormancy release pattern ( F i g . 9) as found with azide ( F i g . 8). Four to 8 hour cyanide treatment resulted i n maximum release of dormancy with the effec t i v e n e s s of treatment d e c l i n i n g (although to a l e s s e r extent than with azide) with longer pulse durations ( F i g . 9) . S i m i l a r i l y maximum 40 Table 7. The e f f e c t of cyanide on percentage and rate of germination i n p r i m a r i l y dormant seeds of l i n e AN-51. Cyanide concentration Percent germination (mM) 2 day 3 day 5 day 0 0a 0a Oa 0.10 0a 0a Oa 0.30 5a 23ab 23ab 0.50 48b 68c 68c 1.0 8a 54c 78c 3.0 0a Oa 38b Seeds were treated with the indicated concentrations of cyanide; germination was recorded 2, 3 and 5 days a f t e r the onset of treatment. Values represent the means of four r e p l i c a t e s . Means within columns followed by the same l e t t e r do not d i f f e r at the p= 0.05 l e v e l according to the SNK MRT. F-values f or the main e f f e c t s of cyanide treatment, observation time and t h e i r i n t e r a c t i o n were s i g n i f i c a n t at the p= 0.05 l e v e l . 41 F i g . 8 E f f e c t of duration of azide treatment on the release of primary dormancy i n seeds of l i n e AN-51. Seeds were treated with 1 mM azide f o r the indicated duration and then transferred to d i s t i l l e d water for the remainder of the experiment. Germination was recorded 14 days a f t e r azide treatment. Values represent the means of 4 r e p l i c a t e s . 42 Table 8. The e f f e c t of duration of azide (1 mM) treatment on the percentage and the rate of germination. Treatment Percentage germination duration (h) 1 day 2 day 4 day 8 day 0 8a 20a 20a 20a 4 63b 95b 95b 95b 8 43c 88b 88b 88b 16 Oa 58c 58c 58c Continuous Oa Oa Oa 93b Primarily dormant seeds of l i n e AN-51 were treated with azide for the s p e c i f i e d period, transferred to d i s t i l l e d water and germination recorded at the indicated i n t e r v a l s . Values are the mean of four r e p l i c a t e s . Means within columns 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 at the p= 0.05 l e v e l according to the SNK MRT. F-values for the main e f f e c t s of azide treatment, observation time and t h e i r i n t e r a c t i o n were s i g n i f i c a n t at the p= 0.05 l e v e l . 43 F i g . 9 E f f e c t of duration of cyanide treatment on the release of primary dormancy i n seeds of l i n e AN-51. Seeds (4 r e p l i c a t e s ) were treated with 1 mM cyanide f o r the indicated duration and then transferred to d i s t i l l e d water for the remainder of the experiment. Germination was recorded 14 days a f t e r cyanide treatment. Values represent the means of 4 r e p l i c a t e s . 44 release of dormancy occurred with continuous 1 mM cyanide treatment (Tab. 7) but, unlike azide, germination was much quicker (Tab. 8). Although 1 mM azide e f f e c t i v e l y released both primary and secondary dormancy i n seeds after-ripened f o r 3 months i n one experiment ( F i g . 8), i t was i n e f f e c t i v e at rele a s i n g seed dormancy i n 2 to 5 month old seeds of AN-51 when applied continuously or as a pulse treatment i n a second experiment (Tab. 9). On the other hand, 0.5 mM cyanide stimulated seed germination regardless of the duration of a f t e r - r i p e n i n g although to d i f f e r e n t extents (Tab. 9). 2. The e f f e c t of SHAM on the release of dormancy by cyanide and azide. SHAM (10 mM) applied simultaneously with 1 mM azide, completely i n h i b i t e d germination of secondarily dormant seeds (Tab. 10). In order f o r SHAM to prevent the release of secondary dormancy by a 6 h azide pulse treatment i t was necessary f o r SHAM to be present co n t i n u a l l y ( i . e . during and a f t e r the 6 h pulse; Tab. 10). SHAM applied subsequent to the azide pulse had l i t t l e e f f e c t on the release of dormancy; likewise, when present only during the 6 h azide pulse, SHAM f a i l e d to i n h i b i t azide-induced germination. SHAM applied simultaneously with cyanide had no e f f e c t on the stimulation of germination by the l a t t e r compound (Tab. 10). 45 Table 9. E f f e c t of a f t e r - r i p e n i n g on the release of dormancy by azide and cyanide i n l i n e AN-51. Treatment Months af t e r - r i p e n i n g 2 3 5 Control * 5a Oa 20d Cyanide 70b 40c 75b Azide Oa Oa Oa Azide - 6 h pulse Oa Oa Oa * Percent germination. Seeds of the indicated ages were treated with ei t h e r continuous 0.5 mM KCN, 1.0 mM azide or a 6 h pulse of 1.0 mM azide followed by water imbi b i t i o n . F i n a l germination percentages were recorded 14 days a f t e r treatment. Values are the mean of four r e p l i c a t e s . Means within columns and rows 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 at the p= 0.05 l e v e l according to the SNK MRT. F-values for the main e f f e c t s of seed age, i n h i b i t o r treatment and t h e i r i n t e r a c t i o n were s i g n i f i c a n t at the p= 0.05 l e v e l . 46 Table 10. The e f f e c t of SHAM on the release of secondary dormancy by azide and cyanide. Treatment Percent germination Control 7a SHAM 7a Azide 87b Cyanide 77b Azide + SHAM Oa Cyanide + SHAM 83b 6 h Azide — > water 97b 6 h Azide — > SHAM 70b 6 h Azide + SHAM — > water 90b 6 h Azide + SHAM — > SHAM 15a Six h i n h i b i t o r treatment was followed by transfe r (—>) to water or SHAM for the remainder of the experiment. Germination was recorded 14 days a f t e r the onset of treatment. I n h i b i t o r concentrations used were 1 mM azide, 0.5 mM cyanide and 10 mM SHAM. Values are the means of 4 r e p l i c a t e s . Means 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 at the p= 0.05 l e v e l according to the SNK MRT. 47 C. E f f e c t of re s p i r a t o r y i n h i b i t o r s on seed germination and oxygen uptake. 1. Stimulation of seed germination and r e s p i r a t i o n by azide and cyanide. Cyanide stimulated seed r e s p i r a t i o n to an extent s i m i l a r to azide over the same treatment period (Tab. 11). Cyanide concentrations ranging from 0.05 mM to 1.0 mM gave comparable release of dormancy ( F i g . 10). Oxygen-uptake increased l i n e a r l y with increasing log cyanide concentration with maximum stimulation occurring at 1 mM cyanide. Respiration dropped sharply at 3 mM cyanide and germination was completely i n h i b i t e d at t h i s concentration ( F i g . 10). Short cyanide pulses (2 and 4 h) stimulated r e s p i r a t i o n to a si m i l a r extent (33 and 45% over the c o n t r o l , r e s p e c t i v e l y - F i g . 11). Germination was stimulated by both treatments and did not d i f f e r according to the SNK MRT. Shorter pulse treatments (30 and 60 min) stimulated r e s p i r a t i o n by 20 to 25% but did not release dormancy (Tab. 12). 2. The nature of azide and cyanide-induced r e s p i r a t i o n . Azide and cyanide stimulated oxygen uptake to a s i m i l a r extent (77-79%) when applied continuously (Tab. 11). SHAM (10 mM) applied concurrently not only prevented the stimulation of r e s p i r a t i o n by azide but also reduced i t f a r below that of the cont r o l (by 68%); SHAM had no e f f e c t on the stimulation of r e s p i r a t i o n by cyanide (Tab. 11). Removing seeds from azide and cyanide a f t e r 24 h and placing them on water for a subsequent 14 h resulted i n lower oxygen uptake than the seeds re c e i v i n g continuous cyanide or azide ( i . e . 50-58% above the 48 F i g . 10. E f f e c t of cyanide on r e s p i r a t i o n and germination of primarily dormant seeds of l i n e AN-51. Oxygen uptake of i n t a c t seeds was recorded 24 h following KCN treatment. Control seeds did, not .germinate and had a respi r a t o r y rate of 0.60 nmol min" seed . Germination was recorded 5 days a f t e r the onset of treatment. Values represent the means of 3 r e p l i c a t e s . 49 HOURS AFTER THE ONSET OF KCN TREATMENT F i g . 11 E f f e c t of cyanide pulse treatments on the r e s p i r a t i o n and germination of primarily dormant seeds of l i n e AN-51. Seeds were treated with 1 mM KCN for 2 or 4 h and then transferred to water; oxygen-uptake was then recorded at the s p e c i f i e d i n t e r v a l s . Germination (recorded 5 days a f t e r the i n t i a l treatment) was: c o n t r o l , 0%; 2 h pulse, 66% ; 4 h pulse, 87%. Values represent the means of 3 r e p l i c a t e s . 50 Table 11. E f f e c t of SHAM on the induction of r e s p i r a t i o n by 24 h azide or cyanide treatment i n primarily dormant seeds of l i n e AN-51. Treatment Oxygen uptake (nmol min ^  seed ^) Percent of co n t r o l Control 0.56a 100 Cyanide 1.00b 179 Azide 1.01b 180 SHAM 0.60a 107 Cyanide + SHAM 1.09b 195 Azide + SHAM 0.18c 32 Inh i b i t o r concentrations used were 1 mM azide and cyanide and 10 mM SHAM. Values are the means of three r e p l i c a t e s . Means 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 at the p= 0.05 l e v e l according to the SNK MRT. 51 Table 12. The e f f e c t of short cyanide pulse treatments on r e s p i r a t i o n and germination i n primarily dormant seeds of l i n e AN-51. Pulse duration Oxygen uptake % Over % Germination (min) (nmol min * seed-''') Control 0 0.67a - 0 30 0.84b 25 0 60 0.80b 19 0 Seeds were treated with 1 mM KCN for 30 or 60 minutes and then transferred to water f o r the duration of the experiment. Oxygen consumption was recorded 24 h a f t e r the onset of the pulse. Values represent the mean of 2 r e p l i c a t e s ; means followed by the same l e t t e r do not d i f f e r at the p= 0.05 l e v e l according to the SNK MRT. 52 Table 13. The nature of azide- and cyanide-induced r e s p i r a t i o n i n prim a r i l y dormant seed of l i n e AN-51. Treatment Oxygen-uptake (nmol seed ^ min ^ ) Percent increase over c o n t r o l Water — > water 0.69a — Cyanide — > water 1.04b 51 Cyanide — > cyanide 1.30de 88 Cyanide — > SHAM 1.09bc 58 Cyanide — > cyanide + SHAM 1.24de 80 Cyanide — > azide + SHAM 1.21cd 75 Cyanide — > azide 1.38e 100 Azide — > water l . l l b c 61 Azide — > cyanide 1.37e 99 Azide — > azide 1.36e 97 Azide — > SHAM 1.33de 93 Azide — > cyanide + SHAM 1.37e . 99 Azide — > azide + SHAM 1.01b 46 Seeds were incubated i n water, azide or cyanide f o r 24 h and then transferred (—>) to the s p e c i f i e d solutions for 14 h and oxygen uptake subsequently recorded. I n h i b i t o r concentrations used were 1 mM azide and cyanide and 10 mM SHAM. Values are the means of two r e p l i c a t e s . Means 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 at the p= 0.05 l e v e l according to the SNK MRT. 53 c o n t r o l ) ; renewing azide and cyanide solutions a f t e r the i n t i a l 24 h treatment resulted i n increased r e s p i r a t i o n i n the following 14 h period (Tab. 13). SHAM (10 mM) applied a f t e r a 24 h cyanide or azide treatment did not i n h i b i t r e s p i r a t i o n either alone or when applied i n combination with azide or cyanide. SHAM did, however, prevent further stimulation of r e s p i r a t i o n by azide when applied i n combination with 1 mM azide for the 14 h period subsequent to the i n i t i a l 24 h azide treatment (Tab. 13). The r e s p i r a t i o n of dormant seed treated with high concentrations of azide (5-20 mM) for 5 h (preliminary experiments showed 5 h to be adequate for maximum i n h i b i t i o n at these azide concentrations) was measured (Tab. 14). Azide reduced oxygen uptake i n i n t a c t seeds by approximately 40% at a l l concentrations. The r e s p i r a t i o n of seeds treated with azide plus 1 or 5 mM SHAM did not d i f f e r s i g n i f i c a n t l y from seeds treated with 5 mM azide alone a f t e r a 5 h incubation (Tab. 15). Applied concurrently with 5 mM azide, 10 and 20 mM SHAM i n h i b i t e d r e s p i r a t i o n below that of seeds r e c e i v i n g 5 mM azide alone or with 1 or 5 mM SHAM (Tab. 15). 3. P a r t i t i o n i n g of azide and cyanide-induced r e s p i r a t i o n . The r e s p i r a t o r y components of embryos excised from non-germinated seeds treated with either 1 mM cyanide or azide for 24 h were determined. SHAM (5 mM) alone did not s i g n i f i c a n t l y i n h i b i t the r e s p i r a t i o n of embryos excised from water, azide or cyanide imbibed seeds (Tab. 16). Respiration of the co n t r o l embryos was i n h i b i t e d 45% by 1 mM cyanide but the r e s p i r a t i o n of embryos excised from azide and 54 Table 14. Dose-response for the inhibition of seed respiration by azide. Azide concentration (mM) Oxygen uptake (nmol seed ^ min ^) 0 0.66a 5 0.41b 10 0.42b 15 0.38b 20 0.41b Seeds were imbibed on an azide solution for 5 h after which oxygen uptake was recorded. Values represent the mean of 3 replicates. Means followed by the same letter do not differ significantly at the p= 0.05 level according to the SNK MRT. F-value for comparison of a l l azide concentrations vs. control was significant at the p= 0.05 level. 55 Table 15. Dose-response f or the e f f e c t of SHAM on the i n h i b i t i o n of seed r e s p i r a t i o n by 5 mM azide. Treatment Oxygen uptake (nmol seed ^ min *) Control (water) 0.82a Azide (5 mM) 0.43b " + SHAM (1 mM) 0.41b " + SHAM (5 mM) 0.46b " + SHAM (10 mM) 0.34c " + SHAM (20 mM) 0.33c Seeds were imbibed f o r 5 h on 5 mM azide with or without the s p e c i f i e d concentrations of SHAM and oxygen uptake was recorded. Values are the mean of 3 r e p l i c a t e s . Means 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 at the p= 0.05 l e v e l according to SNK MRT. 56 Table 16. The e f f e c t of cyanide and azide pretreatment of prim a r i l y dormant i n t a c t seeds on the response of excised embryos to resp i r a t o r y i n h i b i t o r s ( l i n e AN-51). Oxygen uptake (nmol embryo min ) Treatment buffer ** KCN SHAM KCN + SHAM cyt V 1 a l t V a l t only (V ) res' (1) (2) (3) (4) (3 - 4) (1 - 3) (2 - -Control 0.29a 0.16b 0.28a 0.06c 0.22 0.01 0.10 Azide 0.69f 0.61ef 0.60ef O.Ald 0.19 0.09 0.20 Cyanide 0.66ef 0.60ef 0.65ef 0.56e 0.09 0.01 0.04 2 Engaged ( i n the absence of KCN); p o t e n t i a l ( i n the presence of KCN). * Intact seeds were imbibed for 24 h on water, or 1 mM azide or cyanide. Embryos were then excised and t h e i r oxygen uptake recorded i n 50 mM phosphate buffer (pH 6.9) with or without i n h i b i t o r s (1.0 mM KCN and/or 5 mM SHAM) as s p e c i f i e d (**). Values are the means of 3 r e p l i c a t e s . Means within columns and rows 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 at the p= 0.05 l e v e l according to the SNK MRT. F-values f or the main e f f e c t s of pretreatment, res p i r a t o r y i n h i b i t o r s applied during oxygen uptake and t h e i r i n t e r a c t i o n were s i g n i f i c a n t at the p= 0.05 l e v e l . 57 cyanide treated seeds was not strongly i n h i b i t e d (Tab. 16). In the presence of SHAM, cyanide i n h i b i t e d r e s p i r a t i o n i n the co n t r o l (79%) fa r more than i n embryos of ei t h e r cyanide or azide treated seeds (Tab. 16). D. E f f e c t of pH on the action of respi r a t o r y i n h i b i t o r s 1. Respiration studies Substantial pH changes occur when SHAM, cyanide, and azide are dissolved i n d i s t i l l e d water (Tab. 17). Experiments were performed to determine the e f f e c t of pH on the action of re s p i r a t o r y i n h i b i t o r s . The r e s p i r a t i o n of dormant seeds imbibed on water or citrate-phosphate buffer (pH 5 and 7) did not d i f f e r s i g n i f i c a n t l y (Tab. 17). S i m i l a r l y SHAM alone did not e f f e c t r e s p i r a t i o n regardless of pH or buff e r i n g . Cyanide, with or without SHAM stimulated oxygen uptake i n the buffered systems and i n water to the same extent (Tab. 17). On the other hand, pH had a s t r i k i n g e f f e c t on the action of azide with or without SHAM. In water and i n buffer (pH 7), 1 mM azide stimulated seed r e s p i r a t i o n , however at pH 5 r e s p i r a t i o n was strongly i n h i b i t e d (by 85%). At pH 7, azide + SHAM not only f a i l e d to i n h i b i t r e s p i r a t i o n ( c f . the control) but a c t u a l l y stimulated oxygen consumption by over 20% (Tab. 17). On the other hand, at pH 5 azide + SHAM i n h i b i t e d r e s p i r a t i o n by 85%. In dose-response studies, seed r e s p i r a t i o n was stimulated by azide (pH 7) at concentrations ranging from 0.065 to 2.0 mM ( F i g . 12). However, r e s p i r a t i o n was sharply i n h i b i t e d by azide concentrations over 0.125 and 0.5 mM at pH 5 and 6, res p e c t i v e l y ( F i g . 12). The e f f e c t of cyanide on seed r e s p i r a t i o n was not pH s e n s i t i v e ( F i g . 13). 58 Table 17. The e f f e c t of pH on the action of res p i r a t o r y i n h i b i t o r s as they e f f e c t oxygen uptake of primarily dormant seeds of l i n e AN-51. Treatment Solution pH  non-buffered pH 5 pH 7 Control 0.81b1 [5.2] : I 0.85b 0.87b Azide 1.43c [6.2] ( 7 7 ) 3 0.29a (-66) 1.45c (67) Azide + SHAM 0.27a [5.2] (-67) 0.13a (-85) 1.09c (25) Cyanide 1.37c [9.6] (69) 1.45c (71) 1.40c (61) Cyanide + SHAM 1.38c [6.0] (70) 1.34c (58) 1.39c (60) SHAM 0.78b [3.9] (-4) 0.90b (6) 0.79b (-9) Oxygen uptake (nmol seed min ). 2 Values i n brackets represent the pH of i n h i b i t o r solutions dissolved i n d i s t i l l e d water. 3 Values i n parentheses represent % i n h i b i t i o n / s t i m u l a t i o n compared to the c o n t r o l . Seeds were treated with r e s p i r a t o r y i n h i b i t o r s (1 mM azide and cyanide, and 10 mM SHAM) dissolved e i t h e r i n d i s t i l l e d water or c i t r a t e phosphate buffer (pH 5 or 7) for 24 h before recording oxygen consumption. Values represent the means of 2 r e p l i c a t e s ; means 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 at the p= 0.05 l e v e l according to the SNK MRT. F-values for the main e f f e c t s of buffering, i n h i b i t o r treatment and t h e i r i n t e r a c t i o n were s i g n i f i c a n t at the p= 0.05 l e v e l . 59 1.4 0.05 0.1 1 AZIDE CONCENTRATION - mM 12 The effect of pH on azide-induced respiration in primarily dormant seeds of line AN-51. Oxygen uptake of seeds treated with azide (dissolved in citrate-phosphate buffer) was measured 24 h after the onset of treatment. The value of the controls,(indicated by the arrow) at pH 5, 6 and 7 (x = 0.71 nmol min" seed" ) did not differ significantly. Values represent the means of 2 replicates. F-value for interaction of pH X azide concentration was significant at the p= 0.05 level. 60 The e f f e c t of 10 mM SHAM and pH adjustment (citrate-phosphate buffer) on the stimulation of r e s p i r a t i o n by a range of azide concentrations i s shown i n F i g . 14. Respiratory stimulation by azide increased with increasing azide concentration above 0.1 mM. Simultaneous presence of SHAM with azide (> 0.5 mM) resu l t e d i n severe r e s p i r a t o r y i n h i b i t i o n . However, at low azide concentrations (0.1 -0.25 mM) i n unbuffered medium, SHAM not only f a i l e d to i n h i b i t azide-induced r e s p i r a t i o n but a c t u a l l y enhanced i t compared to the co n t r o l ( F i g . 14). When the pH of azide solutions (of d i f f e r e n t concentrations) was adjusted to the pH of 10 mM SHAM plus corresponding azide concentrations, the oxygen uptake response curve was very s i m i l a r to the azide dose-response curve i n the presence of 10 mM SHAM ( F i g . 14). SHAM (10 mM) had l i t t l e e f f e c t on the stimulation of r e s p i r a t i o n by cyanide regardless of cyanide concentration ( F i g . 15). 2. Germination studies Azide (1 mM) stimulated the release of secondary dormancy when the so l u t i o n was unbuffered or buffered at pH 6, 7 or 8 (Tab. 18). Stimulation of germination was maximum at pH 6 and 8. At pH 5 the release of dormancy by azide was completely i n h i b i t e d . On the other hand, cyanide (1 mM) released seed dormancy regardless of medium pH (Tab. 19). Germination was maximum when cyanide was applied i n water or buffered at pH 5 (85 and 90%, r e s p e c t i v e l y ) . When applied at pH 6, 7 or 8, germination was stimulated to a s l i g h t l y l e s s e r extent (60 -68%, Tab. 19). 61 1 KCN CONCENTRATION - mM F i g . 13 The e f f e c t of pH on cyanide-induced r e s p i r a t i o n i n primarily dormant seeds of l i n e AN-51. Oxygen uptake of seeds treated with KCN (dissolved i n citrate-phosphate buffer) was measured 24 h a f t e r the onset of treatment. The value of the con t r o l s . ( i n d i c a t e d by the arrow) at pH 5, 6 and 7 (x = 0.63 nmol min seed ) did not d i f f e r s i g n i f i c a n t l y . Values represent the means of 2 r e p l i c a t e s . pH X KCN concentration i n t e r a c t i o n was not s i g n i f i c a n t at the p= 0.05 l e v e l . 62 F i g . 14 The e f f e c t of SHAM and pH on azide-induced r e s p i r a t i o n i n primarily dormant seeds of l i n e AN-51. Seeds were treated with the s p e c i f i e d concentration of azide dissolved i n ei t h e r : A, d i s t i l l e d water,X , i n d i s t i l l e d water with 10 mM SHAM, or •, i n c i t r a t e -phosphate buffer, the pH of which equalled the pH of the azide + SHAM solu t i o n ( i n d i s t i l l e d water) at any azide concentration. Oxygen-uptake was recorded 24 h a f t e r the onset of treatment. Values represent the means of 2 r e p l i c a t e s . 63 K C N C O N C E N T R A T I O N — mM F i g . 15 The e f f e c t of SHAM on cyanide-induced r e s p i r a t i o n i n dormant seeds of l i n e AN-51. Oxygen uptake of seeds treated with KCN (with or without 10 mM SHAM) was recorded 24 h a f t e r the onset of treatment. Values represent the means of 2 r e p l i c a t e s . Interaction of KCN concentration X +/- SHAM was not s i g n i f i c a n t at the p= 0.05 l e v e l . 64 Table 18. The e f f e c t of pH on the release of secondary dormancy i n seeds of l i n e AN-51 by azide. Solution pH Percent germination co n t r o l 1 mM azide Unbuffered 5a 80b 5 5a 5a 6 5a 93c 7 5a 78b 8 15a 100c Seeds were imbibed i n either 50 mM phosphate (pH 6 to 8) or c i t r a t e phosphate buffer (pH 5) or water with or without 1 mM azide at the s p e c i f i e d pH. Germination was recorded 14 days a f t e r the onset of treatment. Values represent the means of 4 r e p l i c a t e s . Means within columns and rows 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 at the p= 0.05 l e v e l according to the SNK MRT. F-values for the main e f f e c t s of pH, +/- azide and t h e i r i n t e r a c t i o n were s i g n i f i c a n t at the p= 0.05 l e v e l . 65 Table 19. The e f f e c t of pH on the release of secondary dormancy i n seeds of l i n e AN-51 by cyanide. Solution pH Percent germination co n t r o l 1 mM cyanide Unbuffered Oa 85b 5 Oa 90b 6 Oa 68c 7 Oa 65c 8 Oa 60c Seeds were imbibed on 50 mM phosphate (pH 8) or c i t r a t e phosphate buffer (pH 5 to 7) or water with or without 1 mM KCN at the s p e c i f i e d pH. Germination was recorded 7 days a f t e r the onset of treatment. Values represent the means of 4 r e p l i c a t e s . Means within columns and rows 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 at the p= 0.05 l e v e l according to the SNK MRT. F-values for the main e f f e c t s of pH, cyanide treatment and t h e i r i n t e r a c t i o n were s i g n i f i c a n t at the p= 0.05 l e v e l . 66 DISCUSSION A. The induction and release of secondary dormancy. Research on wild oats seed dormancy has been confused by the use of seeds c o l l e c t e d from heterogenous f i e l d populations of wild oats that vary i n t h e i r dormancy behavior due to both genetic (Naylor and Jana, 1976) and environmental (Naylor and Fedec, 1978; Sawhney and Naylor, 1979, 1980) f a c t o r s . Observations on f i e l d populations are often not a p p l i c a b l e to i n d i v i d u a l biotypes and v i s e versa. The r e s u l t s presented here add a new dimension to t h i s problem by showing that g e n e t i c a l l y pure l i n e s d i f f e r with regard to optimal conditions for the induction of secondary dormancy and the degree and duration of the dormancy induced. Hay and Cumming (1959), working with seeds c o l l e c t e d from f i e l d populations, suggested the p o s s i b i l i t y of obtaining a constant supply of uniformly dormant seeds by inducing secondary dormancy i n after-ripened seeds. In l i g h t of the v a r i a t i o n between pure l i n e s i n the induction and expression of secondary dormancy reported i n t h i s t h e s i s , inducing dormancy i n a g e n e t i c a l l y diverse sample of seeds cannot be considered an adequate method for obtaining homogenous research material. V a r i a b i l i t y i n the duration of primary (innate) dormancy has been considered a major fa c t o r i n the s u r v i v a l of wild oats. This v a r i a b i l i t y leads to the maintenance of a large seed bank i n the s o i l rendering the weed l e s s susceptible to c o n t r o l methods aimed at the seedling stage (e.g. c u l t i v a t i o n , h e r b i c i d e s ) . 67 V a r i a b i l i t y i n secondary dormancy behavior provides yet another mechanism i n the o v e r a l l s u r v i v a l strategy of wild oats. A f t e r -ripened seeds of dormant biotypes can acquire secondary dormancy under unfavourable conditions and d i s t r i b u t e germination over time due to differences i n the duration of acquired dormancy. The s i g n i f i c a n c e of secondary dormancy i n the s u r v i v a l of g e n e t i c a l l y nondormant l i n e s i s obvious. To survive the harsh winters of the Canadian P r a i r i e s , these l i n e s must have a mechanism, l i k e secondary dormancy, whereby germination can be delayed u n t i l winter temperatures are low enough to prevent germination. Although i n t h i s study only a short-term dormancy was induced i n nondormant l i n e s ( F i g . 4), i t i s possible that under f i e l d conditions a combination of fa c t o r s ( v i z . temperature, carbon dioxide, anaerobiosis, presence of h u l l s , etc.) could induce a longer-term dormancy. High temperatures are reported to promote the induction of secondary dormancy i n curled dock ( T o t t e r d e l l and Roberts, 1979), l e t t u c e (Vidaver and Hsiao, 1975) and cocklebur (Esashi et a l . , 1978). In the present study greater induction of secondary dormancy was found as the temperature during anaerobiosis increased (Tab. 2). This i s i n contrast to the find i n g s of Hay (1962). This c o n t r a d i c t i o n may be explained by the di f f e r e n c e i n the seed used and by the presence of h u l l s i n the e a r l i e r study. The f a c t that secondary dormancy could be induced i n up to 85% of AN-51 seeds following 4 h of aerobic imbibition ( F i g . 3) suggests that biochemical changes during t h i s period do not i r r e v e r s i b l y commit these seeds to germination. 68 Several chemically unrelated compounds are known to break primary dormancy i n wild oats. These include azide (Fay and Gorecki, 1978; Upadhyaya et a l . , 1982a), ethanol (Adkin et a l . , 1984a) n i t r a t e and n i t r i t e ions (Adkins et a l . , 1984b; Johnson, 1935) and g i b b e r e l l i n s (Simpson, 1978). Cytokinins (isopentenyl adenine, k i n e t i n ) have been shown to stimulate germination of primarily dormant seeds i n red r i c e (Cohn and Butera, 1982) and wild oats (unpublished r e s u l t s , M.K. Upadhyaya). C o l l e c t i v e l y , these chemicals provide a valuable t o o l f o r the comparison of the p h y s i o l o g i c a l c h a r a c t e r i s t i c s of primary and secondary dormancies. I t was found here that azide, n i t r a t e , ethanol, isopentenyl adenine and k i n e t i n stimulated the germination of secondarily dormant seeds (Tab. 4). The s i m i l a r i t y i n response of p r i m a r i l y and secondarily dormant seeds to such diverse chemicals suggests that the two types of dormancies i n wild oats are s i m i l a r at l e a s t i n part i n t h e i r r e g u l a t i o n . The l o s s of secondary dormancy through a f t e r - r i p e n i n g (as occurs with primary dormancy) supports the notion that secondary and primary dormancies are d i f f e r e n t manifestations of the same p h y s i o l o g i c a l s t a t e . B. A l t e r n a t i v e r e s p i r a t i o n and the regulation of secondary dormancy. In order to t e s t the hypothesis that the induction of secondary dormancy i s accompanied by a decline i n the a c t i v i t y of a l t e r n a t i v e r e s p i r a t i o n the r e l a t i v e contribution of CN-sensitive and i n s e n s i t i v e pathways i n embryos excised from seeds before and a f t e r the induction of secondary dormancy was examined. Respiratory components were 69 determined f o r excised embryos rather than i n t a c t seed f o r the following reasons. F i r s t l y , i n order to determine r e s p i r a t o r y components instantaneously, i n h i b i t o r s i n j e c t e d i n t o the oxygen -uptake vessel must e l i c i t an immediate i n h i b i t i o n of r e s p i r a t i o n . Although cyanide alone gave rapid i n h i b i t i o n of r e s p i r a t i o n of i n t a c t seeds (Tab. 5), the lack of a d d i t i o n a l i n h i b i t i o n i n the presence of SHAM in d i c a t e s that SHAM does not e f f e c t i v e l y block the a l t e r n a t i v e pathway of i n t a c t seeds compared to excised embryos presumably because of the permeability b a r r i e r presented by the seed coat. This would make d i s t i n g u i s h i n g r e s i d u a l r e s p i r a t i o n from incompletely i n h i b i t e d a l t e r n a t i v e r e s p i r a t i o n d i f f i c u l t . Secondly, the response of excised embryos and i n t a c t seeds to res p i r a t o r y i n h i b i t o r s i s q u a l i t a t i v e l y the same (Upadhyaya et a l . , 1983); presumably the p a r t i t i o n i n g of embryo r e s p i r a t i o n can be extrapolated to be i n d i c a t i v e of i t s p a r t i t i o n i n g i n i n t a c t seeds. This approach i s commonly used and accepted i n studies i n v o l v i n g seed r e s p i r a t i o n (Leopold and Musgrave, 1980; M i l l e r el; a l . , 1983). F i n a l l y , i n the event that excision does cause an a r t i f a c t u a l change i n the res p i r a t o r y components of embryo ti s s u e , t h i s change would l i k e l y be uniform across treatments, thus enabling treatment e f f e c t s to be distinguished from the e f f e c t s of mechanical i n j u r y . Based on t h e i r f i n d i n g s that several treatments rel e a s i n g secondary dormancy resulted i n a concomitant increase i n the p a r t i c i p a t i o n of the a l t e r n a t i v e pathway, Esashi et a l . (1981) have proposed that secondary dormancy i n cocklebur seeds i s due to t h e i r 70 i n a b i l i t y to perform a l t e r n a t i v e r e s p i r a t i o n . In the current study i t was found that with the exception of azide treatment, SHAM did not i n h i b i t the stimulation of germination by a diverse group of chemicals (Tab. 4). Thus, as with primary dormancy, a SHAM-sensitive process (presumably a l t e r n a t i v e r e s p i r a t i o n ) i s not a pr e r e q u i s i t e f o r the promotion of germination of secondarily dormant wild oat seeds by these chemicals. In wild oats, the a l t e r n a t i v e pathway did not contribute to the r e s p i r a t i o n of excised embryos regardless of dormancy status (Tab. 6). The after-ripened and secondary dormant embryos did however, have the capacity to d i v e r t electrons through the a l t e r n a t i v e pathway when the cytochrome pathway was blocked with cyanide. In a l l cases the maximum f l u x of the a l t e r n a t i v e pathway was much l e s s than the maximum f l u x through the cytochrome pathway (Tab. 6). C l e a r l y , these r e s u l t s do not support the hypothesis that a l t e r n a t i v e r e s p i r a t i o n (or the p o t e n t i a l to perform a l t e r n a t i v e r e s p i r a t i o n ) i s r e l a t e d to dormancy status i n wild oats seeds. The p h y s i o l o g i c a l s i g n i f i c a n c e of the elevated contribution of the a l t e r n a t i v e pathway i n cocklebur seeds during the release of dormancy must be questioned. Rather than determining the actu a l p a r t i c i p a t i o n of the a l t e r n a t i v e pathway ( i . e . indicated by r e s p i r a t o r y i n h i b i t i o n i n the presence of SHAM alone), Esashi et a l . (1982, 1983) measured i t s p o t e n t i a l capacity ( i . e . r e s p i r a t i o n i n the presence of cyanide l e s s r e s i d u a l r e s p i r a t i o n ) . In no case did the authors show that any dormancy-breaking treatment a c t u a l l y activated the a l t e r n a t i v e pathway 71 while the cytochrome pathway was s t i l l f unctioning. Given the f a i l u r e of SHAM to i n h i b i t : i ) the spontaneous release of secondary dormancy i n nondormant l i n e s , i i ) the release of secondary dormancy by several chemicals and i i i ) the r e s p i r a t i o n of embryos excised from both a f t e r -ripened and secondarily dormant seed, i t i s u n l i k e l y that - with the possible exception of azide-induced release of dormancy - a l t e r n a t i v e r e s p i r a t i o n has any p h y s i o l o g i c a l s i g n i f i c a n c e i n the regulation of secondary dormancy i n wild oats. C. The act i o n of azide and cyanide on the release of seed dormancy Although cyanide i s known to release seed dormancy i n several species (Hendricks and Taylorson, 1972) i t s e f f e c t on germination and r e s p i r a t i o n has not been examined i n wild oats. Cyanide and azide i n h i b i t cytochrome-mediated r e s p i r a t i o n at the same s i t e - cytochrome oxidase (Ikuma and Bonner, 1967). The r e s u l t s presented here suggest that the two i n h i b i t o r s also have the same action i n stimulating germination and r e s p i r a t i o n i n wild oat seeds - presumably v i a t h e i r common act i o n on cytochrome oxidase. Although generally speaking cyanide and azide stimulated germination i n the same concentration range and by s i m i l a r treatment durations, some differences between the two compounds were apparent. In one experiment, cyanide was more e f f e c t i v e at r e l e a s i n g dormancy i n seeds with l i t t l e a f t e r - r i p e n i n g ( i . e . 2 months, Tab. 9); azide (1 mM, eit h e r as a continuous treatment or as a 6 h pulse - Tab. 9) was completely i n e f f e c t i v e at rele a s i n g dormancy i n 2 to 5 month old seeds. 72 In a separate experiment, azide (1 mM) released primary dormancy i n 3 month old seeds ( F i g . 8). The inconsistent release of primary dormancy by azide may be explained by d i f f e r e n t rates of a f t e r -ripening observed i n separate harvests of wild oat seed. V a r i a t i o n i n a f t e r - r i p e n i n g rate may a r i s e through d i f f e r i n g storage conditions ( i . e . changes i n temperature and r e l a t i v e humidity). Consequently the p h y s i o l o g i c a l age of the seed ( i n r e l a t i o n to dormancy status) may not be the same i n a l l seeds of the same chronological age. Adkins et a l . (1984 a,b) have proposed that two d i s t i n c t germination blocks e x i s t i n dormant wild oat seeds, one which i s i n s e n s i t i v e to azide and i s overcome during a f t e r - r i p e n i n g and a second block which can be released by azide ( i . e . i n p a r t i a l l y after-ripened seeds). I f cyanide and azide release dormancy by the same mechanism, the f a c t that cyanide could stimulate germination regardless of the duration of a f t e r - r i p e n i n g contradicts t h i s hypothesis. Accordingly, f a i l u r e of azide to induce germination i n f r e s h l y harvested seeds may not be r e l a t e d to i t s mode of a c t i o n per se. I t i s possible that i n order f o r azide to release dormancy i n f r e s h l y harvested seeds i t would have to be applied at concentrations which would completely i n h i b i t subsequent germination. Rather than two d i s t i n c t blocks c h a r a c t e r i z i n g wild oats seed dormancy, i t seems more l i k e l y that the degree of dormancy follows a continuum that declines with a f t e r -r i p e n i n g . Germination rate i n response to cyanide and azide treatments also d i f f e r e d . Although continuous cyanide and azide treatments gave 73 comparable release of dormancy, the l a t t e r caused germination to be delayed 5-10 days (Tab. 8) c f . 48-72 h with cyanide treatment (Tab. 7). Upadhyaya et a l . (1983) found that 1 mM azide had two d i s t i n c t e f f e c t s on wild oat seeds: 1. the release of dormancy and 2. the i n h i b i t i o n of germination. Azide (1 mM) was found to delay the germination of nondormant seeds f o r a period s i m i l a r to the lag found i n the stimulation of germination by azide i n dormant seeds (Upadhyaya et a l . , 1983). The effectiveness of short azide pulses at releasing dormancy without the resultant i n h i b i t i o n of germination supports t h i s idea of two d i s t i n c t e f f e c t s of azide. The release of dormancy by azide probably occurs i n the early i m b i b i t i o n a l stages; the presence of azide a f t e r t h i s time i s i n h i b i t o r y to germination (Tab. 8). Cyanide (0.5 mM) however was not as t o x i c to germination; at higher concentrations ( i . e . 1.0 mM) germination was somewhat delayed (by 3 days, Tab. 7). Although treatment plates were wrapped with Parafilm, the l e s s e r t o x i c i t y of cyanide may be due to the v o l a t i l i z a t i o n and gradual escape of HCN from the plates (Yu et a l . , 1981), r e s u l t i n g i n sub-toxic l e v e l s of t h i s i n h i b i t o r . D. The stimulation of seed r e s p i r a t i o n by azide and cyanide. Cyanide stimulated seed r e s p i r a t i o n to an extent s i m i l a r to that of azide under i d e n t i c a l conditions (24 h treatment at 1 mM) (Tab. 11). Upadhyaya et a l . (1983) found that azide (1 mM continuous treatment) stimulated r e s p i r a t i o n f a r i n advance of germination. However since azide also i n h i b i t s germination following the release of 74 dormancy, i t i s not possible to determine i f r e s p i r a t i o n was enhanced p r i o r to the a c t u a l release of dormancy per se. Because dormancy can be broken by cyanide without an accompanying i n h i b i t i o n of germination, the onset of r e s p i r a t o r y stimulation by t h i s compound was examined. To determine i f the release of dormancy and the stimulation of r e s p i r a t i o n by cyanide could be d i s s o c i a t e d , two experiments were performed. Figure 10 shows the cyanide dose-response of seed germination and r e s p i r a t i o n . The release of dormancy ( i n t h i s experiment) was always accompanied by an increase i n seed r e s p i r a t i o n (measured well i n advance of any v i s i b l e signs of germination). The degree of r e s p i r a t o r y stimulation did not however, r e l a t e q u a n t i t a t i v e l y to the release of dormancy. Although oxygen-uptake was maximum at 1.0 mM cyanide, release of dormancy varied l i t t l e between 0.05 and 1.0 mM cyanide. In a second experiment ( F i g . 11) seed r e s p i r a t i o n and germination i n response to short cyanide pulses were measured. Both 2 and 4 h cyanide pulses stimulated r e s p i r a t i o n (33 and 35% r e s p e c t i v e l y ) and germination (66 and 87%, r e s p e c t i v e l y ) to a s i m i l a r extent. Shorter pulses (30 and 60 min) also enhanced r e s p i r a t i o n (19 and 25% r e s p e c t i v e l y ) but did not release dormancy (Tab. 12). C l e a r l y , while the release of seed dormancy by cyanide i s i n e v i t a b l y preceded by an increase i n oxygen uptake, cyanide-induced r e s p i r a t i o n does not appear to be r e l a t e d to germination process(es)_p_er se. Adkins et a l . (1984b,d) have recently reported that n i t r a t e , n i t r i t e and ethanol stimulate r e s p i r a t i o n of dormant wild oat seeds i n 75 advance of germination. Whether or not these compounds have the same mode of action i s not known. The f a c t that several chemically d i f f e r e n t dormancy-breaking compounds a l l stimulate pre-germination r e s p i r a t i o n i s suggestive of a causal r e l a t i o n s h i p between the two phenomena. However, a non-causal r e l a t i o n s h i p between r e s p i r a t o r y enhancement and the release of seed dormancy cannot be ruled out with the a v a i l a b l e data. E. The e f f e c t of SHAM on the release of dormancy by azide and cyanide. Although SHAM i s known to i n h i b i t seed germination i n the presence of azide (Upadhyaya et a l . , 1983; Adkins et a l . , 1984 c,d) the physiology of t h i s i n h i b i t i o n i s not understood. Upadhyaya et a l . (1983) suggested (as one p o s s i b i l i t y ) that the i n h i b i t o r y e f f e c t of SHAM may be due to the complete i n h i b i t i o n of ATP production i n the presence of azide. Accordingly, a l t e r n a t i v e r e s p i r a t i o n i s necessary f o r supplying ATP i n the presence of azide and may not be the cause of dormancy release per se. The r e s u l t s i n Tab. 10 suggest that SHAM does not act i n t h i s manner. When 10 mM SHAM was present during and a f t e r a 6 h azide pulse, the release of dormancy was i n h i b i t e d ; however, i f SHAM was applied only a f t e r the 6 h azide treatment germination proceeded with l i t t l e i n h i b i t i o n . S i m i l a r i l y when SHAM was present only during the 6 h azide pulse, germination was not reduced. I f SHAM blocks azide-induced germination by h a l t i n g ATP generation v i a the a l t e r n a t i v e pathway one would expect SHAM applied a f t e r the azide pulse also to i n h i b i t germination since i t i s u n l i k e l y that s u f f i c i e n t 76 ATP could be generated i n t h i s 6 h period to support subsequent germination when both pathways are blocked. I t i s possible that SHAM applied during, but not a f t e r the azide treatment f a i l e d to i n h i b i t germination because the seeds were not i n contact with the chemical long enough to absorb an i n h i b i t o r y dose or that SHAM taken-up by the seed i s metabolized to an i n a c t i v e form. Given the s i m i l a r i t y of cyanide and azide a c t i o n on seed dormancy and r e s p i r a t i o n , i t was unexpected to f i n d that SHAM (applied concurrently) f a i l e d to i n h i b i t the release of dormancy by cyanide as i t had i n the case of azide (Tab. 10). To tes t the p o s s i b i l i t y that the lack of i n h i b i t i o n by SHAM i n the case of cyanide was due to slow uptake of SHAM by the seed, SHAM was applied to seeds 24 h before cyanide treatments as well as during cyanide treatment. However there was s t i l l no i n h i b i t i o n of germination (data not shown). I t i s therefore u n l i k e l y that the release of dormancy by cyanide i n wild oat seeds i s dependant on a SHAM-sensitive process ( a l t e r n a t i v e r e s p i r a t i o n ) . The differe n c e i n the e f f e c t of SHAM on cyanide and azide action i s i n cont r a d i c t i o n to the notion that the two i n h i b i t o r s have the same mode of action i n re l e a s i n g seed dormancy. F. The nature of azide and cyanide stimulated r e s p i r a t i o n . 1. The e f f e c t of SHAM on the stimulation of resp i r a t o n by azide and cyanide. The SHAM-sensitivity of the induction of seed r e s p i r a t i o n by azide and cyanide p a r a l l e l e d the r e s u l t s found with the release of dormancy. 77 SHAM did not i n h i b i t the induction of r e s p i r a t i o n by cyanide as i t did i n the case of azide (Tab. 11). This would suggest that the mechanism f o r the induction of r e s p i r a t i o n by the two compounds i s d i f f e r e n t -azide action r e q u i r i n g a SHAM-sensitive process while cyanide action does not. I f SHAM i n h i b i t s the induction of r e s p i r a t i o n by i n h i b i t i n g a l t e r n a t i v e r e s p i r a t i o n (stimulated by azide) then cyanide treatment must not induce t h i s pathway. 2. The nature of azide and cyanide induced r e s p i r a t i o n . To further i n v e s t i g a t e the SHAM s e n s i t i v i t y of cyanide and azide induced r e s p i r a t i o n , the nature of the induced oxygen uptake (as opposed to the nature of the induction of r e s p i r a t i o n ) was examined. a) A l t e r n a t i v e (SHAM-sensitive) r e s p i r a t i o n . S u r p r i s i n g l y , a f t e r seeds were removed from dishes containing azide, induced r e s p i r a t i o n could not be i n h i b i t e d by the subsequent a p p l i c a t i o n of SHAM (Tab. 13). Taken alone, these r e s u l t s might suggest that the r e s p i r a t i o n of these seeds was cytochrome-mediated (presuming the seeds recovered from the previous i n h i b i t i o n of t h i s pathway). This however, does not appear to be the case since SHAM applied i n combination with azide or cyanide subsequent to res p i r a t o r y stimulation f a i l e d to i n h i b i t cyanide-induced r e s p i r a t i o n and had only a small e f f e c t on azide-induced r e s p i r a t i o n (Tab. 13). In t h i s s i t u a t i o n one would expect both pathways to be blocked thus a s h i f t 78 from the a l t e r n a t i v e pathway to the cytochrome pathway could not occur. I t was of concern that SHAM might be entering the seeds i n the presence of azide but not when applied alone or i n combination with cyanide due to e f f e c t ( s ) azide might have on membrane permeability. I f t h i s was the case, then t h i s could explain the lack of i n h i b i t i o n of cyanide-induced germination and r e s p i r a t i o n by SHAM (Tabs. 10 and 11); i n h i b i t i o n of cytochrome-mediated r e s p i r a t i o n by cyanide might increase the f l u x of electrons through the a l t e r n a t i v e pathway but t h i s would not be detected i f SHAM i s unable to penetrate the seed and i n h i b i t a l t e r n a t i v e r e s p i r a t i o n . For t h i s reason, SHAM was applied i n combination with azide to seeds that had been pre-treated with cyanide or azide (Tab. 13). No res p i r a t o r y i n h i b i t i o n r e s u l t e d . C l e a r l y , the SHAM-insensitivity of cyanide and azide-induced r e s p i r a t i o n cannot be due to the i n a b i l i t y of SHAM to penetrate seeds. SHAM has been used i n many seed p h y s i o l o g i c a l studies without any reported penetration problems (Esashi et a l , 1981b, 1982; Yu et a l . , 1979; Yentur and Leopold, 1976). These r e s u l t s suggest that while the induction of r e s p i r a t i o n by azide appears to be a SHAM-sensitive process (Tab. 11), once induced, cyanide and azide-stimulated r e s p i r a t i o n are not SHAM-se n s i t i v e (Tab. 13) and thus not a l t e r n a t i v e . The s e n s i t i v i t y of induced r e s p i r a t i o n to i n h i b i t o r s was more accurately determined by p a r t i t i o n i n g the r e s p i r a t i o n of embryos excised from seeds previously treated with azide or cyanide (Tab. 16). As i n i n t a c t seeds (Tab. 13), the actu a l c o n t r i b u t i o n of the 79 a l t e r n a t i v e pathway was e s s e n t i a l l y n e g l i g i b l e i n con t r o l s , and i n azide or cyanide treated seeds. C l e a r l y , azide and cyanide treatment did not cause the a c t i v a t i o n of the a l t e r n a t i v e pathway. b) Residual r e s p i r a t i o n . I t i s c l e a r from Table 16 that the stimulation of r e s p i r a t i o n by azide and cyanide i s expressed through large increases i n r e s i d u a l r e s p i r a t i o n . This source of oxygen uptake i s generally considered to be nonmitochondrial i n nature (e.g. Theologis and L a t i e s , 1978a). Unfortunately, attempts to i s o l a t e mitochondria from seed ti s s u e s were not successful and therefore the p o s s i b l i l i t y that some or a l l of the re s i d u a l r e s p i r a t i o n was of mitochondrial o r i g i n could not be e n t i r e l y discounted, although i t i s highly u n l i k e l y given our understanding of mitochondrial r e s p i r a t i o n pathways. Two enzyme systems u t i l i z i n g molecular oxygen (catalase and lipoxygenase) have lead to some confusion i n i n t e r p r e t i n g r e s p i r a t o r y data obtained i n seed systems. Lipoxygenase (LOX) can account f o r a large proportion of t o t a l r e s p i r a t i o n i n some ti s s u e s (e.g. soybean cotyledons, Theologis and L a t i e s , 1978a). I t i s u n l i k e l y however that t h i s i s the case i n azide/cyanide treated wild oat seeds since: i ) LOX does not e x i s t to an appreciable extent i n wild oat seeds (Upadhyaya et a l . , 1983) and i i . ) LOX i s i n h i b i t i e d by SHAM (Parri s h and Leopold, 1978) which f a i l e d to i n h i b i t cyanide and azide induced r e s p i r a t i o n i n wild oats (Tabs. 13 and 16). 80 Hendricks and Taylorson (1975) found that at low concentrations cyanide i n h i b i t e d catalase but not r e s p i r a t i o n i n seeds of several species. They a t t r i b u t e d the promotion of germination by cyanide to elevated l e v e l s of peroxide. Others (Esashi et a l . , 1979a) have found no evidence of peroxide accumulation i n cyanide or azide treated seeds. In wild oats i t i s u n l i k e l y that catalase i n h i b i t i o n could account for the observed increase i n r e s p i r a t i o n upon cyanide or azide treatment. Since catalase a c t i v i t y evolves oxygen, catalase i n h i b i t i o n would be recorded polarographically as an increase i n oxygen consumption by the seeds due to change i n the concentration of dissolved oxygen. In t h i s s i t u a t i o n , oxygen uptake would be i n h i b i t e d by cyanide since r e s p i r a t i o n would s t i l l be cytochrome-mediated. Table 16 shows however, that cyanide at concentrations s u f f i c i e n t to completely i n h i b i t cytochrome-mediated r e s p i r a t i o n i n embryos excised from water-imbibed seeds had l i t t l e e f f e c t on the r e s p i r a t i o n of embryos excised from azide or cyanide treated seeds. c) Cytochrome-mediated r e s p i r a t i o n . I n t e r e s t i n g l y , cytochrome-mediated r e s p i r a t i o n of embryos excised from azide and cyanide treated seeds was s i m i l a r to that of the c o n t r o l (Tab. 16), implying that 1 mM azide or cyanide treatment (under the given conditions) did not i r r e v e r s i b l y i n h i b i t cytochrome oxidase. Although i t could be argued that during excision and s t i r r i n g of the embryos i n the oxygen uptake vessel, azide and cyanide 81 are leached from the t i s s u e , t h i s i s improbable since, i f the i n h i b i t o r s were l o s t during the measurement of oxygen uptake then one would expect to see a gradual increase i n r e s p i r a t i o n as the i n t e r n a l i n h i b i t i o r concentration decreased rather than a constant rate of r e s p i r a t i o n as was observed. A d d i t i o n a l l y , i n preliminary experiments where embryos were excised and placed i n P e t r i dishes containing azide, r e s p i r a t i o n was i n h i b i t e d and t h i s i n h i b i t i o n did not decrease during the measurement of oxygen uptake. S i m i l a r l y , r e s p i r a t i o n induced by azide or cyanide treatment i n i n t a c t seeds was not s e n s i t i v e to 1 mM azide or cyanide (applied following the i n i t i a l 24 h treatment - Tab. 13); i n f a c t renewing azide or cyanide s o l u t i o n further stimulated oxygen uptake by an a d d i t i o n a l 30 - 40%. Although concentrations of azide and cyanide used i n t h i s study give good i n h i b i t i o n of cytochrome-mediated r e s p i r a t i o n i n most tissues (Theologis and L a t i e s , 1978a,b), the seed coat may present a permeability b a r r i e r rendering the i n h i b i t o r concentrations used here i n s u f f i c i e n t to i n h i b i t cytochrome-mediated r e s p i r a t i o n . Taylorson and Hendricks (1973) found that cyanide and azide did not i n h i b i t cytochrome-mediated r e s p i r a t i o n at concentrations which promoted germination i n dormant l e t t u c e seed (0.1 mM). G. The e f f e c t of pH on the a c t i o n of azide and cyanide on seeds. During the course of t h i s study i t was reported that the a c t i v i t y 82 of cyanide and azide on seeds was pH-dependent (Cohn, 1985). Azide and cyanide are weak acids (hydrazoic a c i d , HN^ - pKa 4.7 and hydrocyanic a c i d , HCN - pKa 8.9 r e s p e c t i v e l y - James, 1953) upon d i s s o l u t i o n . Accordingly, decreasing pH w i l l s h i f t the molecule/ion equilibrium, increasing the proportion of undissociated molecules i n the s o l u t i o n . I t i s the undissociated molecule that i s able to passively d i f f u s e through the phospholipid phase of the plasraalemma (Simon and Beevers, 1952). Consequently, an increase i n the concentration of undissociated molecule i n the external medium w i l l r e s u l t i n a corresponding increase i n the i n t e r n a l concentration of the i n h i b i t o r . Once i n the cytoplasm, azide and cyanide both i n h i b i t cytochrome oxidase as undissociated molecules (James, 1953). Thus decreasing external pH w i l l enhance the i n h i b i t i o n of cytochrome oxidase by f a c i l i t a t i n g i n h i b i t o r d i f f u s i o n i n t o the c e l l by creating a steep concentration gradient and by increasing the concentration of the a c t i v e (undissociated) form of the i n h i b i t o r within the cytoplasm. 1. The e f f e c t of pH on the stimulation of r e s p i r a t i o n by azide and cyanide. Since d i s s o l u t i o n of azide, cyanide and SHAM i n d i s t i l l e d water r e s u l t s i n s u b s t a n t i a l pH change (Tab. 17), i t was of concern that some of the e f f e c t s of the r e s p i r a t o r y i n h i b i t o r s could be due i n part to i n d i r e c t e f f e c t s of pH changes rather than to d i r e c t e f f e c t s of these compounds on r e s p i r a t i o n . Experiments examining pH e f f e c t s were thus carried-out. Although seed r e s p i r a t i o n per se was uneffected by 83 the pH of the i m b i b i t i o n a l medium (pH 5-8, F i g s . 12 and 13), the e f f e c t of azide on seed r e s p i r a t i o n was strongly pH-dependent ( F i g . 12). At pH 5 and 6, azide concentrations greater than 0.0625 and 0.25 mM, r e s p e c t i v e l y , not only f a i l e d to stimulate seed r e s p i r a t i o n (as occurred at pH 7) but a c t u a l l y reduced oxygen uptake f a r below that of the c o n t r o l . Lower concentrations of azide were e f f e c t i v e at stimulating r e s p i r a t i o n at pH 5 and 6. Simon and Beevers (1952) found that the i n h i b i t i o n of r e s p i r a t i o n by hydrogen f l u o r i d e (pK =3.2) was extremely pH s e n s i t i v e . They a found that i n h i b i t i o n of r e s p i r a t i o n by t h i s compound could be a l t e r e d by i ) maintaining a constant concentration while a l t e r i n g pH or by i i ) changing i n h i b i t o r concentration while maintaining a constant pH (the i n h i b i t i o n curves f o r both were sigmoidal). The authors concluded that i t was the undissociated HF molecule that i n h i b i t e d r e s p i r a t i o n since low pHs increased i t s i n h i b i t o r y a c t i v i t y . An analogous s t i t u a t i o n appears to e x i s t with azide a c t i v i t y on seed r e s p i r a t i o n . I t seems highly l i k e l y that the i n h i b i t i o n of r e s p i r a t i o n by azide ( F i g . 12) i s due to i n h i b i t i o n of cytochrome-mediated r e s p i r a t i o n , implying that azide concentrations stimulating seed r e s p i r a t i o n do not i n h i b i t cytochrome oxidase. At pH 5 the undissociated azide concentration would be higher than at pH 6, thus where 0.25 mM azide stimulates r e s p i r a t i o n at pH 6, at pH 5 t h i s concentration i s high enough to i n h i b i t cytochrome oxidase and thus oxygen uptake. At pH 7, azide did not i n h i b i t seed r e s p i r a t i o n at concentrations as high as 2.0 mM; c l e a r l y azide i s a much l e s s potent i n h i b i t o r of cytochrome oxidase at t h i s pH. 84 In contrast to azide, the induction of r e s p i r a t i o n by cyanide was not strongly effected by change i n pH (5-7, F i g . 13). This i s expected since pH values used i n t h i s experiment were a l l below pKa f o r cyanide (8.9), consequently the external equilibrium was strongly i n favor of the undissociated HCN molecule. At low concentrations (0.065 to 0.25 mM) cyanide a c t i v i t y was enhanced somewhat by low pH. However, t h i s e f f e c t disappears at higher cyanide concentrations where the cyanide concentration was already s u f f i c i e n t l y high to give maximum res p i r a t o r y stimulation. The f a i l u r e of cyanide to i n h i b i t r e s p i r a t i o n at even the lowest pH and highest concentration may be due to the gradual l o s s of cyanide from the treatment plates (Yu et a l . , 1981). Consequently cyanide concentration may not have remained high enough for s u f f i c i e n t time to i n h i b i t cytochrome-mediated r e s p i r a t i o n . This i s probable since low pHs are known to favor the formation of gaseous HCN (Merck Index, 1983). Applied at pHs exceeding the pKa value of cyanide, i t i s l i k e l y that a decrease i n effectiveness (at stimulating seed r e s p i r a t i o n ) would have been observed at low concentrations. 2. Comparison of the e f f e c t of SHAM and pH on the action of azide and cyanide. Further examination of the e f f e c t of pH and buffe r i n g on azide a c t i v i t y revealed that at pH 5, 1 mM azide i n h i b i t e d r e s p i r a t i o n to the same extent as 1 mM azide + 10 mM SHAM i n d i s t i l l e d water (pH 5.2 - Tab. 17). This suggests the p o s s i b i l i t y that the i n h i b i t o r y e f f e c t of SHAM on the induction of r e s p i r a t i o n by azide i n an unbuffered 85 system may be an a r t i f a c t r e l a t e d to i t s capacity to a c i d i f y the so l u t i o n (Tab. 17) (thus increasing the concentration of undissociated azide) rather than through the i n h i b i t i o n of SHAM-sensitive processes (in c l u d i n g a l t e r n a t i v e r e s p i r a t i o n ) . I t i s possible that SHAM lowers pH to an extent such that the undissociated azide concentration i s increased to the point where 1 mM azide i s s u f f i c i e n t to i n h i b i t cytochrome-mediated r e s p i r a t i o n i n imbibing seeds ( i . e . as i n F i g . 12) This hypothesis would explain why, when buffered at pH 7, SHAM (applied simultaneously) had no e f f e c t on the stimulation of seed r e s p i r a t i o n by t h i s compound (Tab. 17). To tes t the hypothesis that SHAM e f f e c t s the stimulation of r e s p i r a t i o n by azide i n the same manner as d e c l i n i n g pH, the e f f e c t of SHAM and pH on the induction of seed r e s p i r a t i o n by d i f f e r e n t concentrations of azide were compared ( F i g . 14). I f the e f f e c t of SHAM on the induction of r e s p i r a t i o n by azide i s purely a pH e f f e c t then SHAM should e f f e c t the response of seeds to various azide concentrations d i f f e r e n t l y since r e s p i r a t o r y stimulation occurs only within a narrow concentration range of azide (Upadhyaya et a l . , 1983; F i g . 12). Concentrations of azide too low to increase r e s p i r a t i o n would become stimulatory since the concentration of the undissociated molecule would increase while concentrations stimulating r e s p i r a t i o n could become i n h i b i t o r y as the concentration of undissociated i n h i b i t o r becomes s u f f i c i e n t l y high to i n h i b i t cytochrome oxidase. The r e s u l t s i n F i g . 14 show t h i s to be the case. Azide ( i n d i s t i l l e d water) was e f f e c t i v e i n stimulating r e s p i r a t i o n at concentrations of 0.75 and 1.0 mM; SHAM (10 mM) i n h i b i t e d t h i s 86 stimulation. However, lower concentrations of azide (0.1 - 0.25 mM) that alone did not stimulate seed oxygen uptake, increased r e s p i r a t i o n i n the presence of 10 mM SHAM ( F i g . 14). This can be explained by pH changes. In d i s t i l l e d water, 0.25 mM azide i s sub-optimal for r e s p i r a t o r y stimulation ( F i g . 14), however when applied with 10 mM SHAM the s o l u t i o n pH declines (Tab. 17) r e s u l t i n g i n concentration of undissociated azide high enough to give maximal stimulation of oxygen-uptake. At azide concentrations higher than 0.25 mM, azide + 10 mM SHAM cause r e s p i r a t i o n to be i n h i b i t e d ( F i g . 14). Here d e c l i n i n g pH r e s u l t s i n dissociated azide concentrations s u f f i c i e n t l y high to competely i n h i b i t cytochrome oxidase. D i r e c t comparison of the e f f e c t of SHAM and of pH (alte r e d such that the pH of each azide s o l u t i o n was equal to that of azide + SHAM at a given azide concentration) on azide action supports t h i s hypothesis. When the pH of an azide s o l u t i o n was adjusted to mimic the drop i n pH incurred with simultaneous d i s s o l u t i o n of 10 mM SHAM, the change i n azide a c t i v i t y was p r a c t i c a l l y i d e n t i c a l to that observed with concurrent a p p l i c a t i o n of 10 mM SHAM. The displacement of the azide (pH altered) and azide + SHAM curves along the abcissa ( F i g . 14) could be due to small changes i n pH occurring i n the unbuffered azide + SHAM solutions over the 24 h incubation period, which would i n turn, a f f e c t azide a c t i v i t y . Given the lack of s e n s i t i v i t y of the induction of r e s p i r a t i o n by cyanide ( F i g . 13) at low pH, i t was not su r p r i s i n g that 10 mM SHAM applied concurrently with cyanide had no e f f e c t on the induction of 87 r e s p i r a t i o n by cyanide regardless of i t s concentration ( F i g . 15). Any increase i n the concentration of undissociated cyanide afforded by SHAM's e f f e c t on medium pH was not s i g n i f i c a n t with respect to the a c t i v i t y of cyanide on seed oxygen consumption. I t i s known that the undissociated and the i o n i c forms of azide and cyanide have d i f f e r e n t e f f e c t s on c e l l physiology. For example while the undissociated molecules of azide and cyanide i n h i b i t cytochrome oxidase, the ions of both i n h i b i t cytochrome c (James, 1953). The dual a c t i v i t y of azide and cyanide on wild oat seeds ( i . e . stimulating and i n h i b i t i n g seed r e s p i r a t i o n ) could conceivably be explained by the d i f f e r i n g actions of the ions and molecules of these i n h i b i t o r s . However, since decreasing pH (by SHAM addition, F i g . 14) enhances the stimulation of r e s p i r a t i o n by low concentrations of azide and i n h i b i t s i t by higher concentrations, the undissociated molecule of azide and cyanide appears to be responsible for both e f f e c t s . 3. The e f f e c t of pH on the release of seed dormancy by azide and cyanide. Upadhyaya et a l . , (1983) found that when azide was given to seeds at concentrations high enough to completely i n h i b i t r e s p i r a t i o n , the release of dormancy (and/or germination) was also i n h i b i t e d (Upadhyaya et a l . , 1982a). S i m i l a r l y , cyanide at concentrations high enough to i n h i b i t r e s p i r a t i o n does not stimulate germination ( F i g . 10). The i n h i b i t i o n of germination by 1 mM azide + 10 mM SHAM i s undoubtedly 88 due to the increase i n azide concentration afforded by SHAM; not nece s s a r i l y by blockage of the a l t e r n a t i v e pathway. This i s supported by the lack of germination of seeds treated with 1 mM azide at pH 5 where no blockage of the a l t e r n a t i v e pathway occurs (Tab. 18). A d d i t i o n a l l y , at pH 7, 10 mM SHAM f a i l e d to i n h i b i t the release of dormancy by 1 mM azide (data not shown). The f a i l u r e of SHAM to i n h i b i t the release of dormancy by cyanide can also be explained by pH. Since the the a c t i v i t y of cyanide i n rele a s i n g seed dormancy i s not strongly pH dependent (pH range 5 to 8, Tab. 19), a c i d i f i c a t i o n of the media that occurs when SHAM i s dissolved w i l l not de l e t e r i o u s l y a f f e c t the a b i l i t y of cyanide to promote seed r e s p i r a t i o n . The release of seed dormancy by cyanide, as with the stimulation of seed r e s p i r a t i o n , does not require a l t e r n a t i v e r e s p i r a t i o n . I t appears therefore, that the stimulation of r e s p i r a t i o n and germination by cyanide and azide requires cytochrome-mediated r e s p i r a t i o n regardless of whether or not the a l t e r n a t i v e pathway i s blocked by SHAM. This can be deduced from Tables 17 and 18. When azide i s applied to seeds at concentrations high enough to i n h i b i t cytochrome-mediated r e s p i r a t i o n ( i . e . 1 mM at pH 5, Tab. 17), the stimulation of r e s p i r a t i o n i s prevented as i s the release of seed dormancy (Tab. 18). Thus, i t appears that the mechanism(s) whereby r e s p i r a t i o n and germination are stimulated i s not rela t e d to the i n h i b i t i o n of cytochrome oxidase by azide and cyanide. 89 H. General discussion. What then, i s the status of the a l t e r n a t i v e pathway i n wild oat seeds? Although a l t e r n a t i v e r e s p i r a t i o n does not appear to be involved i n the regulation of seed dormancy i n wild oats, i n t a c t seeds (as well as excised embryos, Tab. 6, 16) have the a b i l i t y to d i v e r t electrons v i a t h i s pathway when the cytochrome pathway i s blocked. This can be deduced by examining Tables 14 and 15. Azide (5 mM) + 10 mM SHAM i n h i b i t e d r e s p i r a t i o n more than 5 mM azide alone. Since i n the absence of SHAM, increasing azide concentration beyond 5 mM did not further i n h i b i t r e s p i r a t i o n (Tab. 14) i t can be concluded that maximum i n h i b i t i o n of cytochrome r e s p i r a t i o n occurs at 5 mM. Thus SHAM did not increase the i n h i b i t i o n of r e s p i r a t i o n by 5 mM azide by causing a pH decline and a consequent increase i n the undissociated azide concentration. Therefore, a l t e r n a t i v e r e s p i r a t i o n can occur i n wild oat seeds when the cytochrome pathway i s completely blocked; the maximal f l u x of the a l t e r n a t i v e pathway i n these seeds i s however, only 15-20% of the CN-sensitive pathway (Tab. 16). Others have also found a s i m i l a r i l y low capacity of the a l t e r n a t i v e pathway i n seeds ( M i l l e r et a l . , 1983). C l e a r l y , the i n t e r p r e t a t i o n of r e s u l t s obtained using SHAM as an i n h i b i t o r of a l t e r n a t i v e r e s p i r a t i o n must be made with caution. Not only does SHAM i n h i b i t enzymes such as lipoxygenase, peroxidase and tyrosinase (Rich et a l . , 1978) but a recent study (Upadhyaya, 1986) shows that SHAM at concentations frequently used i n seed studies i n h i b i t s the GA^-induced <x-amylase production and the subsequent release of reducing sugars. The i n h i b i t o r y e f f e c t of SHAM alone on 90 seed germination (Yentur and Leopold, 1976; Yu et al.,1979; Esashi et al.,1980) may not be e n t i r e l y due to the i n h i b i t i o n of the a l t e r n a t i v e oxidase but may involve some secondary e f f e c t s as w e l l . The r e s u l t s of t h i s study h i g h l i g h t the need to challenge several assumptions often made i n studies i n v o l v i n g seeds and resp i r a t o r y i n h i b i t o r s . Cyanide and azide concentrations frequently employed to i n h i b i t cytochrome oxidase i n bulky s l i c e d t i s s u e s (Theologis and La t i e s , 1978 a,b) may not be s u f f i c i e n t to i n h i b i t cytochrome oxidase i n i n t a c t seed. Levels of cyanide and azide stimulating r e s p i r a t i o n and germination do not appear to i n h i b i t cytochrome-mediated r e s p i r a t i o n i n wild oat seeds; conclusions based on seed studies using s i m i l a r i n h i b i t o r concentrations must be reevaluated (Adkins et al_., 1984c,d; Zagerski and Lewak, 1983). When using azide and cyanide at concentrations know to e f f e c t other enzymes ( i . e . formic dehydrogenase, uricase, catalase and peroxidase; James, 1953), caution must be taken not to a t t r i b u t e a l l of the e f f e c t s of these i n h i b i t o r s to the i n h i b i t i o n of cytochrome oxidase. Respiratory i n h i b i t i o n by azide i n the presence of SHAM when neither compound i n h i b i t s r e s p i r a t i o n i n d i v i d u a l l y ( i n an unbuffered system), may erroneously be taken as evidence of a l t e r n a t i v e r e s p i r a t i o n occurring i n the presence of azide alone. Adkins et a l . (1984 c,d), using an unbuffered system, found that neither 1 mM azide or 10 mM SHAM i n h i b i t e d the induction of r e s p i r a t i o n by ethanol and n i t r a t e i n wild oat seeds. However, when azide and SHAM were applied i n combination, r e s p i r a t i o n was strongly i n h i b i t e d (>90%). The authors explanation was as follows: ethanol and n i t r a t e ions can 91 stimulate seed r e s p i r a t i o n v i a the cytochrome or the a l t e r n a t i v e pathway, eit h e r providing the ATP necessary to support germination. When azide (1 mM) i s applied simultaneously with ethanol or n i t r a t e , r e s p i r a t i o n proceeds v i a the a l t e r n a t i v e pathway; on the other hand, when SHAM (10 mM) i s applied concurrently with ethanol or n i t r a t e , r e s p i r a t i o n i s e n t i r e l y cytochrome-mediated. Consequently, when both azide and SHAM are applied with n i t r a t e or ethanol, r e s p i r a t i o n cannot proceed because both re s p i r a t o r y pathways are blocked. In l i g h t of the r e s u l t s of the present study, i t seems highly probable that the r e s p i r a t i o n induced by n i t r a t e and ethanol was i n h i b i t e d by a combination of azide and SHAM by v i r t u e of complete blockage of the cytochrome pathway ( i . e . by increasing the undissociated azide concentration) and that any blockage of the a l t e r n a t i v e pathway was immaterial to the observed r e s p i r a t o r y i n h i b i t i o n . I f t h i s e x p l a i n t i o n i s corr e c t , then i t appears that, as with azide and cyanide, cytochrome-mediated r e s p i r a t i o n i s probably necessary f o r the stimulation of germination and r e s p i r a t i o n by ethanol and n i t r a t e . Adkins el: a l . (1984 c,d) f a i l e d to d i s t i n q u i s h between the nature of the induction of r e s p i r a t i o n by n i t r a t e and ethanol and the nature of the induced r e s p i r a t i o n . Although azide and SHAM applied simultaneously with ethanol or n i t r a t e i n h i b i t e d r e s p i r a t i o n , whether azide, SHAM or a combination of both would have i n h i b i t e d the stimulated oxygen uptake ( i . e . applying r e s p i r a t o r y i n h i b i t o r s subsequent to the a p p l i c a t i o n of n i t r a t e or ethanol) was not studied. 92 Given the s i m i l a r i t y ( i . e . very s i m i l a r time course and s e n s i t i v i t y to r e s p i r a t o r y i n h i b i t o r s ) of the stimulation of wild oat seed germination and r e s p i r a t i o n by azide, cyanide, ethanol and n i t r a t e i t seems highly probable that the o r i g i n of oxygen uptake stimulated by these chemically diverse compounds i s the same. I f t h i s i s the case i t would be tempting to put f o r t h the hypothesis that while the mode of action of these chemicals i n r e l e a s i n g seed dormancy might d i f f e r , t h e i r d i r e c t or i n d i r e c t stimulation of some nonmitochondrial source of oxygen-consumption might ultimately be responsible (or necessary) f o r the subsequent release of seed dormancy i n wild oats. At t h i s time the nature of the induced, nonmitochondrial oxygen-consumption remains a mystery. I t would be valuable to pinpoint i t s source i n order to further e s t a b l i s h a l i n k between i t and the release of seed dormancy i n wild oats and i n other species. 93 CONCLUSIONS A. The induction and release of secondary seed dormancy. 1. As with primary dormancy, pure l i n e s (both nondormant and dormant) of wild oat exhibit genetic v a r i a b i l i t y i n t h e i r secondary dormancy behaviour and factors l i k e temperature can modify the expression of t h i s t r a i t . 2. Treatments e f f e c t i v e at releasing primary dormancy ( i . e . chemicals, af t e r - r i p e n i n g ) also released secondary dormancy, suggesting that the two types of dormancy are s i m i l a r , at l e a s t i n part, i n t h e i r regulation. 3. A l t e r n a t i v e (SHAM-sensitive) r e s p i r a t i o n i s not involved i n regulating the induction or release of primary and secondary dormancy. B. The e f f e c t r e s p i r a t o r y i n h i b i t o r s on seed r e s p i r a t i o n and the release of dormancy. 1. Azide and cyanide stimulate seed r e s p i r a t i o n and germination at si m i l a r concentrations and treatment durations and appear to act by the same mode of action. Cyanide, however, was more e f f e c t i v e at r e l e a s i n g dormancy i n f r e s h l y harvested seeds; azide was impotent at releasing primary dormancy i n these seeds. 2. Although the release of dormancy by cyanide i s always preceded by an increase i n seed oxygen consumption, a causal r e l a t i o n s h i p between the two could not be established. 3. The induction of seed r e s p i r a t i o n by azide (but not by cyanide) was completely i n h i b i t i e d by simultaneous a p p l i c a t i o n of SHAM. However, once induced, both azide and cyanide stimulated 94 oxygen consumption were i n s e n s i t i v e to SHAM. Thus, the stimulated r e s p i r a t i o n i s not a l t e r n a t i v e and was found to be r e s i d u a l i n nature. C. The e f f e c t of pH on the stimulation of r e s p i r a t i o n and germination by r e s p i r a t o r y i n h i b i t o r s . 1. The action of azide on seed r e s p i r a t i o n and germination i s extremely pH-sensitive. Decreasing pH increases the concentration of undissociated (active) azide which i n turn e f f e c t s the action of azide on seeds. A si n g l e concentration of azide can be either stimulatory or i n h i b i t o r y to r e s p i r a t i o n and germination depending on the pH of the so l u t i o n . Evidence suggests that azide and cyanide at concentrations stimulating seed r e s p i r a t i o n and germination, do not i n h i b i t cytochrome oxidase. 2. I n h i b i t i o n by SHAM of the stimulation of r e s p i r a t i o n and germination by azide was due to a c i d i f i c a t i o n of the medium upon d i s s o l u t i o n of 10 mM SHAM. This increases the concentration of undissociated azide molecules, r e s u l t i n g i n i n h i b i t i o n of both cytochrome oxidase and consequently, germination. Any blockage of the a l t e r n a t i v e pathway i s i n c i d e n t a l to the f a i l u r e of azide to release seed dormancy or stimulate r e s p i r a t i o n i n the presence of SHAM. 3. 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