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Embryo-independent mobilization of endosperm starch in cereal seeds Konesky, David William 1990

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EMBRYO-INDEPENDENT MOBILIZATION OF ENDOSPERM STARCH IN CEREAL SEEDS by DAVID WILLIAM KONESKY A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Dept. of Plant Science) We accept th is thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA ©Dave Konesky, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. 1 further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) i i ABSTRACT Endosperm mobi l izat ion studies in cereal seeds are t y p i c a l l y based on the view tha t a-amylase s y n t h e s i s in the a l eu rone t i s s u e i s c o n t r o l l e d by g i b b e r e l l i n s from the germinat ing embryo. However, a-amylase i s often produced by de-embryonated endosperm segments in the absence of added g i b b e r e l l i n s . Two s p e c i f i c systems exh ib i t ing th is phenomenon were examined; 1) the a b i l i t y of amino ac ids to promote a-amylase p roduc t i on in de-embryonated w i l d oat segments and, 2) autonomous starch hydrolys is ( in the absense of exogenous GA3 or amino acids) in de-embryonated barley endosperm halves. S p e c i f i c technical problems were addressed p r io r to the onset of these s tud ies. Captan (66 /zM) contro l led fungal contamination in the incubat ion medium without i n h i b i t i n g GA^-induced sugar re lease, which occurs fo l lowing seed s t e r i l i z a t i o n in hypochlor i te and ethanol . The Nelson-Somogyi reducing sugar assay was not su i tab le for quant i fy ing sugar leve ls in incubation solut ions containing amino acids as cyste ine, c y s t i n e , s e r i n e , tryptophan and ty ros ine in te r fe red with the assay. Absorbance (540 nm) increased as concentrations increased from 0.1 to 1 mM; simultaneous add i t i ons of amino ac ids with glucose resu l ted in absorbance values higher than glucose alone. MnC^ (0.5 mM) inh ib i ted absorbance in the presence of g lucose and the amino ac ids se r i ne , cys t ine , and tryptophan. The a b i l i t y of i n d i v i d u a l amino ac i ds to promote a-amylase product ion in w i l d oat endosperm halves i s yet unc lea r . Although cer ta in amino acids were shown to enhance enzyme production, the level i i i was of ten qu i te d i f f e r e n t between rep l i ca ted experiments. However, i n c u b a t i o n of endosperm h a l v e s in a m i x tu re of 18 amino a c i d s cons is tent ly promoted a-amylase synthesis ; enzyme production was further . g enhanced i f a leve l of GA 3 (10 M), which was too low to promote a-amylase synthesis alone, was included wi th in the amino acid mixture. Autonomous endosperm mobi l izat ion (AEM) was var iab le in d i f fe rent barley c u l t i v a r s . High sugar re lease cor re la ted wel l with a-amylase production; the leve ls were s im i l a r among indiv idual cu l t i va rs harvested in two d i f fe rent seasons. The onset of AEM was delayed, as the majority of sugar was released in the second day of incubat ion. AEM was great ly reduced by i n h i b i t o r s o f RNA ( 6 - m e t h y l p u r i n e ) and p r o t e i n (cycloheximide) synthesis suggesting that AEM was a resu l t of the de novo synthesis of a-amylase. Incubation condi t ions g rea t l y a f fected AEM. Al though AEM was high at a c i d i c pH (4 .6 -5 .6 ) , i t was g rea t l y reduced at basic pH (7 .6 -8 .6 ) . AEM increased as the temperature was raised to 28°C. Low leve ls of C a 2 + (0.25-0.5 mM) enhanced AEM whereas higher amounts (0.5-1 mM) were i nh ib i t o ry . I n c u b a t i o n of w i l d oat endosperm h a l v e s i n s o l u t i o n s of pre-emergence herbic ides affected GA^-induced sugar release to varying extents when applied at f i e l d app l icat ion l e v e l s . Only t r i a l l a t e (22% reduction) and t r i f l u r a l i n (21% reduction) prevented sugar release (only - 5 at 3x10 M); no i n h i b i t i o n was seen f o l l o w i n g incubat ion in EPTC (5x l0~ 5 , 5 x l 0 " 6 M), metribuzin ( 5 x l 0 " 6 , lxlO~ 6M) and oryza l in (3x l0~ 5 , 3x10 ^ M). However, the h igher c o n c e n t r a t i o n s of each h e r b i c i d e e f fec t i ve l y inh ib i ted the development of wi ld oat seedl ings. i v TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES vi LIST OF FIGURES v i i ACKNOWLEDGEMENT . . ix INTRODUCTION 1 CHAPTER 1 ENHANCEMENT OF a-AMYLASE PRODUCTION BY AMINO ACIDS IN CEREAL ENDOSPERM HALVES 1.1 Introduction 1.1.1 Enhancement of a-amylase production by amino acids in cereal endosperm halves 10 1.1.2 Technical problems associated with endosperm mobi l iza t ion studies 13 1.1.2.1 Control of seed contamination 13 1.1.2.2 Amino acid a c t i v i t y in the Nelson-Somogyi glucose assay 14 1.1.2.3 Inh ib i t ion of a-amylase production by prol ine 15 1.2 Mater ia ls and methods 1.2.1 Seed source 16 1.2.2 Seed incubation 16 1.2.3 Measurement of release of reducing sugars 18 1.2.4 Extract ion and assay of a-amylase 19 1.2.5 S t a t i s t i c a l analys is 20 1.3 Results 1.3.1 Seed s t e r i l i z a t i o n techniques 21 1.3.2 Amino acid a c t i v i t y in the Nelson-Somogyi glucose assay 21 1.3.3 Prol ine interference in a-amylase production 29 1.3.4 Enhancement of a-amylase production by indiv idual amino acids .29 1.3.5 Interact ions between GA^ and amino acids 29 1.4 Discussion 35 V CHAPTER 2 AEM IN BARLEY ENDOSPERM TISSUE 2.1 Introduction 42 2.2 Mater ia ls and methods... 46 2.3 Results 2.3.1 V a r i a b i l i t y of AEM among barley cu l t i va rs 49 2.3.2 Inh ib i t ion of RNA and protein synthesis 49 2.3.3 Effect of pH and temperature changes on AEM 55 2.3.4 Time course of onset of AEM 55 2.3.5 Effect of calcium on AEM 55 2.4 Discussion " 61 CHAPTER 3 EFFECT OF RESIDUAL HERBICIDES IN SOIL ON GA,- INDUCED a-AMYLASE PRODUCTION . 3.1 Introduction 67 3.2 Mater ia ls and methods 3.2.1 Measurement of release of reducing sugars 69 3.2.2 Growth studies 70 3.3 Results 3.3.1 Effect of herbicides cn sugar release 71 3.3.2 Effect of herbicides on seedling growth 71 3.4 Discussion 79 CONCLUSIONS 81 LITERATURE CITED 84 APPENDIX A 91 v i LIST OF TABLES CHAPTER 1 Page Table 1.1 Table 1.2 Effects of various methods of seed s t e r i l i z a t i o n on reducing sugar release and length of fungal- f ree incubation period in wi ld oat ( vAN51') Effect of MnCl ? on co lor formation in the presence of glucose and some amino acids Table 1.3 Time of amino acid addi t ion in the Nelson-Somogyi reaction procedure Table 1.4 Effect of pro l ine on amino acid-promoted a-amylase production in wi ld oat ( vAN51') Table 1.5 Effect of serine and aspar t ic acid on reducing sugar release in wi ld oat (*AN51') Table 1.6 a-amylase release fo l lowing incubation with a mixture of 18 amino acids with or without GA, in wi ld oat and barley 22 27 28 30 31 33 CHAPTER 2 Table 2.1 AEM among d i f fe ren t barley cu l t i va r s Table 2.2 Effect of exogenous GA, on reducing sugar release in barley endosperm halves Table 2.3 a-amylase production and reducing sugar release from x Klages ' endosperm t issue Table 2.4 Effect of RNA and protein synthesis inh ib i to rs on autonomous reducing sugar release in 'K lages ' 50 51 53 54 CHAPTER 3 Table 3.1 Effect of 5 herbicides on GA,-induced reducing sugar release ( vAN51') Table 3.2 Effects of various concentrations of t r i a l l a t e and t r i f l u r a l i n on GA,-induced reducing sugar release ( vAN51') * 72 73 LIST OF FIGURES CHAPTER 1 Figure 1.1 Figure 1.2 Figure 1.3 Figure 1.4 Figure 1.5 CHAPTER 2 Figure 2.1 Figure 2.2 Figure 2.3 Figure 2.4 Figure 2.5 Figure 2.6 Embryo and scutellum l o c a l i z a t i o n in wi ld oat ( XAN51') fo l lowing incubation in tetrazol ium Color development in the presence of 0.5 mM amino acids Standard curves for co lor development in the presence of selected amino acids Glucose standard curves in the presence of cysteine and serine Reducing sugar release in wi ld oat fol lowing incubation in d i f fe rent GA., concentrations ('AN51') * Embryo and scutellum l o c a l i z a t i o n in barley ( 'K lages ' ) fo l lowing incubation in tetrazol ium Autonomous reducing sugar release among barley cu l t i va rs harvested in two d i f fe rent growing seasons Effect of pH on autonomous reducing sugar release Effect of temperature and pH on autonomous reducing sugar release Time course of autonomous reducing sugar release Effect of calcium on autonomous reducing sugar release v i i i CHAPTER 3 Page Figure 3.1 Inh ib i t ion of wi ld oat growth by t r i a l l a t e 74 Figure 3.2 Inh ib i t ion of wi ld oat growth by EPTC 74 Figure 3.3 Inh ib i t ion of wi ld oat growth by metribuzin 74 Figure 3.4 Inh ib i t ion of wi ld oat growth by t r i f l u r a l i n 74 Figure 3.5 Inh ib i t ion of wi ld oat growth by oryza l in 74 Figure 3.6 Wild oat emergence fo l lowing appl icat ion of t r i a l l a t e 75 Figure 3.7 Wild oat emergence fo l lowing appl icat ion of 3x10° M t r i a l l a t e 75 Figure 3.8 Wild oat emergence fo l lowing appl icat ion of metribuzin 77 Figure 3.9 Delayed necrosis of wi ld oat seedlings fol lowing appl icat ion of metribuzin 77 Figure 3.10 Inh ib i t ion of wi ld oat seedling growth fol lowing appl icat ion of d i f fe rent t r i a l l a t e concentrations 78 Figure 3.11 Inh ib i t ion of wi ld oat seedling growth fol lowing appl icat ion of d i f fe rent t r i f l u r a l i n concentrations 78 i x ACKNOWLEDGEMENTS I wish to express my sincere appreciat ion to Dr. M.K. Upadhyaya for his assistance and guidance throughout the course of th is study. Special a p p r e c i a t i o n is also extended to Dr. I .E .P . Taylor (Department of Botany, U . B . C ) , Dr. M.B. Isman (Department of Plant Science, U . B . C ) , and Dr. R . J . Copeman (Chairman) for the i r presence on my thesis committee. I wish to thank Dr. Upadhyaya for f inanc ia l support rec ieved through his National Research Council of Canada grant. I am espec ia l l y gratefu l for the encouragement and emotional support expressed by my fami ly and f r iends without which t h i s work would have ended prematurely. 1 INTRODUCTION An immense body of information ex is ts on endosperm mobi l izat ion in c e r e a l g r a i n s . Most s t u d i e s are based on the p r e m i s e t h a t embryo-produced hormones promote enzyme product ion in the aleurone t i s sue . This perspective arose fo l lowing pioneering work by Paleg (1960 a,b) and Yomo (1960) in which i t was demonstrated that g i b b e r e l l i c acid (GAg) could promote l e v e l s of endosperm m o b i l i z a t i o n in ha l f - seeds ( f o l l ow ing removal of the embryo) s i m i l a r to those in en t i re seeds during germination. A rapid and almost complete hydro lys is of s tarch resul ted and reducing sugars were released into the external medium. The work of Varner et a l . (1965) led to the important discovery that GA 3 c o n t r o l l e d the de. novo syn thes is of a-amylase and o ther hydro lases ( i . e . proteases, RNA-ases, and acid phosphatases) in the aleurone l a y e r , which hydro lyzed endosperm storage r e s e r v e s . They hypothesized that hormones were t ranspor ted from the embryo to the aleurone c e l l s , where they stimulated enzyme production. a-Amylase and protease a c t i v i t y in the aleurone were dependent on de novo synthesis of protein which in turn depended on synthesis of new mRNA (Varner 1964; Chrispeels and Varner 1967a). G ibbere l l i n (GA) was considered a general t r i gge r i ng agent for t h i s i n i t i a l synthesis of p ro te ins in aleurone c e l l s . Several l i nes of evidence substantiated the f indings of Varner et a l . (1965). De novo synthesis could be prevented by i n h i b i t o r s of DNA-dependent RNA syn thes is wh i le newly formed a-amylase could be " f inger p r in ted" by supplying the aleurone with r a d i o - l a b e l l e d amino 2 acids and GAg (Varner and Chandra 1964). Further proof was obtained fol lowing density l a b e l l i n g of the enzyme in GAg-treated barley aleurone 1 g layers cu l tured in D20 or H20 (F i lner and Varner 1967; Jacobsen and Varner 1967). The resu l ts suggested that newly synthesized enzyme was der ived from the amino ac ids in pools produced by the breakdown of reserve p ro te ins . Newer approaches aimed at understanding hormonal cont ro l of endosperm mob i l i za t ion included examination of the d i rec t e f fect of GA on a-amylase mRNA synthesis. GA treatment increased mRNA s p e c i f i c fo r a-amylase (Jacobsen and Zwar 1974; Ho and Varner 1976); increased l e v e l s of GA-dependent mRNA were cor re la ted with a-amylase synthesis (Higgins et a l . 1976). A number of GAs were synthesized in bar ley seeds fo l lowing the onset of ge rmina t ion , r a i s i n g GA l e v e l s from the r e l a t i v e l y smal l amounts in the mature embryo (Bi lderback 1971; Jacobsen and Chandler 1987). GAj was reported t o ' be the prominent GA in bar ley embryos, however, small amounts of GAg, GAj^, GAjg, GAg^, and GA^ g also occurred wi thin the seed (Macleod and Palmer 1966; Radley 1967; Gaskin et a l . 1984). When appl ied ex te rna l l y to dormant seeds, the most e f fec t ive g ibbere l l i ns in inducing endosperm mobi l izat ion were GAg, GA^, and GA^ (Mayer and Poljakoff-Mayber 1989). Although i t i s gene ra l l y accepted that g i b b e r e l l i n s induce the production of enzymes responsible for endosperm degradation, the s i t e of hormone synthesis i s unclear. Indirect evidence from ear ly experiments suggested that GA from the embryo induced enzyme syn thes is in the scutellum and aleurone t issues (Varner et a l . 1965). The ' t ranspor t ' of th is hormone was considered to occur by d i f fus ion in f ree space or by 3 symplastic movement v ia piasmodesmata to the aleurone t i ssue . However, more recent work using highly sens i t i ve radio-immunoassays for hormones chal lenged the hypothesis that g i bbe re l l i n s were transported from the embryo to the aleurone (Weiler and Wieczorek 1981). These r e s u l t s suggested that GA^ synthesis occurr ing in the bar ley aleurone t issue d i r e c t l y c o n t r o l l e d the s y n t h e s i s o f a - a m y l a s e . However, t h i s hypo thes is of aleurone l o c a l i z e d syn thes is of GA requ i res f u r t he r substant iat ion before the or ig ina l concept of embryonic control can be set aside or modified (Atzorn and Weiler 1983c). Reserve degradation and mobilization During ear ly seedling development, stored reserves in the endosperm and aleurone (prote in, carbohydrate, l i p i d , and mineral complexes) are degraded by enzymes from the s c u t e l l u m and a leurone t i s s u e s and t rans fe r red v ia the scutel lum to the develop ing s e e d l i n g . A la rge number of enzymes respond to GAs in the germinating cereal seed. They have been categor ized in to four groups (Jacobsen 1983). The f i r s t g roup , f o r which t h e r e i s l i t t l e i n f o r m a t i o n c o n c e r n i n g t h e i r regu la t ion , includes enzymes involved in phospholipid metabolism. These are character ized by a rapid change in a c t i v i t y , with l i t t l e synthesis of new enzyme. The second group includes enzymes, such as a-amylase and protease, whose synthesis and secret ion are stimulated by GA. The th i rd group includes /3-glucanase, acid phosphatase, and r ibonuclease enzymes whose a c t i v i t i e s increase by new synthesis in the absence of added GA and which may show addi t ional a c t i v i t y i f GA is present. F i n a l l y , there are some enzymes (xylopyranosidase and arabinosidase) whose leve ls are constant in the absence of GA, but increase in the presence of GA. 4 The enzymes r e s p o n s i b l e f o r endosperm p r o t e i n and s t a r c h mobi l izat ion have been studied most extens ive ly . Reserve proteins are most abundant in the starchy endosperm (Mikola 1983) and are mobil ized through a concerted action of many pro teo ly t i c enzymes act ing in three phases. F i r s t , there i s an i n i t i a l hydrolysis of storage proteins in the scutellum and aleurone layers which provides f ree amino acids fo r the synthesis of new hydro ly t ic enzymes to be secreted into the starchy endosperm. The second phase, which involves the hydrolysis of endosperm p ro te ins , i s fo l lowed by the t h i r d phase in which small peptides and amino ac ids are taken up by the scu te l l um. Some amino ac ids are incorpora ted in to new enzymic prote in whi le others are t rans located without fur ther metabolism from the scutellum to the growing seedl ing. Starch hydrolysis proceeds through the action of several d i f fe rent enzymes which break down amylose and amylopect in in s ta rch g ra ins (MacLeod et a l . 1969). I n i t i a l degradation of amylose and amylopectin involves a-amylase, which hydrolyzes a ( l -4 ) l inkages to y i e l d D-glucose, maltose and d e x t r i n . The two l a t t e r sugar forms are not hydrolyzed fu r ther by a-amylase, which cannot attack branchpoint a ( l -6 ) l inkages. However, a ( l -6 ) glucosidase and l i m i t dextr inase (debranching enzymes) expose another t i e r of a ( l -4 ) l inkages to a-amylase fo l lowing hydrolysis of these branch l inkages. The combined actions of these enzymes convert amylose and amylopect in to g lucose and a small amount of maltose (degraded by maltase). In contrast to a-amylase, a-glucosidase, l i m i t dextr inase, and maltase,- which are a l l synthesized in the aleurone layer fo l lowing germinat ion, ^-amylase e x i s t s in the endosperm of the dry grain as a resu l t of synthesis during development of the grain (Tronier and Ory 1970). It c leaves amylopectin in to maltose un i ts which are 5 degraded to glucose by maltase. Genetic control of a-amylase Exogenous GAg induces barley aleurone t issue to synthesize various isozymes of a-amylase which can be c l a s s i f i e d into two d i s t i n c t groups (Jacobsen and Higgins 1982). A low pl group appears f i r s t followed by a high pl group which accumulates rap id ly to become the dominant isozyme g r o u p . Low p l i s o z y m e s a p p e a r a t l ow GA c o n c e n t r a t i o n and show r e l a t i v e l y l i t t l e response to increasing GA l e v e l s , while high pl forms - 8 are not p resen t at low GA (10" M) c o n c e n t r a t i o n s but i n c r e a s e subs tan t ia l l y at higher GA concentrat ions. These forms of a-amylase are not contained in dry gra ins . Seed endosperm mobi l iza t ion studies in w i ld oat Understanding the complexity of embryonic control i s very important in weedy spec ies such as w i l d oa t . In Canada, w i l d oat p resents g rea tes t problems in A l b e r t a , Saskatchewan and Manitoba where 17.3 m i l l i on hectares of arable land are infested (Alex 1966). Annual crop losses and herb ic ide costs a t t r ibu tab le to wi ld oat in Western Canada alone have been estimated at $280 m i l l i on (Dew 1978). Wild oat seeds usua l ly mature before the crop and become mixed among the crop seeds reducing the value of the product. Many new seeds are added to the s o i l seed bank each year . Although a large number germinate in the f i r s t year a f ter production, the extended v i a b i l i t y of those seeds remaining in the s o i l seems to ar ise from features involv ing 6 the hormonal control of endosperm metabolism (Naylor 1969). In w i l d oat , seed dormancy i s a major su rv iva l fac to r (Simpson 1978). In those caryopses remaining v i a b l e , one requirement f o r s u r v i v a l i s the preservat ion of endosperm reserves un t i l the embryo becomes capable of germination e i ther through loss of dormancy or some environmental change. There must be a strong physio logical mechanism which renders the synthesis of h y d r o l y t i c enzymes in the endosperm r igorous ly dependent on hormones produced by the embryo during the onset of germination (Naylor 1969). Unl ike bar ley, the structure and function of w i l d oat endosperm and aleurone have not been wel l i nves t iga ted . However, the ro les of these t issues appear s im i l a r in bar ley and wi ld oat. Further understanding of seed dormancy and endosperm mobi l izat ion in wi ld oat seeds may suggest improved control methods for the weed. Seed endosperm mobi l izat ion studies in barley Barley i s commonly used for endosperm mobi l izat ion studies because the seeds have been w e l l c h a r a c t e r i z e d b i o c h e m i c a l l y and are economica l l y important in the brewing i n d u s t r y . The embryonic , endosperm and aleurone t i ssues are c l e a r l y d i s t i n g u i s h a b l e , with the aleurone t issue exhib i t ing high a-amylase production in response to GAs. In brewing, the pr inc ipa l aim of the malting process is to act ivate and increase production of enzymes present in the bar ley ke rne l . Barley endosperm mobi l izat ion must be car r ied out in a contro l led environment (Marschal l et a l . 1982; Enari and Sopanen 1986). Hydro l y t i c enzyme production and grain degradation are prec ise ly monitored to maintain the qua! i ty of beer. 7 Gibberel l in- independent production of a-amylase The concepts expla in ing control of endosperm mobi l izat ion suggest that enzyme synthesis in the aleurone and scutel lum is con t ro l l ed by hormones produced in the embryo. However, the fac t that c e r t a i n c u l t i v a r s e x h i b i t i nc reased p roduc t i on of a-amylase by i s o l a t e d endosperm t issue in the absence of exogenously applied GA (Naylor 1966) suggests that other mechanisms, not d i r e c t l y re la ted to the embryo, c o n t r o l t h i s endosperm h y d r o l y s i s . Th i s autonomous endosperm mob i l i za t i on (AEM), which i s widespread and v a r i a b l e among c e r t a i n species (Naylor 1969; MacGregor 1976; MacGregor et a l . 1983; N icho l ls et a l . 1986), forces one to reassess the ro le of the embryo in the control of endosperm mob i l i za t ion . The a v a i l a b i l i t y of f ree amino acids i s important in AEM because addit ions of exogenous amino acids often enhance endosperm hydro lys is wi thout added GA (Naylor 1966). Iso la ted w i ld oat aleurone t i ssue incubated in a mixture of the 20 protein amino acids showed increased a-amylase a c t i v i t y (Naylor 1969) suggesting that the level of avai lab le amino acids affected AEM. The understanding of how GA^ and amino acids in f luence t h i s embryo-independent enzyme production i s very important not only to explain the complex processes of endosperm mobi l izat ion but because of the negative impl icat ions (see sect ion 2.1) that AEM has for cer ta in industr ies ( i . e . bread-making, brewing). Two spec i f i c systems ( i . e . wi ld oat, barley) which exh ib i t AEM were inves t iga ted in my study to determine the extent of t h i s phenomenon among bar ley c u l t i v a r s and the importance of GA, and amino ac id 8 a v a i l a b i l i t y for promoting th is endosperm mob i l i za t ion . The a b i l i t i e s of both i n d i v i d u a l and mixtures of amino ac ids to increase enzyme product ion in w i ld oat and barley endosperm t issue were examined. In add i t ion , a number of barley cu l t i va rs were screened to determine the extent of AEM wi th in one spec ies . Incubation condi t ions under which th is c h a r a c t e r i s i t i c was expressed were also examined. I n h i b i t i o n of endosperm m o b i l i z a t i o n i s a t a rge t f o r c e r t a i n herb ic ides as a number of pre-emergence herb ic ides (see sect ion 3.1) a f fec t ear ly enzyme synthesis in bar ley. This i nh ib i t i on of GAg-induced enzyme product ion (Rao and Duke 1976) often reduces seedl ing v igo r . Herbicides may also reduce endosperm mobi l izat ion in weedy species such as w i l d oat which e x h i b i t s i m i l a r mechanisms of s to rage reserve d e g r a d a t i o n . However, the a f f e c t s on endosperm m o b i l i z a t i o n of herbic ides commonly used to control wi ld oat have not yet been examined. The object ives of th is study were to determine; 1. the a b i l i t y of i nd i v idua l and mixtures of amino acids to promote a-amylase synthesis in wi ld oat and bar ley, 2. the in te rac t ions between GAg and amino acids for enhanced enzyme production in wi ld oat and bar ley, 3. AEM among 17 barley c u l t i v a r s , 4. incubation condit ions for detect ion of AEM in barley c u l t i v a r s , 5. the e f f e c t s of s o i l - a p p l i e d res i dua l herb ic ides on GAg-induced a-amylase production 9 CHAPTER 1 ENHANCEMENT OF a-AMYLASE PRODUCTION BY AMINO ACIDS IN CEREAL ENDOSPERM HALVES 10 1.1 Introduction 1.1.1 Enhancement of a-amylase production by amino acids in cereal endosperm halves The enzyme-inducing a b i l i t y of GA3 has been studied extensively in cerea l aleurone layers (Akazawa and Miyata 1982; Black et a l . 1982; Ashford and Gubler 1984). While i t i s c l e a r tha t GA 3 s t i m u l a t e s a - a m y l a s e p r o d u c t i o n , G A - i ndependent s y n t h e s i s has a l s o been demonstrated (oats, bar ley , and wheat). Naylor (1966) observed that a-amylase product ion in aleurone t i ssue of oat cv 'To rch ' increased a f ter 70 hours of incubation in the absence of exogenous GA3 ( lag time was shortened to about 12 hours when GA3 was added to the incubation medium). Surpr is ing ly , the time period was shortened to about 22h i f a mixture of 20 amino acids was added to the incubation medium. Exogenous amino ac i ds a l s o enhanced a-amylase product ion in aleurone t i ssue of wi ld oat cv 'Montana ' . However, the response was d i f f e r e n t from that of oat cv ' T o r c h ' . There were no d i f fe rences between maximum accumulated enzyme in GA 3 - and amino a c i d - t r e a t e d aleurone in ' T o r c h ' , whi le 'Montana' aleurone was less responsive to amino acids than to GA and showed no increase in a-amylase production in the absence of exogenous amino acids or GA. These resu l ts suggested that free amino acid a v a i l a b i l i t y was important fo r enzyme production although the synthesis was not t o t a l l y GA-dependent. A l t e r a t i o n s in seed germination c h a r a c t e r i s t i c s during breeding programs may explain why wi ld oat 'Montana' and domestic 'To rch ' show 11 di f fe rent responses to amino ac ids. Natural se lect ion in wi ld oat seems to have operated to r e t a i n a p h y s i o l o g i c a l mechanism in which the synthesis of hydro ly t ic enzymes in the endosperm is r igorously dependent on hormones produced by the embryo during the onset of germinat ion . This func t ions to preserve the endosperm starch reserves un t i l the embryo becomes capable of germination through e i ther loss of dormancy or some environmental change (Naylor 1969). This s e l e c t i v e pressure is re laxed or absent in cu l t i va ted species as the contro l of endosperm m o b i l i z a t i o n by the embryo i s not as c r i t i c a l f o r t h e i r s u r v i v a l . Cu l t i va ted oat v a r i e t i e s of ten d i s p l a y l ess dormancy. Dormancy i s recessive in wi ld oat and i s often not expressed in the newer cu l t i va rs (Pawloski 1959). Interpretat ion of GAg-independent enzyme synthesis in w i ld oat i s made d i f f i c u l t due to the incons is ten t resu l t s achieved in separate exper imen ts . A smal l l e v e l of a-amylase s y n t h e s i s occu r red i n 'Montana' , however, i t was only seen in some aleurone t issue (Naylor 1969), whi le 'SH267' exh ib i ted va r iab le l eve l s of enzyme product ion among d i f f e r e n t e x p e r i m e n t s (Upadhyaya et a l . 1982 ) . These i n c o n s i s t e n c i e s f u r t he r compl icate the understanding of endosperm mob i l i za t ion . Enhancement of GA-independent enzyme production by amino acids is not res t r i c ted to oat and wi ld oat. Amino acids also enhance a-amylase production in bar ley aleurone t i ssue in the absence of exogenous GAg (Chrispeels and Varner 1967b). Enzyme production increased fo l lowing addit ion of a mixture of 16 amino acids to the incubation medium. As with 'Montana' aleurone (Naylor 1969), the level of enzyme induced was 12 lower than with GA. Although the enzyme-promoting a b i l i t y of amino ac id mixtures is c l ea r , the ef fect of ind iv idual amino acids on th is production has not been determined. Aspartate and glutamate were shown to induce a-amylase production in bar ley (Galsky and L i p p i n c o t t 1969); at 0.1 mM these compounds induced 40% as much a-amylase as GA3 a f ter 24 hours incubation and were equa l l y e f f e c t i v e wi th longer i n c u b a t i o n p e r i o d s . Th is induct ion was pH-dependent with the greatest enzyme production between pH 4.8 and 6.6 (Galsky and L ipp inco t t 1971). I t was proposed that aspartate and glutamate caused the bypass of a l im i t i ng step act ivated by GA during regulat ion of a-amylase b iosynthesis. The precise s i t e of act ion of these amino acids between the i n i t i a l events of GA action and the ult imate t ranscr ip t ion of the a-amylase st ructura l gene is unclear because the. enzyme-inducing a b i l i t y of i n d i v i d u a l amino ac ids was incons is tan t between separate experiments despi te using s im i l a r seed batches. a-Amylase (units per ha l f seed) induced by glutamate (pH 4.8) ranged from 2 - 7 4 among 8 separate experiments. No explanat ion was provided by the authors for these inconsistent values. Although i t was apparent that aleurone t issue was the major s i te of a-amylase synthesis in cereal seeds (Naylor 1966; Chrispeels and Varner 1967a,b) , the p h y s i o l o g i c a l r o l e of. the endosperm in GA^-tr iggered a-amylase production had not yet been determined. Studies to re late the i n d u c t i o n of wheat a-amylase in embryoless ha l f - seeds to that of aleurone t i ssue (La l l et a l . 1988) showed that synthesis of GA3~ induced a-amylase was s i g n i f i c a n t l y h igher in ha l f - seeds than in exc ised aleurone suggest ing that some f a c t o r in the endosperm t i s s u e was 13 contr ibut ing to maximal enzyme a c t i v i t y . This hypothesis was confirmed by adding an endosperm extract plus GAg to the excised aleurone layers . Although GAg-induced a-amylase a c t i v i t y was enhanced fo l lowing th i s add i t ion , the to ta l a c t i v i t y of a-amylase was lower than that observed in the hormone treated embryoless ha l f seeds. However, upon addit ion of a mixture of 19 amino acids into the incubation medium along with the exc ised wheat aleurones and GAg, the leve l of a-amylase induced was s im i la r to that induced by GAg in in tact endosperm t i ssues . Amino acid pools were much higher in the endosperm suggesting that the endosperm t issue plays a dominant ro le in providing a r e l a t i v e l y high pool of free amino acids for enzyme synthesis . Although free amino acid a v a i l a b i l i t y i s important in cereal seed endosperm mob i l i za t ion , there have been only prel iminary studies on th is top ic . A number of technical problems as well as the lack of consistent r e s u l t s between e x p e r i m e n t s have s l o w e d p r o g r e s s i n t h e s e invest iga t ions . 1.1.2 Technical problems associated with endosperm mobi l izat ion studies 1.1.2.1 Control of seed contamination B a c t e r i a l and fungal contamination are a major concern in seed incubat ion s t u d i e s . The la rge re lease of sugars fo l l ow ing s ta rch hydrolysis provides a favorable medium for growth of these contaminants. B a c t e r i a are e a s i l y c o n t r o l l e d by a d d i t i o n of s t r e p t o m y c i n and p e n i c i l l i n , while fungal contamination may be minimized by d is in fec t ion of seeds in hypoch lo r i t e s o l u t i o n s (Ch r i spee l s and Varner 1967a). 14 However, hypoch lo r i t e i n h i b i t s GAg-induced a-amylase product ion by iso la ted half-seeds (Goudey et a l . 1987; T i t t l e et a l . 1988) and a l te rs dormancy charac te r i s t i cs of wi ld oat (Hsiao and Quick 1985). Although solut ions of hypochlori te have been used frequently to d i s i n fec t cereal seeds, the p h y s i o l o g i c a l changes caused by hypochlor i te have, un t i l recent ly , been large ly ignored. These i n h i b i t o r y e f fec ts necess i ta te the use of other ant imicrobial agents for d i s in fec t i on of cereal seeds. 1.1.2.2 Amino acid a c t i v i t y in the Nelson-Somogyi glucose assay The level of reducing sugars released i s a convenient measure of a-amylase a c t i v i t y in s tarch hydro lys is s tud ies . The Nelson-Somogyi reducing sugar assay u t i l i z e s copper reagent (Somogyi 1952) and Nelson's arsenomolybdate reagent (Nelson 1944) to measure the reduced copper. Because of i t s s i m p l i c i t y and high s e n s i t i v i t y , t h i s method has been widely used in the determinat ion of blood sugar (Nelson 1944) and of reducing sugars in plant extracts and plant hydrolyzates (Upadhyaya et a l . 1986). Although the assay i s general ly thought to be unaffected by interference from other substances present in the b io log ica l material or extracts (Breui l and Saddler 1985), bor ic ac id , CI" (Coruslu and Pekin 1984), c i t r i c ac id (Pa leg 1959) , M n 2 + (Rutman et a l . 1964) and t r i c h l o r o a c e t i c acid (Sampathnarayanan et a l . 1967) have been reported to i nh ib i t the formation of the blue co lor complex. The interference by manganese has been suggested to occur at the C u 2 + reduct ion s tep, e i ther from the reduction of M n 4 + by glucose and i t s reoxidat ion by oxygen, or by the c a t a l y t i c reox idat ion of Cu + by 15 Mn (Rutman et a l . 1964). In a study of the induction of a-amylase in de-embryonated seed segments of wi ld oat by amino ac ids , i t was found t ha t c e r t a i n amino a c i d s s t r o n g l y a f f e c t e d the reduc ing sugar measurement by the Nelson-Somogyi assay. Therefore, invest igators using t h i s assay for glucose determinat ion must cor rec t fo r the e f fec t of amino ac ids and/or manganese ions in the r e a c t i o n m ix tu re when in terpret ing the i r r esu l t s . 1.1.2.3 Inh ib i t ion of a-amylase production by pro l ine Reserve proteins in germinating seeds are hydrolyzed to free amino acids p r io r to new enzyme synthesis or transport to growing t i ssues . In barley gra ins , prol ine i s one of the most abundant of these free amino acids (Shewry et a l . 1978). Although exogenous amino acid mixtures enhance a-amylase production in iso la ted endosperm t issue (Naylor 1966, Chrispeels and Varner 1967a), pro l ine must, be excluded since i t i nh ib i t s th is enzyme production (Freudenrich and Dashek 1983). The object ives of th i s study were: 1. to develop methods of con t ro l l i ng fungal contamination in incubation solut ions which do not i nh ib i t GA^-induced sugar re lease, 2. to determine the extent of amino acid a c t i v i t y in the Nelson-Somogyi glucose assay, 3. to determine the a b i l i t y of ind iv idual and mixtures of amino acids to promote a-amylase synthesis in wi ld oat and bar ley, 4. to study the in teract ions between GA3 and amino acids for enhanced enzyme production in wi ld oat and bar ley. 16 1.2 Mater ia ls and methods 1.2.1 Seed source Wild oat seeds (Avena fatua L. AN51) were germinated in 10 cm petr i d ishes (conta in ing two Whatman No. 1 f i l t e r papers wetted with 5 ml water) fo r 48 hours. The seeds were t ransferred to 1 gal lon pots and grown (3 plants/pot) to maturity in cont ro l led growth chambers at 20°C, at a l i g h t i n t e n s i t y of about 125 Wm ( S y l v a n i a wide spectrum f lorescent tubes) on a 16 hr photoperiod. Rigorous control of growing condit ions for plants was very important to ensure predictable dormancy behaviour of seeds (Sexsmith 1969; Sawhney and Naylor 1979; Sawhney and Naylor 1980). Development of w i ld oat seeds was very sens i t i ve to temperature, l i g h t i n t e n s i t y , and moisture l e v e l s . Primary seeds (proximal p o s i t i o n in s p i k e l e t ) were used w i t h i n 3-6 months a f t e r harvest ing. 1.2.2 Seed incubat ion Wild oat endosperm segments (3mm long) were excised t ransverse ly from the d i s ta l port ion of dehulled seeds. The locat ions of the embryo and scu te l lum in sample seeds were de te rm ined , f o l l o w i n g 48 hour i n c u b a t i o n of i n t a c t seeds in 0.1% t e t r a z o l i u m , to ensure t o t a l e x c l u s i o n of the s c u t e l l u m ( F i g . 1 .1 ) . Endosperm segments were incubated in e i ther 25 ml Erlenmeyer f lasks sealed with paraf i lm or 10 cm petr i dishes containing two Whatman No. 1 f i l t e r papers for 48 hours (3 r e p l i c a t e s ) . Endosperm segments (15) were added to each f lask or petr i dish pr io r to the addit ion of 5 ml of a so lut ion containing e i ther 17 Made in England O Z : 0 9 O S O t / O C QZ O I WW ULLLL in i ml in i iniiiii AN51 + TETRAZOLIUM FIG. 1.1 Embryo and scutel lum l o c a l i z a t i o n in wi ld oat ( XAN51') fo l lowing incubation in tet razol ium 18 GAg (0.5 mM), ind iv idual L-amino acids (5 mM), or a mixture of 18 amino acids (a lanine, arg in ine, asparagine, aspar t ic ac id , cyste ine, glutamic a c i d , g l u tam ine , g l y c i n e , h i s t i d i n e , i s o l e u c i n e , l e u c i n e , l y s i n e , methionine, phenylalanine, ser ine, threonine, tryptophan, v a l i n e ; at 5 mM each) d issolved in 25 mM phosphate buf fer , pH 4 .8 , containing 13 /iM streptomycin, 56 /zM p e n i c i l l i n (an t i -bac te r ia l agents) and 66 /JM captan ( fungic ide) . The i n h i b i t o r y e f f e c t s of hypoch lo r i te on GAg-induced reducing sugar release ( T i t t l e et a l . 1988) necessitated the use of a l te rnat ive control procedures to prevent fungal contamination within the incubation f l a s k s . These included ethanol s t e r i l i z a t i o n , the add i t ion of captan (N- t r i ch lo romethy l t h io -4 -cyc lohexane-1 ,2 - dicarboximide) or benomyl [methyl-1-(butylcarbamoyl)-2- benzimidazolecarbamate] to the incubation media. L - p r o l i n e , was excluded from the amino ac id mixture s ince L-prol ine i nh ib i t s a-amylase production (Freudenrich and Dashek 1983). The f lasks were shaken in the dark (54 o s c i l l a t i o n s per minute at 25°C) throughout the 48 hour incubat ion per iod whi le the pe t r i dishes were incubated in a 25°C environmental chamber. A l l chemicals were obtained from Sigma Chemical Company (St. Louis , Mo.) unless otherwise spec i f i ed . 1.2.3 Measurement o f release o f reducing sugars Following 48 hour incubation of the endosperm segments in f l a s k s , the solut ion was centr i fuged in 13 x 100 mm test tubes at 4000 x g for 10 minutes (IEC c l i n i c a l cen t r i fuge) . Reducing sugars were assayed with Nelson and Somogyi reagents us ing g lucose standards (Witham et a l . 1971). Af ter bringing the sample volume to 1 ml with water, 1 ml of 19 Somogyi reagent was added. The mixture was heated in a 25°C water bath f o r 5 min fo l l ow ing add i t i on of 1 ml of N e l s o n ' s arsenomolybdate reagent . A f t e r adding 5 ml water, absorbance (at 540 nM) was read (Baush & Lomb Spectronic 21 spectrophotometer). Ac t i v i t y of indiv idual amino acids in the Nelson-Somogyi assay was determined by adding 1 ml of the copper reagent p r i o r to the heat ing step to a l i quo t s (1 ml) of glucose or L-amino ac ids. 1.2.4 Extract ion and assay of a-amylase a-Amylase leve ls were measured in endosperm segments incubated in both petr i dishes and Erlenmeyer f l a s k s . Endosperm segments from petr i d ishes were homogenized in 5 ml sodium acetate buf fer (50 mM sodium acetate, 6 mM C a C ^ , 4 mM NaCl , pH 5.3) at 4°C. The homogenate was cent r i fuged at 10,000 x g fo r 10 min (Sorva l l RC2-B Centr i fuge, SS34 rotor) at 4°C. The supernatant was incubated in a water bath at 70°C f o r 15 min ( to i n a c t i v a t e / 3 -amy lase ) , coo led on i c e , and then centr i fuged (IEC c l i n i c a l centr i fuge) at 4000 x g for 10 min. The f i na l supernatant was used for the a-amylase assay. This extracted supernatant or the incubation medium from the f lasks was assayed according to Shuster and Gi f ford (1962). Enzyme ext rac ts (0.2 ml) were added to 1 ml of starch so lut ion (reagent grade, soluble potato s tarch, 0.125% w/v in homogenization buf fer ) . The react ion was stopped with 1 ml of a c i d i f i e d iodine reagent (90 mg iodine and 1.59 g KI mixed with 400 ml of 0.25 N HC1), 5 ml of water was added and the absorbance was read at 620 nM. 20 1.2.5 S t a t i s t i c a l analysis A one-way analysis of variance was performed on a l l data (p>0.05). D i f f e r e n c e s between the r e s u l t s i n T a b l e s were de te rm ined by Newman-Keuls s t a t i s t i c a l ana lys is ( indicated by lower case l e t t e r s ) . Dif ferences among treatments in Figures were determined by regression ana lys i s ; s t a t i s t i c a l data i s l i s t e d in appendix A. 21 1.3 Results 1.3.1 Seed s t e r i l i z a t i o n techniques Wi ld oat endosperm halves were of ten heav i l y contaminated by m ic robes . Glassware and water s t e r i l i z a t i o n p r i o r to s o l u t i o n p repara t ion d id not con t ro l t h i s contaminat ion suggesting that the microbes were introduced with the seed. Bac te r ia l growth was e a s i l y i nh i b i t ed with streptomycin and p e n i c i l l i n while fungal contamination was c o n t r o l l e d by s t e r i l i z a t i o n w i th h y p o c h l o r i t e or e t h a n o l , or i nco rpo ra t i on of fung ic ides (captan or benomyl) in to the incubat ion medium. A l l methods succesfu l ly inh ib i ted contamination for up to 84 hours (Tab le 1 . 1 ) , however, h y p o c h l o r i t e and ethanol i n h i b i t e d GAg-induced re lease of sugars in the endosperm ha l ves . Captan and benomyl d isso lved in the buffer were more useful since they prevented fungal contamination in the incubation media containing wi ld oat (up to 15 days of seed incubation at 25°C) or barley endosperm (data not shown) without subsequent reduction in 48 hour GAg-induced sugar re lease. 1.3.2 Amino acid a c t i v i t y in the Nelson-Somogyi glucose assay During induction studies of a-amylase by amino acids in w i ld oat endosperm halves, reducing sugar release into the incubation medium was monitored using the Nel son-Somogyi assay. High absorbance va lues , i n d i c a t i v e of a-amylase product ion, were observed in the presence of cer ta in amino ac ids . However, t h i s increase in absorbance was l a t e r found to be due to the a c t i v i t y of amino acids in the sugar assay; no a-amylase was detected in the t i ssue extract (data not shown). 22 Table 1.1 Ef fects of various methods of seed s t e r i l i z a t i o n on reducing sugar release and length of fungal- f ree incubation period in wi ld oat ( XAN51') Treatment V i s i b l e Fungal Reducing Sugar Infect ion (hr)^ Release (ug/segment)^ : Control (-GAg) 96 18. ,42 + 1 .63 a Control + hypochlori te ("GAg) 96 19. .23 + 1 .42 a GA3 72 290. .38 + 16 .42 d GAg + hypochlori te 96 214. ,35 + 12 .32 b GAg + ethanol 84 209. ,61 + 14 .63 b Ethanol + hypochlori te (+GAg) 84 215. .85 ± 16 .32 b Captan (+GAg) * 302. .43 + 20 .41 d Captan + hypochlori te (+GAg) * 263. .94 + 18 .36 c Benomyl (+GAg) * 300. ,63 + 14 .83 d Benomyl + hypochlori te (+GAg) * 286. ,42 + 22 .43 cd Two separate experiments Mean of three rep l ica tes ± S.D. *no v i s i b l e in fect ion seen a f ter 15 days Notes: Di f ferent l e t te rs fo l lowing values in each column indicate s ign i f i can t d i f ferences (p>0.05). 23 Of the twenty amino ac ids t e s t e d , c y s t e i n e , c y s t i n e , s e r i n e , tryptophan, and tyrosine caused an increase in absorbance values in the Nelson-Somogyi assay (F ig . 1.2); A ^ Q values for these amino acids were 5-40 % of the absorbance value fo r an equimolar concent ra t ion of g lucose. A lan ine , a rg in ine , aspa r t i c a c i d , g lutamic a c i d , g l y c i n e , h i s t i d i n e , hyd roxyp ro l i ne , i s o l e u c i n e , l euc ine , l y s i n e , methionine, pheny la lan ine , p r o l i n e , t h reon ine , and v a l i n e showed l i t t l e or no a c t i v i t y . A ^ Q increased to vary ing degrees as cys te ine , cys t i ne , ser ine , tryptophan, and tyrosine concentrat ions increased from 0.1 to 1.0 mM (F ig . 1.3). Regression analys is (see Appendix A) showed l i near re la t ionsh ip with r values ranging from 0.74 to 0.99. The absorption spectra (not shown) of the react ion products formed in the presence of cyste ine, cys t ine , ser ine, tryptophan and tyrosine were s im i la r to that for g lucose; absorpt ion spectrum fo r the co lo r complex formed in the presence of glucose was s im i l a r to that reported by Marais et a l . (1966) with an absorption maximum at 750 nM. Addit ions of amino acids with glucose resul ted in absorbance values h igher than those wi th g lucose alone ( F i g . 1 . 4 ) . M n C l 2 (0.5 mM) i nh ib i t ed co lo r development in the presence of glucose and the amino acids ser ine, cys t ine, and tryptophan (Table 1.2). Add i t ion of amino acids (1 mM cys te ine , c y s t i n e , ser ine, tryptophan, or tyrosine) af ter heating with Somogyi reagent but p r io r to the addit ion of Nelson reagent did not form color (Table 1.3) suggesting that amino acids react at the 2+ Cu reduction step of th is assay. 24 A L A -\ A R G A S P C Y S CYS) 2 G L U G L Y HIS H Y P ILL: L E U LYS M E T P H E P R O S E R T H E T R P T Y R VAL a a I ab f Jab Sab Bab 3 a b a a ^ ab ab ab mmmmzsm c 0.0 0.1 0.2 0.3 0.4 0.5 0.6 A B S O R B A N C E AT 540 nm 0.7 FIG. 1.2 Color development in the presence of 0.5 mM amino acids. (Cys)2 = cystine; all other abbreviations are as in Bender (1985). Note: Different letters following values in each column indicate significant differences (p>0.05). 25 FIG. 1.3 Standard curves for co lor development in the presence of selected amino ac ids : (V) cys te ine ; (j^ ) cys t i ne ; (A) ser ine ; ( • ) tryptophan; and (Si) t y ros ine . 26 0 0.10 0.15 0.20 0.25 0.30 0.35 G L U C O S E CONCENTRATION (mM) FIG. 1.4 Glucose standard curves in the presence of cysteine and se r ine : (A) control (glucose on ly ) ; ( • ) glucose + 0.2 mM cys te ine; (9) glucose + 0.4 mM cys te ine ; (V) glucose + 0.2 mM ser ine; (Y) glucose + 0.4 mM ser ine . 27 TABLE 1.2 Effect of MnC^ on color formation in the presence of glucose and some amino acids Absorbance (540 nm) Change in Treatment 0 mM MnCl„ 0.5 mM MnCl 0 absorbance Control 0, .003 + 0, .002* a 0, .000 + 0. .000 a 0. 003 Glucose (0.25 mM) 0. .958 ± 0, .006 e 0. ,683 + 0. .014 d 0. 320 Serine (0.25 mM) 0. .176 + 0, .008 b 0. .004 + 0. .002 a 0. 172 Tryptophan (0.25 mM) 0. ,184 + 0. .006. b 0. ,003 + 0. .002 a 0. 181 Cystine (0.25 mM) 0. ,353 + 0. .006 c 0. .205 + 0. .011 b 0. 148 Mean of three rep l ica tes ± S.D. Dif ference in A ^ Q with or without 0.5 mM MnCl 2 Note: Di f ferent l e t t e r s fo l lowing values in each column indicate s ign i f i can t di f ferences (p>0.05). TABLE 1.3 Time of amino acid addit ion in the Nelson-Somogyi react ion procedure Amino Acid (ImM) Addit ion Time A b s 5 4 0 t Cystine Before heating^ 1.491 ± .015 g Af ter heating 0.002 ± .002 a Cysteine Before heating 0.813 ± .009 f Af ter heating 0.361 ± .008 d Serine Before heating 0.400 ± .006 e Af ter heating 0.002 ± .001 a Tryptophan Before heating 0.244 ± .001 c Af ter heating 0.001 ± .001 a Tyrosine Before heating 0.124 ± .004 b Af ter heating 0.002 + .002 a Mean of three rep l ica tes ± S.D. Amino acid added pr ior to heating of so lut ion i . e . Somogyi's + amino acid » heat + cool + Nelson's Amino acid added af ter heating so lut ion i . e . Somogyi's + heat + cool » amino acid + Nelson's Note: Di f ferent l e t te rs fo l lowing values in each column indicate s ign i f i can t di f ferences (p>0.05). 29 1.3.3 Pro l ine inter ference in a-amylase production Pro l ine inh ib i ted amino ac id - and GA^-enhanced a-amylase production in wi ld oat endosperm halves (Table 1.4); the level of enzyme produced was measured i ns tead of sugar r e l e a s e because of the amino a c i d inter ference in the glucose assay (see previous sec t ion) . The a b i l i t y of amino acid mixtures to promote a-amylase production was inh ib i ted by 66% when pro l ine was included in the amino acid mixture. 1.3.4 Enhancement of a-amylase production by ind iv idual amino acids The enzyme-promoting c a p a b i l i t i e s of ind iv idua l amino acids were fo l lowed in a s e r i e s of experiments (data not shown) to determine whether they c o u l d promote a-amylase p roduc t i on l e a d i n g to the subsequent re lease of reducing sugars in w i ld oat endosperm ha l ves . A l though autonomous and GA^-promoted sugar re lease was cons i s ten t throughout the experiments, the a b i l i t y of amino acids to promote th is sugar re lease was extremely var iab le . Studies with aspart ic acid and ser ine (Table 1.5) demonstrate the wide va r i a t i on in r esu l t s between exper iments . A l t e r i n g var ious f ac to r s (seed ba tch , a f t e r - r i pen ing per iod, buf fer , pH, incubation, time, temperature), did not improve the consistency (data not shown). 1.3.5 Interact ions between GA^  and amino acids Wild oat and barley endosperm segments incubated with, a mixture of 18 amino acids (pH 4.8 for 48 hours) cons i s t en t l y promoted a-amylase TABLE 1.4 Effect of pro l ine on amino acid-promoted a-amylase production in wi ld oat ('AN51') Treatment a-amylase (abs/min/ml) Control 0.002 + 0.001 a 10" 8 M GAg 0.100 + 0.003 b 10" 8 M GA3 + aa§ (+proline) 0.399 + 0.092 c 10 " 8 M GAg + aa (-prol ine) 0.596 + 0.026 d Mean of 3 rep l ica tes ± S.D. 18 amino acid mixture Note: Di f ferent l e t t e r s fol lowing values in each column indicate s ign i f i can t di f ferences (p>0.05). 31 TABLE 1.5 Effect of serine and aspar t ic acid on reducing sugar release in wi ld oat ('AN51') Experiment Reducing Sugar Release Number (ug/segment)^ Control GA3 Asp Ser (0.05 mM) (5 mM) (5 mM) 1 33.23 291.84 170.18 169.65 2 19.68 225.23 14.32 10.65 3 25.21 282.12 227.04 149.16 4 27.60 302.64 275.84 260.97 5 32.42 322.32 58.58 72.31 6 30.66 250.43 142.63 14.63 7 19.20 263.87 246.30 201.42 8 21.56 259.54 248.31 220.63 9 19.36 295.38 14.63 21.42 10 19.80 235.13 193.68 212.57 Interference by amino acids in the Nelson-Somogyi's assay was subtracted from the means 32 production (Table 1.6). a-Amylase was again assayed (instead of sugar l e v e l s ) because many of the 18 amino ac i ds i n t e r f e r e d w i t h the Nelson-Somogyi assay ( F i g . 1 .2 ) . Amino ac id -enhanced a-amylase production was fur ther enhanced when GA3 was also added. When a level q of GA3 which was too low to promote enzyme synthesis (10 M GA^; F i g . 1.5) was added in combination with the amino ac ids , increased a-amylase p roduc t i on occur red in ba r ley (21% i n c r e a s e ) . Fur ther inc reases occurred at higher GA, leve ls (87% increase) . TABLE 1.6 a-amylase release fo l lowing incubation with a mixture of 18 amino acids with or without GA3 in wi ld oat and barley Species Treatment a-amylase (abs/min/ml) Wild oat Control 0, .007 + 0.002 a ( XAN51') 10" 9 M GA3 0, .006 + 0.001 a 10" 8 M GA3 0, ,146 + 0.023 c aa§ alone 0. .091 + 0.018 b 10" 9 M GA3 + aa 0, .105 + 0.009 b 10" 8 M GA3 + aa 0. .670 ± 0.044 d Barley Control 0. .034 + 0.011 a ( 'V i rden ' ) 10" 9 M GA3 0. .056 + 0.010 a 10" 8 M GA3 0. .576 + 0.111 d aa alone 0. ,112 + 0.035 b 10" 9 M GA3 + aa 0. ,141 + 0.021 c 10" 8 M GA3 + aa 0. 830 + 0.091 e Mean of three rep l ica tes ± S.D. 18 amino acid mixture Note: Di f ferent l e t te rs fo l lowing values in each column indicate s ign i f i can t di f ferences (p>0.05). 34 — 5 0 0 CD E CO C o n c e n t r a t i o n (M) F i g . 1.5 Reducing sugar re lease in wi ld oat fo l lowing incubation in d i f fe ren t GA., concentrat ions ( 'AN51') . 35 1.4 Discussion Exogenous l y -app l i ed GAs have been shown to promote a-amylase production and the subsequent release of reducing sugars in endosperm halves of cereal seeds (Akazawa and Miyata 1982; Black et a l . 1982; Ashford and Gubler 1984). a-Amylase production a lso occurs in bar ley and wi ld oat endosperm segments in the absence of GA^ when amino acids mixtures are included in the incubation medium (Naylor 1966; Chrispeels and Varner 1967a). However, the demonstration of th is phenomenon in wi ld oat endosperm t i ssue has been clouded by techn ica l problems and inconsistent r esu l t s . Some of the technical problems encountered in my studies included 1) fungal contamination, 2) amino acid ac t i v i t y in the reducing sugar assay and, 3) the inh ib i to ry ef fect of p ro l ine . Sur face s t e r i l i z a t i o n of seeds wi th hypoch lo r i t e s o l u t i o n i s commonly used to control fungal contamination in endosperm mobi l izat ion s tud ies (Chr ispeels and Varner 1967a; Moll and Jones 1982). Fungal spores are f requent ly seed borne, e i the r on the surface of seeds or between the seed coat and the embryo. Although hypochlori te e f fec t i ve l y i n h i b i t s funga l c o n t a m i n a t i o n d u r i n g i n c u b a t i o n , r e d u c t i o n i n GA^-induced a-amylase synthesis by iso la ted aleurone layers also occurs (Goudy et a l . 1987; T i t t l e et a l . 1988). However, t h i s i n h i b i t o r y e f f e c t of h y p o c h l o r i t e was not cons idered in many of the e a r l i e r endosperm mobi l izat ion s tud ies . A l t e rna t i ves (ethanol s t e r i l i z a t i o n , f u n g i c i d a l treatment with captan or benomyl) were sought to contro l fungal growth without i nh ib i t i ng sugar release (Table 1.1). Captan and benomyl, commonly used seed protectant fungicides (Sinha et a l • 1988), were e f fec t ive up to 15 days without r e s t r i c t i n g sugar re lease . As a 36 r esu l t , captan (66 /zM) was included in subsequent endosperm mobi l izat ion s tud ies. a-Amylase l e v e l s in endosperm and aleurone t i s s u e are usua l l y quant i f ied by the d i rec t measurement of enzyme l e v e l s . However, enzyme ex t rac t ion and assay are both t ime-consuming and labour i n t e n s i v e , l im i t i ng treatment and rep l i ca t i on s i z e s . As a r esu l t , the hydrolysis of starch was determined by measuring the re lease of reducing sugars (Nelson-Somogyi assay) because sugars can be measured on a much larger sca le . During studies of reducing sugar release in de-embryonated seed segments of wi ld oat, cer ta in amino acids appeared to enhance reducing sugar re lease . Upon fu r ther i n v e s t i g a t i o n , i t was apparent that a number of these amino acids did not promote sugar re lease, as there was no measurable a-amylase a c t i v i t y in the t issue ex t rac t ; the amino acids instead increased co lor imet r ic a r t i f a c t s in the sugar assay. Of the twenty amino ac ids t e s t e d , c y s t e i n e , c y s t i n e , s e r i n e , tryptophan and tyrosine caused an increase in absorbance values in the Nelson-Somogyi assay ( F i g . 1.2). This e f fec t was greater at higher amino ac id concentrat ions ( F i g . 1.3) and increased the absorbance reading when in combination with glucose (F ig . 1.4). This ef fect can occur at amino ac id concentrat ions as low as 0.1 mM. Although the mechanism of amino acid a c t i v i t y is beyond the scope of th is thes i s , I 2+ propose that amino acids reacted at the Cu reduc t ion step of the assay, since addit ions of these amino acids af ter heating with Somogyi reagent but p r io r to the addit ion of Nelson reagent did not a l te r co lor development (Table 1.3). It i s i n te res t i ng to note that , while high absorbance va lues were ob ta ined in the p resence o f s e r i n e , the 37 s t ruc tu ra l l y s im i la r threonine did not have the same e f fec t . The color formation by both glucose and the d i f fe ren t amino acids in the assay was inh ib i ted by MnCl 2 (Table 1.2), an important trace metal often included in gluconeogenesis studies (Rutman et a l . 1964). This i nh ib i t i on also 2+ appears to occur at the Cu reduction step. It is therefore, important that invest igators using th i s assay for g lucose de te rm ina t i on c o n s i d e r the e f f e c t of amino ac ids and/or manganese ions in the reac t ion mixture when in terpret ing the resu l t s . Since interact ions of cer ta in amino acids in the Nelson-Somogyi assay prevented use of t h i s procedure when quant i fy ing sugar leve ls in the amino acid s tud ies, a-amylase was d i r e c t l y assayed (Shuster and Gi f ford 1962), because th is analysis was not affected by most free amino ac ids. Endosperm m o b i l i z a t i o n s tud ies wi th amino ac ids were f u r the r complicated by an i n h i b i t o r y e f f e c t of L -p ro l i ne in the incubat ion mix tu re . This was not cons idered in e a r l i e r s tudies (Naylor 1966; Chrispeels and Varner 1967a). L-prol ine i nh ib i t s GA 3-induced a-amylase re lease (Table 1.4). The i n h i b i t o r y e f f ec t of pro l ine is large (66% reduction) suggesting that the responses seen in the e a r l i e r studies were s u b s t a n t i a l l y masked. This i nh ib i t i on by pro l ine is probably an a s s a y a r t i f a c t r e s u l t i n g f r o m t h e f o r m a t i o n o f an enzyme-substrate-pro l ine complex s ince p ro l i ne reduces e x t r a c e l l u l a r a-amylase a c t i v i t y w i thout a f f e c t i n g i t s i n t r a c e l l u l a r a c t i v i t y (Freudenrich and Dashek 1983). Although e a r l i e r studies have demonstrated that amino acid mixtures promote a-amylase production in iso la ted aleurone t issue of both barley 38 and wild oat (Naylor 1966; Chrispeels and Varner 1967), the ability of individual amino acids to enhance this enzyme production is unclear. Galsky and Lippincott (1969) reported that aspartate and glutamate increased a-amylase levels in barley endosperm tissue. This production was highly pH dependent, especially between 4.8 and 6.6 (Galsky and Lippincott 1971). However, the abilities of amino acids to cause this increase were very inconsistent among experiments. Although the authors concluded that these amino acids induced a-amylase, no explanations were provided for their inconsistent results. The a b i l i t y of individual amino acids to promote a-amylase production in the absence of exogenous GAg was also highly variable in wild oat endosperm tissue (Table 1.5). Although enzyme production was consistently promoted by amino acid mixtures (Table 1.6), individual amino acids exhibited variable and inconsistent effects in this regard. Many experimental factors were altered to correct this inconsistency ( i . e . seed batch, incubation period, incubation temperature, pH, buffer), however, the reason for this anomaly was undetermined. These variable results seen with wild oat (Table 1.5) and barley (Galsky and Lippincott 1971) prevent me from concluding that these individual amino acids are inducing a-amylase as suggested in earlier studies (Galsky and Lippincott 1971). The i n c o n s i s t e n t r e s u l t s c o u l d be r e l a t e d to the environmentally-sensitive nature of wild oat dormancy, as there is evidence for considerable variation in both the presence and depth of dormancy depending on growth conditions (Sawhney and Naylor 1979; Sawhney and Naylor 1980). As well, many factors affect free amino acid 39 l e v e l s in endosperm t i s s u e ; changes can occur w i th in the seed during maturat ion, a f t e r - r i p e n i n g , or during exper imentat ion. However, the c o n s i s t e n t a b i l i t y of f r ee amino ac id mixtures to promote enzyme production (Table 1.6) emphasizes that an avai lab le source of free amino acids is necessary for further synthesis of a-amylase. The fact that amino acids can pa r t l y subs t i tu te fo r GA^ suggests that one of the c o n t r o l l i n g f ac to r s in the degradat ion of the endosperm may be the presence of proteases, which are a l so induced by GA^ (Jacobsen and Varner 1967). Proteases hydrolyze storage proteins to free amino acids providing a necessary substrate for enzyme synthesis. In te res t ing ly , l eve l s of GA3 too low to induce enzyme synthesis -9 (10 M; F i g . 1.5) were synerg is t ic to the ef fects of free amino acids in barley (Table 1.6). Sharp increases in enzyme production occurred as higher l eve l s of hormone were included wi th in the amino acid mixture. This suggests that the leve ls of both hormone and free amino acids were important for maximal enzyme production. One of the major ro les of the endosperm t i ssue i s to provide a r e l a t i v e l y enriched pool of free amino acids necessary for a high rate of pro te in synthesis ( La l l et a l . 1988). This enzyme production i s mediated by GA which is synthesized in e i ther the embryo (Chrispeels and Varner 1967a,b) or endosperm t issues (Weiler and Wieczorek 1981; Atzorn and Weiler 1983c), but is often quite low in dormant seeds. Despite the presence of adequate amino acid l e v e l s , a cer ta in endogenous level of hormone must be present to t r igger th is production as low leve ls of GA3 great ly enhanced amino acid-promoted enzyme production (Table 1.6). The low leve ls of a-amylase produced in the presence of added amino acids 40 alone could be due to endogenous endosperm g ibbe re l l i ns which promote enzyme synthesis fol lowing appl icat ion of free amino ac ids . CHAPTER 2 AEM IN BARLEY ENDOSPERM TISSUE 42 2.1 Introduction The most widely held view of cereal endosperm mobi l izat ion places a heavy emphasis on the germinating embryo in the contro l of endosperm hydro lys is (Paleg 1960 a ,b ; Yomo 1960). G ibbe re l l i ns are thought to induce a-amylase production in the aleurone and scutellum layers p r i o r to the hydrolysis of stored s tarch . However, in several cereal species, some a-amylase is also produced by endosperm segments which have been deembryonated to remove tha GA source (Naylor 1966; Harvey and Oaks 1974; MacGregor 1983; N i c h o l l s 1983) . Th is autonomous endosperm m o b i l i z a t i o n (AEM), which i n v ivo often resu l t s in premature starch hydro lys is in mature g ra ins , depends on the environmental condi t ions dur ing development ( N i c h o l l s 1982, 1983). I t i s of much i n t e res t because i t plays a major ro le in endosperm mobi l izat ion of a number of important weedy and cu l t iva ted species (e .g . wi ld oat, bar ley, wheat). Seed dormancy i s a major fac tor in the surv ival of wi ld oat, a weed causing large economic losses in the p r a i r i e provinces of Canada (Dew 1978). In some biotypes, caryopses may remain v iable for several years in the s o i l . Survival i s dependent on the preservat ion of endosperm s ta rch reserves for extended per iods u n t i l the embryo can germinate a f te r e i t he r emergence from dormancy or some environmental change. C lea r l y , there i s a physio logical mechanism which makes the synthesis of endosperm hyd ro l y t i c enzymes dependent on hormones produced by the embryo during the onset of germination (Naylor 1969). However, the high incidence of AEM in cer ta in biotypes (Naylor 1966; Upadhyaya et a l . 1982) points to the existence of some l ess rigorous con t ro l , with or without GA, of endosperm mob i l i za t ion . 43 AEM i s a l s o common i n c u l t i v a t e d c e r e a l s . C u l t i v a r s of economically important species such as barley and wheat often show high l e v e l s o f AEM, an u n d e s i r a b l e c o n d i t i o n f o r both brewing and bread-making indus t r ies . During brewing, malting requires the synthesis and ac t iva t ion of hydro ly t ic enzymes, breakdown of protein and c e l l wall material within the endosperm and hydrolys is of starch granules (Briggs 1978). This hydro lys is must be performed in a contro l led environment (Enari and Sopanen 1986) and invo lves a-amylase (Sandstedt 1955), an enzyme which inc reases r a p i d l y dur ing malt ing (Briggs 1968). When hydro ly t ic enzyme synthesis, and grain structure degradation have reached the des i red stage, the germination process i s in terrupted by dry ing. The brew master must have optimal cont ro l of t h i s malt ing process to produce h igh q u a l i t y beer . As a r e s u l t , the g r a i n q u a l i t y and biochemical un i formi ty must be cons is ten t to achieve the appropriate leve l of endosperm mob i l i za t ion . Batch qua l i t y of gra in is often d i f f i c u l t to maintain due to the prevalence of AEM. Although barley embryos remain dormant during grain development, AEM may occur in response to cer ta in environmental s t i m u l i . Since large quant i t ies of barley are processed in the brewing industry, farmers of ten attempt to extend growing seasons to increase y i e l d s . This resu l t s in pre-harvest s p r o u t i n g , which i s common dur ing l ess favo rab le c o n d i t i o n s , e .g . moist , coo ler condi t ions of ear ly autumn (Brookes 1979), and i s accompanied by increased starch hydro lys is and decreased grain qua l i t y . Heavy losses due to th is pre-mature endosperm m o b i l i z a t i o n f r e q u e n t l y o c c u r ; r e p o r t s of 30-50% damage are not uncommon. Bar ley i s unsu i tab le fo r mal t ing when the inc idence of pre-germinated grains exceeds 5% (Brookes 1979). 44 Pre-harvest sprouting also has negative e f fec ts in the bread-making industry a f f ec t i ng the b read-p repara t ion process and bread q u a l i t y (Buchanan and Nicholas 1979). Affected wheat grains have higher sugar contents and produce a h i g h l y c o l o u r e d , s t i c k y crumb l o a f which adversely af fects the cut t ing operation of bread s l i c e r s because a gummy crumb deposit adheres to the high speed blades. Pre-harvest sprouting of bread-making wheat grain has been a major problem in England where a number of domestic c u l t i v a r s exhib i t high a-amylase a c t i v i t y during dormancy (Gale et a l . 1983). I r on i ca l l y , th is problem was made worse by a breeding program previously undertaken in England to. improve the q u a l i t y of the Eng l i sh g r a i n . A number of cu l t i va rs introduced during the breeding program or ig inated from the cv 'Professor Marcha l ' , which showed high leve ls of pre-harvest sprout ing. The i n h e r i t a n c e of the un favo rab le c h a r a c t e r i s t i c , which was not understood at the t ime, was not considered during the breeding programs. High leve ls of AEM also occur during storage of the seed. Since i t i s very d i f f i c u l t and c o s t l y to maintain su i tab le storage condit ions (e.g. control moisture, temperature leve ls ) for a large bulk of seeds, storage losses are often very high. Spec i f i c propert ies of seeds must be monitored to avoid use of substandard materials (Duffus and Slaughter 1980). AEM in bar ley has not been given much recogn i t ion in previous studies in sp i te of i t s widespread occurrence in many barley c u l t i v a r s . High leve ls of a-amylase occurred in the absence of exogenously-appl ied GA, or amino acids (chapter 1). 'H imalaya ' , a barley c u l t i v a r commonly 45 used in previous endosperm mob i l i za t i on studies (e .g . Chr ispeels and Varner 1967a,b; Ranki and Sopanen 1984), apparent ly exh ib i t ed high l e v e l s of AEM. However, these studies made l i t t l e reference to th is phenomenon, preferr ing to assume that the enzyme production was en t i re l y induced by embryo-synthesized GA^. Experiments reported in th is chapter were an attempt to determine the extent of AEM in se lec ted bar ley c u l t i v a r s and the optimum i n c u b a t i o n cond i t i ons under which t h i s cha rac te r i s t i c is expressed. 46 2.2 Mater ia ls and methods The loca t ions of the embryo and scutellum in in tact barley seeds (Hordeum vulqare L.) were determined by incubating seeds for 48 hours on two Whatman No. 1 f i l t e r papers in 10 cm petr i dishes containing 5 ml of 0.1% tetrazol ium (F ig . 2 .1) . Barley endosperm segments (4 mm long) were cut to exclude embryonic t i s s u e , incubated in 25 ml Erlenmeyer f lasks (15 segments/f lask; 3 r e p l i c a t e s ) , sealed with paraf i lm, and shaken in the dark for 48 hours (54 o s c i l l a t i o n s per minute) at 25°C p r i o r to determination of reducing sugar leve ls (see sect ion 1.2.3) or a-amylase production (see sect ion 1 .2 .4) . Consistency in sugar release between experiments was only poss ib le i f incubat ion temperature, incubat ion time, and shaker speed were p rec ise ly monitored and cont ro l led . Solut ions in each f l ask (5 ml) contained 25 mM phosphate buffer pH 4.8 with GAg (0.5 mM), 18 amino acids alone or in mixture (a lan ine, arg in ine, asparagine, aspar t ic ac id , cyste ine, glutamic ac id , glutamine, g l y c i n e , h i s t i d i n e , i s o l e u c i n e , l e u c i n e , l y s i n e , m e t h i o n i n e , phenylalanine, ser ine, threonine, tryptophan, va l i ne ; 5 mM each), C a C ^ (0.25, 0 .5, 0 .1 , 0.25, 0.5, 1, 5, 10 mM), 6-methyl purine (10 mM), and cycloheximide (35 /zM). The buffer contained 13 /zM streptomycin, 56 /zM p e n i c i l l i n , and 66 juM captan. A l l chemicals l i s t e d were obtained from Sigma Chemical Company unless otherwise spec i f i ed . Seeds of both t rad i t i ona l and experimental va r ie t i es were obtained from A g r i c u l t u r e Canada Research S t a t i o n , R e g i n a , Saskatchewan ( h a r v e s t e d 1986 and 1988) and were grown i n e i t h e r Reg ina 47 • • • . KLAGES + TETRAZOLIUM FIG. 2.1 Embryo and scutel lum l o c a l i z a t i o n in barley ( 'K lages ' ) fo l lowing incubation in tetrazol ium 48 /Compana ' , *Hannchen' / H i m a l a y a ' , ' K l a g e s ' / K lond ike ' / L i o n ' , ' S t e p t o e ' , ' T r e b i ' / V a n t a g e ' ) or Ind ian Head (' Chapai s ' / J a c k s o n ' / V i r d e n ' , v A B 7 8 - l ' / A B 7 9 - 1 7 ' / B T 6 3 1 ' / T R 2 2 6 ' / 8 2 - R C - B B 1 3 ' ) , Saskatchewan. 49 2.3 Results 2.3.1 V a r i a b i l i t y of AEM among barley cu l t i va rs There was substant ia l va r ia t ion in AEM by barley cu l t i va rs af ter 48 hours of incubat ion (Table 2 .1 ) . The extremes were 'H ima laya ' and ' V i r d e n ' , which e x h i b i t e d an 18 f o l d d i f f e r e n c e ( i . e . 'H ima laya ' released 523 ug/segment while 'V i rden ' released 29 ug/segment). This re lease was fu r ther enhanced by exogenous GAg (Table 2 .2 ) . AEM was s im i l a r among ind iv idual cu l t i va rs harvested in two d i f f e ren t seasons ( F i g . 2 .2 ) . High leve ls of sugar release in 'K lages ' correlated well with a-amylase production (Table 2 .3) . Secreted a-amylase (measured in the incubation solut ion) and extracted a-amylase (from endosperm t issue) were monitored; fu r ther amounts of extracted a-amylase were re leased fo l lowing addit ion of Tr i ton X-100, which enhances the release of bound a-amylase in endosperm segments fo l low ing d i s rup t ion of the membrane (Locy and Kende 1978). 2.3.2 Inh ib i t ion of RNA and protein synthesis I nh ib i t o r s of RNA (6-methyl purine) and prote in (cycloheximide) synthesis were included in the incubation medium to deduce whether AEM i s due to de novo synthesis of a-amylase, i . e . not from pre-ex is t ing enzyme carr ied over during seed development. AEM, l i k e the mobi l izat ion by GAg (Varner et a l . 1965), was s i g n i f i c a n t l y i n h i b i t e d by both cycloheximide and 6-methyl purine (Table 2 .4 ) . a-Amylase l eve l s were reduced 97% by cycloheximide and 82% by 6-methyl purine. TABLE 2.1 AEM among d i f fe ren t barley cu l t i va rs Harvest S i te Var iety Reducing Sugar Release (ug/segment)^ Regina Indian Head 'Compana' 55 .35 + 9 .84 ab 'Hannchen' 216 .64 + 34 .09 d 'Himalaya' 534 .54 + 32 .82 g 'K lages ' 523 .93 + 39 .70 g 'K lond ike ' 137 .04 + 33 .11 c ' L i o n ' 33 .64 + 7. .49 a 'Steptoe ' 154, .89 + 42, .64 c ' T r e b i ' 272, .27 + 15, .59 e 'Vantage' 211, .81 + 15, .80 d 'Chapais ' 66, ,60 + 5, .56 ab 'Jackson' 373. ,90 + 3. .64 f 'V i rden ' 29. ,14 + 5. ,31 a 'AB78-1' 70. ,14 ± 10. ,71 ab 'AB79-17' 46. ,50 + 9. ,41 ab 'BT631' 41. 36 + 1. .21 ab 'TR226' 384. ,84 + 13. .87 f '82-RC-BB13' 93. ,78 + 7. ,49 b Mean of three rep l i ca tes ± S.D. Note: Di f ferent l e t t e r s fo l lowing values in each column indicate s ign i f i cance di f ferences (p>0.05). 51 TABLE 2.2 Effect of exogenous GA^ on reducing sugar release in barley endosperm halves Cu l t i va r Presence of GA- Reducing Sugar Release (ug/segment)* 'Klages ' 695.37 ± 12.87 C 1111.75 ± 28.48 d v V i rden ' 33.98 ± 1.19 a 168.86 ± 36.63 b *Mean of three rep l i ca tes ± S.D. Note: Di f ferent l e t te rs fo l lowing values in each Column indicate s ign i f i canc t d i f ferences (p>0.05). 52 CD CO CD jCD CD CD CO ID CO CO cz "o ID "O CD . CC 600 500 £ 400 CD E O J 300 CD CO go 200 100 0 Himalaya Steptoe Compana Seed Cultivar a IBl a a Lion 1986 1988 FIG. 2.2 Autonomous reducing sugar release among barley c u l t i v a r s harvested in two d i f fe ren t growing seasons. Note: D i f ferent l e t t e r s fo l lowing values in each column ind icate s i gn i f i can t d i f ferences (p>0.05). 53 TABLE 2.3 a-amylase production and reducing sugar release from 'K lages ' endosperm t issue Fract ion Presence of a-amylase Reducing Sugar ,t Tr i ton X-100 (abs/min/ml) a Release (ug/segment) medium - 0.516 + 0.228 c 525.32 + 42.80 homogenate - 0.188 + 0.066 a homogenate + 0.380 ± 0.183 b Mean of three rep l ica tes ± S.D. Note: Di f ferent l e t t e r s fol lowing values in each column indicate s ign i f i can t d i f ferences (p>0.05). 54 TABLE 2.4 Effect of RNA and protein synthesis inh ib i to rs on autonomous reducing sugar release in v Klages ' Treatment Reducing Sugar Release (ug/segment)* Control 457, .31 + 85, .20 c GAg (0.5 mM) 1520, .16 + 53. .66 d Cycloheximide 13, .89 + 0. ,76 a GAg + cycloheximide 137, ,43 + 18. ,55 b 6-methyl purine 56. ,88 + 7. .10 a GAg + 6-methyl purine 71. .38 + 7. ,29 a Mean of three rep l ica tes + S.D. Note: Di f ferent l e t t e r s fol lowing values in each column indicate s ign i f i can t di f ferences (p>0.05). 55 2.3.3 Effect of pH and temperature changes on AEM AEM in b a r l e y endosperm t i s s u e was pH dependent ( F i g . 2 . 3 ) ; x Klages ' endosperm released high leve ls of sugar between pH 4.6 and 5.6. However, at h igher pH (pH 5.6 to pH 8 .6 ) , l ess sugar was re leased . However, AEM with ' V i r d e n ' , a low sugar producing c u l t i v a r , was not s i g n i f i c a n t l y d i f fe ren t (p>0.05) at pH 4.8 and pH 6.6. Higher i n c u b a t i o n temperatures enhanced AEM in ' K l a g e s ' and ' V i r d e n ' ( F i g . 2 .4 ) . AEM was very low at 12°C, i r r espec t i ve of the i n c u b a t i o n pH or c u l t i v a r . Al though sugar re lease from ' K l a g e s ' endosperm was enhanced by higher temperatures at both pH 4.8 and 6.6 , the increase was subs tan t ia l l y greater at pH 4.8. The largest increase in sugar release occurred with an increase from 20 to 28°C (at pH 4 .8) . V i rden , which exh ib i ted no increased sugar release at 25°C (F ig . 2.3) released higher leve ls at 28°C. 2.3.4 Time course of onset of AEM The time course study of onset of AEM showed that only smal l amounts of sugar were released in the f i r s t 24 hours of incubation (F ig . 2 .5 ) . In ' K l a g e s ' endosperm most of the increase occurred a f te r 24 hours. However V i rden , exhib i ted l i t t l e sugar release af ter 48 hours incubation (pH 4 .8 , 25°C; Table 2.2) 2.3.5 Effect of calcium on AEM AEM in 'V i rden ' endosperm was not af fected by calcium ions ( F i g . 56 FIG. 2.3 Ef fect of pH on autonomous reducing sugar re lease : ( • ) Klages; (A) Vi rden. 57 FIG. 2.4 Ef fect of temperature and pH on autonomous reducing sugar re lease. Note: D i f ferent upper case l e t t e r s above bars ind icate s i g n i f i c a n t d i f ferences between Virden treatments; d i f f e ren t lower case l e t t e r s indicate s i g n i f i c a n t d i f ferences between Klages treatments (p>0.05). 58 FIG. 2.5 Time course of autonomous reducing sugar re lease: ( • ) Klages; (/\) Vi rden. 59 2 .6 ) . However, low l eve l s of calc ium (0.25 - 0.5 mM) promoted sugar release in ' K l a g e s ' , higher amounts (0.5 - 1 mM) reduced t h i s re lease and above 1 mM calcium almost completely inh ib i ted sugar re lease. 60 FIG. 2.6 Ef fect of calcium on autonomous reducing sugar re lease: ( • ) Klages; (A) V i rden. 61 2.4 Discussion Autonomous endosperm mobi l izat ion (AEM) is common among cereals and has nega t i ve i m p l i c a t i o n s f o r both the brewing and bread-making indust r ies . Although th is phenomenon has been recognized for many years (Naylor 1966), most s tudies have focused on pre-harvest sprout ing in wheat. Although there have been i n i t i a l repor ts of AEM in barley (MacGregor 19.76; MacGregor et a l . 1983; N i cho l l s et a l . 1986), l i t t l e information is ava i lab le on i t s frequency among d i f fe ren t cu l t i va rs and the optimum condit ions under which th is cha rac te r i s t i c i s expressed. Barley c u l t i v a r s d i f f e r e d s u b s t a n t i a l l y in t h e i r l e v e l of AEM (Table 2.1) , however, leve ls were consistent wi th in cu l t i va rs regardless of growing seasons (F ig . 2 .2) . 'H imalaya ' , a c u l t i v a r used extensively in endosperm mobi l izat ion s tud ies, showed AEM independent of the time of harvest (1986 or 1988). On the other hand, ' L i o n ' exhibi ted low leve ls in both growing seasons. Although th is enzyme production was further enhanced fol lowing addi t ions of GA^ (Table 2 .2 ) , the leve l of AEM in 'Himalaya' and other cu l t i va rs was often high. In sp i te of t h i s , not many studies have focused on th i s phenomenon (e .g . Chrispeels and Varner 1967a,b; Ranki and Sopanen 1984). Most a -amy lase i s produced f o l l o w i n g ge rm ina t ion of ba r l ey seedl ings. ' K l a g e s ' , a c u l t i v a r commonly used in beer making because of good malting qua l i t y and rapid germination (MacGregor 1978), showed less AEM a f te r add i t ion of i n h i b i t o r s of both RNA (6-methyl pur ine) and protein (cycloheximide) synthesis into the incubation medium. Thus, I 62 deduced that AEM was synthesized de novo (Table 2.4) as i s the case with GA^-induced enzyme production (Chrispeels and Varner 1967a,b). Environmental condit ions inf luence the development of AEM. Both temperature and l i gh t qua l i ty af fect grain maturation (Nichol ls 1983); the extent of AEM may vary considerably as the grain matures both on the plant and in dry storage. Expression of AEM in mature seeds also var ies with changes in incubat ion c o n d i t i o n s . This makes i t d i f f i c u l t to choose seeds free of AEM when se lec t ing cu l t i va rs for breeding programs. 2+ AEM was highly dependent on changes in pH, temperature, and Ca . Incubation media in endosperm mobi l iza t ion studies are normally buffered at pH 4 . 8 , a l e v e l where GA^- induced enzyme p roduc t i on i s h i gh . However, ce r ta in c u l t i v a r s ( i . e . ' K l a g e s ' ) e x h i b i t e d high a-amylase production (Table 2.3) and sugar release (Table 2.1) in the absence of exogenously appl ied GA3 at t h i s pH. AEM in ' K l a g e s ' was h i gh l y pH dependent; i t decreased at more b a s i c i n c u b a t i o n pH ( F i g . 2 . 3 ) . However, pH changes a f fec ted c u l t i v a r s d i f f e r e n t l y ( e . g . ' V i r d e n ' responded only s l i g h t l y to r i s i n g pH.) AEM was h i g h l y temperature s e n s i t i v e ( F i g . 2 . 4 ) . ' K l a g e s ' endosperm, which cons is tent ly released high amounts of sugar at 25°C, released very l i t t l e at 12°C. However, a r i se in temperature enhanced AEM i r respect ive of the incubation pH (4.8 or 6 .6) . In ' V i r d e n ' , AEM was also temperature dependent as sugar release increased at 28°C (pH 4.8) . AEM was not apparent un t i l the second day of incubat ion ( F i g . 2.5) and occurred la te r than GA,-induced release which occurs a f ter 12 63 hours of incubation (Varner et a l . 1965). 2+ Ca s t i m u l a t e s the s y n t h e s i s and s e c r e t i o n of a-amylase in i so la ted bar ley aleurone l aye rs that have been p re t rea ted wi th GAg (Jones and Jacobsen 1983; Jones and Carbonell 1984). Although i t does 2+ not induce enzyme production, Ca i s required in the incubation medium fo r maximal enzyme syn thes is and sec re t i on (Chr i spee l s and Varner 1967b). Aleurone layers of 'Himalaya' synthesize and secrete at least four a-amylase isozymes belonging to two groups (Jacobsen and Higgins 1982). Treatment with GAg alone produces a low p i group wh i le the 2+ presence of Ca r e s u l t s in product ion of a second high p i group. 2+ Studies from iso la ted protoplasts of barley aleurone suggest that Ca d i r ec t l y af fects the process of enzyme synthesis and t ransport , however, i t i s not the i n i t i a l ac t iva tor (Jones and Jacobsen 1983). In th is study, calcium enhanced sugar release in barley c u l t i v a r s which already exh ib i ted AEM. ' K l a g e s ' , a high AEM c u l t i v a r , showed increased sugar re lease fo l low ing addi t ions of low l e v e l s of calc ium (0.025-0.25 mM), whereas, higher l eve l s of calcium (0.5-10 mM) were inh ib i to ry (F ig . 2 .6) . However, sugar release in ' V i r d e n ' , a non AEM c u l t i v a r , was not enhanced fo l lowing addit ion of calcium. Therefore, the presence of ca lc ium enhanced AEM, as w i th GAg- induced enzyme synthesis (Chrispeels and Varner 1967a,b), but did not i n i t i a t e enzyme synthesi s. Expression of AEM by c e r t a i n c u l t i v a r s could be due to h igher endogenous GA l e v e l s in the endosperm. Attempts to demonstrate GA re lease by i s o l a t e d embryos have produced c o n t r a d i c t o r y r e s u l t s . 64 However, more recent sens i t i ve and GA-select ive immunoassays (Weiler and Wieczorek 1981; Atzorn and Weiler 1983a,b) enabled the re - inves t iga t ion of the ro le of endogenous GAs in a-amylase formation in bar ley. These s tud ies in ' H i m a l a y a ' suggested tha t the GA necessary to induce a-amylase was not embryo-synthesized because GA^, the predominant g ibbere l l i n synthesized p r io r to a-amylase, was produced p r ima r i l y in the aleurone t i s s u e . Therefore, i t was un l i ke ly that the ro le of the embryo was to provide g ibbe re l l i ns for induction in the aleurone layer . Th is cha l l enged the accepted concept of embryo-cont ro l led enzyme induction opening more questions as to the precise ro le of the embryo in endosperm mob i l i za t ion . Th is recent evidence prov ides a bas is f o r the exp lana t ion of widespread occurrence of AEM among cerea ls . However, the o r ig ina l view (not invo lv ing aleurone GA synthes is) should not be d iscarded, since others have not been able to f ind GA^ in germinating bar ley endosperm (Yamada 1982; Gaskin et a l . 1984). The di f ferences observed may be a consequence of the seed batch used in each experiment as AEM l e v e l s often vary among the same c u l t i v a r (Finn and Kende 1974; Shroeder and Burger 1978). The mechanisms of control wi th in a seed may depend not only on the spec ies and c u l t i v a r , but a lso the i nd i v idua l harvest . These changes may occur in response to d i f f e r e n t growing cond i t i ons (Nichol ls 1982, 1983). This increased enzyme production could also be due to some other f ac to r ( s ) , and may be t o t a l l y GA-independent. Di f ferent control mechanisms between cu l t i va rs may be a resu l t of d i f fe rences in breeding programs. Natural se lec t ion in cer ta in weeds operates to re ta in a phys io log ica l mechanism which renders the synthesis 65 of hydro ly t ic enzymes r igorous ly dependent on hormones produced by the embryo during the onset of germination (Naylor 1969). In c u l t i v a t e d cereals on the other hand, th i s se lec t i ve pressure on the genotype often occurs in lower frequency (Pawloski 1959). For th is reason, mutations contr ibut ing to AEM may be expected to accumulate in the gene pools from which modern va r ie t i es have emerged. Therefore, many of the cu t l i va r s developed through breed ing programs may e x h i b i t h igher l e v e l s of embryo-independent endosperm mobi l iza t ion which must be i den t i f i ed p r io r to use of the seeds. CHAPTER 3 EFFECT OF RESIDUAL HERBICIDES IN SOIL ON GA,-INDUCED a-AMYLASE PRODUCTION 67 3.1 Introduction Herbicides appl ied to cu l t i va ted crops to control weeds are often incorporated in to s o i l s p r i o r to seedl ing emergence s ince germinated seeds are normally more suscept ib le to herbicides than mature p lan ts . They may remain in the s o i l fo r periods ranging from a few weeks to several months a f fec t ing seedl ing growth. Certain of these weeds (e .g . w i l d oat) are d i f f i c u l t to con t ro l due to t h e i r complex dormancy patterns from which only a small number of the seeds emerge to germinate each year (Hsiao 1987). Thus, h e r b i c i d e s which t a r g e t me tabo l i c processes in the seedling (Ashton and Crafts 1981) s t i l l al low a v iab le seed bank to survive over many years. One of many poss ib le target processes fo r he r b i c i des which are absorbed by seeds (Scot t and P h i l l i p s 1971) i s the mob i l i za t i on of endosperm reserves. Any compound that in ter feres with th is process w i l l r e s t r i c t seedling growth by lowering the flow of food mater ia ls to the growing embryo (Dev l in and Cunningham 1970). Ce r ta in h e r b i c i d e s , inc lud ing barban, CIPC, 3-chlorophenyl (Mann et a l . 1967), diphenamid (Yung and Mann 1967), a l a c h l o r , propachlor , chloropham, d i c h l o b e n i l , methoxymethyl (Devl in and Cunningham 1970), CDAA (Jaworski 1970), and prynachlor (Rao and Duke 1976), i nh ib i t GAg-induced a-amylase production in i s o l a t e d bar ley aleurone l a y e r s and h a l f seeds and lower ea r l y seedling v igor . Since GAs also induce endosperm mob i l i za t i on in weed grasses such as w i l d o a t , i t i s reasonab le to assume tha t these he rb i c ides may a lso i n h i b i t t h e i r endosperm reserve m o b i l i z a t i o n . However, there have been few studies to determine i f th i s mechanism can be exploi ted to control weedy species. 68 A number of pre-emergence h e r b i c i d e s ( e . g . t r i a l l a t e , EPTC, metr ibuzin, t r i f l u r a l i n , o ryza l in ) are commonly used to control w i ld oat (Anon. 1990), a major weed in Canada's p r a i r i e provinces (Dew 1978). The ef fects of these substances on GA^-induced reducing sugar release in wi ld oat endosperm halves were examined in th is study to determine i f they i nh ib i t a-amylase production and the subsequent release of reducing sugars thus in te r fe r ing with g i bbe re l l i n control of a-amylase synthes is . 69 3.2 Mater ia ls and methods 3.2.1 Measurement of release of reducing sugars Wild oat endosperm segments (3 mm long) (seed supply described in chapter 1) were incubated in 25 ml Erlenmeyer f l a s k s sea led w i th paraf i lm for 48 hours p r i o r (shaken at 54 o s c i l l a t i o n s per minute at 25°C) and the supernatant so lut ion was assayed for reducing sugars (see sec t ion 1 .2 .3 ) . Each f l a s k conta ined e i t h e r 5 ml of GA^ (0.5 mM) solut ion alone or in combination with i nd i v idua l herb ic ide s o l u t i o n s . A l l s o l u t i o n s were prepared in 25 mM phosphate b u f f e r (pH 4.8) containing 13 /zM streptomycin, 56 zzM p e n i c i l l i n , and 66 /zM captan. The herb ic ide solut ions were t r i a l l a t e (3x10 , 1x10 , 3x10 , 1x10 M), EPTC (5x l0~ 5 , 5 x l 0 " 5 M), metr ibuzin (5x l0~ 6 , l x l O " 6 M), t r i f l u r a l i n ( 3 x l 0 " 5 , l x l O " 5 , 3 x l 0 " 6 , l x l O " 6 M) and oryza l in ( 3 x l 0 " 5 , 3 x l 0 " 6 M). Herbic ides were generously donated as fo l lows : t r i a l l a t e [AVADEX BW, S - ( 2 , 3 , 3 - t r i c h l o r o a l l y l ) d i i sop ropy l thiocarbamate] from Monsanto Chemical Company, S t . Lou is , M i s s o u r i ; EPTC (EPTAM, S-ethyl dipropyl thiocarbamate) from Chipman Chemical Company; me t r i buz i n [SENCOR, 4-amino-6- ter t -buty l -3-(methyl th io)-as- t r iaz in-5(4H)-one] from Chemagro, Kansas C i t y , M issour i ; t r i f l u r a l i n (TREFLAN, a a a - t r i f l u o r o - 2 , 6 - d i n i t r o -N - N - d i p r o p y l - p - t o l u i d i n e ) and o r y z a l i n (RYZELAN, 3 , 5 - d i n i t r o - N , N -dipropyl sul fani lamide) from L i l y Laborator ies, Indianapol is , Indiana. 3.2.2 Growth studies Dehul led w i l d oat seeds 'AN51 ' were p lan ted i n d i v i d u a l l y (4 70 r e p l i c a t e s ) in 150 ml styrofoam cups (perforated at the bottom for drainage) containing about 100 g of s o i l . Water or herbicide solut ion (50 ml) was applied to each cup af ter seeding. The cups were watered regular ly and the plant heights measured every three days for up to 18 days af ter seedling emergence. 71 3.3 Results 3.3.1 Effect of herbicides on sugar release I n c u b a t i o n o f w i l d oat endosperm t i s s u e i n s o l u t i o n s o f pre-emergence herbicides affected GA^-induced reducing sugar release to varying extents when appl ied at f i e l d appl icat ion leve ls (Tables 3 .1 , 3 .2) . Only t r i a l l a t e (25% reduction) and t r i f l u r a l i n (22% reduct ion) _5 prevented sugar re l ease (only at 3x10 M); no i n h i b i t i o n was seen fo l lowing incubation in EPTC, metr ibuzin, and o r yza l i n . 3.3.2 Effect of herbicides on seedling growth Herbicide solut ions prepared for sugar release studies were applied to s o i l a f t e r p l an t i ng w i l d oat seeds (but p r i o r to emergence) to e s t a b l i s h the e f f e c t of these he rb i c i de concen t ra t ions on growth. Higher concentrations of each herbicide inh ib i ted the development of the wi ld oat seedl ings. T r i a l l a t e reduced seedling growth at both herbicide appl icat ion leve ls (100% at 3 x l 0 " 5 M, 31% at 3 x l 0 " 5 M; F igs . 3 . 1 , 3.10). This i nh ib i t i on occurred fo l lowing emergence of the ep ico ty l from the s o i l (F igs . 3 .6 , 3 .7) . However, the inh ib i t i on ,of growth at 3x10"^ M t r i a l l a t e was not accompanied by a corresponding reduct ion in sugar re lease (Tables 3 . 1 , 3 . 2 ) . I n h i b i t i o n of seed l i ng growth by EPTC -5 occurred only at the higher app l ica t ion leve ls (39% inh ib i t i on at 5x10 M EPTC; F i g . 3 .2) . Growth patterns of w i l d oat seedl ings fo l low ing app l i ca t i on of 5x10 ^ M metr ibuzin were qu i te d i f f e r e n t . Regular growth occurred 72 TABLE 3.1 Effect of herbicides on GA,-induced reducing sugar release ('AN51') Treatment Herbicide Reducing Sugar % of Concentration Release Control (M) (ug/segment)^ Control 21.03 + 1.92 a GA3 alone (5 mM) 565.08 + 24.98 d GA3 + t r i a l l a t e 3 x l 0 " 5 423.72 + 19.60 b 75 3 x l 0 " 5 512.15 + 8.72 c 91 GA3 + EPTC 5 x l 0 " 5 521.97 + 44.45 cd 92 5 x l 0 " 5 518.86 + 7.48 cd 92 GA3 + metribuzin 5 x l 0 " 6 545.58 ± 6.42 cd 97 l x l O " 6 550.81 + 7.01 cd 97 GA3 + t r i f l u r a l i n 3 x l 0 " 5 442.89 ± 12.98 b 78 3 x l 0 " 6 512.62 + 17.90 c 91 GA3 + o ryza l in 3 x l 0 " 5 526.71 + 11.25 cd 93 3 x l 0 " 6 557.95 + 13.06 cd 99 Mean of three rep l ica tes + S.D. Note: Di f ferent l e t te rs fo l lowing values in each column indicate s ign i f i can t d i f ferences (p>0.05). 73 TABLE 3.2 Ef fects of various concentrations of t r i a l l a t e and t r i f l u r a l i n on GAg-induced reducing sugar release CAN51') Treatment Herbicide Reducing Sugar % of Concentration Release Control (M) (ug/segment)*. Control 44.65 + 1.93 a GAg alone (5 mM) 469.77 + 5.27 c GAg + t r i a l l a t e 3 x l 0 " 5 368.67 + 46.81 b 77 l x l O " 5 425.78 + 35.49 c 90 3 x l 0 " 6 424.67 + 23.50 c 90 l x l O " 6 439.12 + 19.22 c 93 GAg + t r i f l u r a l i n 3 x l 0 " 5 407.78 + 22.90 b 86 l x l O " 5 429.57 + 8.39 c 90 3 x l 0 " 6 437.89 + 15.70 c 93 • l x l O " 6 439.25 + 8.58 c 93 Mean of three rep l ica tes ± S.D. Note: Di f ferent l e t te rs fo l lowing values in each column indicate s ign i f i can t d i f ferences (p>0.05). 74 oe 6 9 10 11 12 13 11 15 16 17 18 Growth Period (days) FIG. 3.1 Inhibition of wild oat growth by t r i a l l a t e : ( • ) cont ro l ; (A) 3 x l o ' S H t r i a l l a t e ; (^ 7) 3 x l O ' 6 M t r a l l a t e . 6 7 8 9 10 11 12 13 14 15 16 17 18 Growth Period (days) FIG. 3.3 Inhibition of wild oat growth by metribuzin: ( • ) contro l ; (A) 5xlO" 6 M metribuzin; ( V ) l x l O " 6 M metribuzin. 7 8 9 10 11 12 13 14 15 16 17 18 Growth Period (days) FIG. 3.2 Inhibit ion of wild oat growth by EPTC: ( • ) contro l ; ( A ) SxlO" 5 N EPTC; ( V ) SxlO" 6 H EPTC. 8 9 10 11 12 13 14 15 16 17 18 Growth Period (days) FIG. 3.4 Inhibit ion of wild oat growth by t r i f l u r a l i n : ( Q ) cont ro l ; ( A ) 3x lO ' 5 M t r i f l u r a l i n ; ( V ) 3xl0" { H t r i f l u r a l i n . 0^ 6 7 8 9 10 11 12 13 14 15 16 17 Growth Period (days) FIG. 3.5 Inhibition of wild oat growth by oryza l ln : ( • ) con t ro l ; ( A ) 3xlO" 5 M oryzal ln; (V ) 3 x l 0 ' 6 M oryza l ln . 75 FIG. 3.6 Wild oat emergence following application of t r i a l l a t e FIG. 3.7 Wild oat emergence following application of 3x10" M t r i a l l a t e 76 for 10-12 days fo l low ing app l i ca t ion (F ig . 3 .3) , however, necrosis of the plant occurred over the next 6 days (F ig . 3 .8 ,3 .9 ) . Lower leve ls of - 6 metribuzin (1x10" M) had no inh ib i to ry e f fect on growth (F ig . 3 .3 ,3 .8 ) . _5 Oryzal in and t r i f l u r a l i n t o t a l l y i nh i b i t ed growth (3x10 M; Figures 3 .4 ,3 .5 ) , however, a corresponding reduction in sugar release was only _5 apparent with t r i f l u r a l i n (only at 3x10 M; Tables 3 .1 ,3 .2 ) . 77 FIG. 3.8 Wild oat emergence fo l lowing appl icat ion of metribuzin FIG. 3.9 Necrosis of wi ld oat seedlings fol lowing appl icat ion of metribuzin 6 7 8 9 10 11 12 13 14 15 16 17 18 Growth Period (days) FIG. 3.10 I n h i b i t i o n o f w i ld oat seed l ing growth fo l low ing a p p l i c a t i o n o f d i f f e r e n t t r i a l l a t e c o n c e n t r a t i o n s : ( • ) c o n t r o l ; (A) 3 x l 0 " 5 M t r i a l l a t e ; (V) l x l O " 5 M t r i a l l a t e ; (A) 3 x l 0 " 6 M t r i a l l a t e ; (f) l x l O " 5 H t r i a l l a t e . 350 | 1 1 | ^ I I I I I I ^ 6 7 8 9 10 11 12 13 14 15 16 17 18 Growth Period (days) FIG. 3.11 I n h i b i t i o n of wi ld oat seedl ing growth fo l lowing a p p l i c a t i o n of d i f f e r e n t t r i f l u r a l i n c o n c e n t r a t i o n s : ( O ) c o n t r o l ; ( A ) 3 x l O " 5 M t r i f l u r a l i n ; (V) l x l O " 5 M t r i f l u r a l i n ; (A ) 3 x l O " 6 M t r i f l u r a l i n ; ( • ) l x l O " 6 M t r i f l u r a l i n . 79 3.4 Discussion Endosperm mob i l i za t i on i s one of many poss ib le target s i t es for he rb ic ide a c t i o n . Al though a l l pre-emergence h e r b i c i d e s s t u d i e d i n h i b i t e d ea r l y growth of w i l d oat seedl ings when appl ied at f i e l d app l i ca t ion rates ( F i g . 3.1 - 3 . 5 ) , only t r i a l l a t e and t r i f l u r a l i n decreased GA^-induced sugar re lease (Tables 3 .1 , 3 .2) . Although th is inh ib i t i on was only pa r t i a l (25% and 22% respec t i ve ly ) , the lower sugar l e v e l s cou ld a f f e c t the compe t i t i veness of the weed by lower ing resources avai lable for growth. Although s o i l app l i ca t i on may reduce the ac tua l he rb ic ide concen t ra t i on reach ing the seed l i ng ( e . g . by absorption to so i l c o l l o i d s ) , there were more ef fects than on a-amylase a c t i v i t y suggest ing that other ta rge t s i t e s may be involved in the inh ib i t i on of seedling growth. T r i a l l a t e and EPTC are thiocarbamate type herbicides and are known to i n h i b i t shoot ra the r than root growth in germinat ing seed l i ngs fol lowing emergence from the s o i l (Ashton and Cra f ts 1981; F i g . 3 .7 ) . However, the lower s t a r c h h y d r o l y s i s (Tables 3 . 1 , 3 . 2 ) p o i n t s to endosperm mobi l izat ion as another target s i t e . Metr ibuzin, a t r i a z i n e type herbic ide, i nh ib i t s photosynthesis in developing seedlings (P f i s t e r et a l . 1979). Seed endosperm mob i l i za t i on and ear l y seedl ing growth were not af fected ( F i g . 3 . 3 ) ; seedl ing necros is d id not occur un t i l about 12 days of growth (F igs . 3 .8 ,3 .9 ) . U n l i k e t h i o c a r b a m a t e - and t r i a z i n e - t y p e h e r b i c i d e s , the d i n i t r o a n i l i n e herb ic ides used ( i . e . t r i f l u r a l i n , o r yza l i n ) a f fec ted ear ly seedling growth p r i o r to emergence of the ep ico ty l ( F i g s . 3 .4 , 80 3.5). This was expected as ear ly l a te ra l root growth and c e l l d i v i s i on are inh ib i ted thus prevent ing emergence of the seed l ing (Ashton and Crafts 1981). T r i f l u r a l i n also targets endosperm mobi l izat ion as shown by the p a r t i a l i n h i b i t i o n of s t a r c h h y d r o l y s i s occur red (Tab les 3 .1 ,3 .2) . Although reduction of starch hydrolysis in wi ld oat i s not a major target s i t e f o r these h e r b i c i d e s , the i n h i b i t i o n caused by t r i a l l a t e and t r i f l u r a l i n may a f f ec t i ve l y reduce the vigor of the seeds. 81 CONCLUSIONS W i l d oat and b a r l e y endosperm h a l v e s were o f t e n h e a v i l y contaminated by microbes. Sur face s t e r i l i z a t i o n w i th e thano l or hypochlor i te was unsat is fac tory , as these agents inh ib i ted GA^-induced sugar re lease. However, captan (66 /iM) successfu l ly cont ro l led fungal contamination without a f f ec t i ng sugar re lease . Reducing sugars were assayed rather than a-amylase in the amino acid s tud ies. However, high absorbance values in the Nelson-Somogyi assay, i n d i c a t i v e of sugar re lease, were subject to a c t i v i t y of amino acids in the assay. Of the twenty amino acids tes ted , cys te ine , c y s t i n e , se r i ne , tryptophan and tyrosine caused an increase in absorbance values; the increase occurred at varying degrees throughout a concentration range from 0.1 to 1 mM. Addi t ions of amino acids along wi th g lucose r e s u l t e d in absorbance values higher than glucose alone. The amino acids appeared to react at 2+ the Cu reduction step of the assay as addit ions of the react ing amino acids fol lowing heating with Somogyi reagent but p r i o r to the addi t ion of Nelson reagent did not form co lo r . MnCl^ (0.5 mM) i n h i b i t e d co lo r development in the presence of g lucose and the amino acids se r i ne , cyst ine and tryptophan. The a b i l i t y of i n d i v i d u a l amino ac ids to enhance a -amy lase product ion in w i l d oat endosperm halves i s yet unc lea r . Although cer ta in amino acids were shown to promote enzyme production, the leve l was of ten qu i te d i f f e r e n t between rep l i ca ted experiments. A l t e r i n g var ious fac tors (seed batch, a f t e r - r i p e n i n g , b u f f e r , pH, i ncuba t ion time, temperature) did not improve the consistency. However, incubation wi th a mixture of 18 amino ac ids c o n s i s t e n t l y promoted a-amylase 82 produc t ion in both w i l d oat and. bar ley endosperm t i s s u e . Enzyme -9 production was further enhanced i f a leve l of GA^ (10 M) which was too low to promote enzyme product ion was included wi th in the amino ac id m ix tu re . The re fo re , the presence of both f r e e amino a c i d s and g ibbere l l i ns are necessary for maximal enzyme production. Endosperm h a l v e s f rom s e l e c t e d b a r l e y c u l t i v a r s d i f f e r e d subs tan t ia l l y in the i r leve l of autonomous endosperm mobi l izat ion (AEM). Sugar re lease among c u l t i v a r s was extremely var iab le often exh ib i t ing 1 8 - f o l d d i f f e r e n c e s . High l e v e l s of sugar r e l e a s e in ' K l a g e s ' correlated well with a-amylase product ion; the leve ls were s im i la r among i n d i v i d u a l c u l t i v a r s harvested in two d i f f e r e n t seasons. AEM was g r e a t l y reduced by i n h i b i t o r s of RNA (6-methyl purine) and pro te in (cycloheximide) synthesis suggesting that AEM was a r e s u l t of the de novo synthesis of a-amylase. Temperature and pH changes great ly affected AEM. Although sugar leve ls in 'K lages ' endosperm were high at ac id i c pH (pH 4 . 6 - 5 . 6 ) , the release was great ly reduced at basic pH (pH 7.6-8 .6) . However, 'V i rden ' exhibi ted no AEM throughout the incubation per iod i r r e s p e c t i v e of the solut ion pH (pH 4 .6 -8 .6 ) . AEM was very low at 12°C in both v Klages ' and 'V i rden ' endosperm halves. However, t h i s leve l was enhanced in both cu l t i va rs as the incubation temperature was increased to 28°C. The onset of AEM in VK1ages' endosperm halves was delayed as only a small amount of sugar was released in the f i r s t 24 hours of incubat ion. However, large increases occurred a f ter t h i s . Addit ion of low leve ls of C a 2 + (0.25-0.5 mM) to the incubation medium enhanced sugar re lease in 83 VK1ages' whereas higher amounts (0.5-1 mM) were i nh ib i to ry . However, C a 2 + d id not i n c r e a s e sugar r e l e a s e in ' V i r d e n ' , a c u l t i v a r not 2+ exhib i t ing AEM at 25°C. Therefore, the presence of Ca enhances AEM, as with GA.j-induced enzyme s y n t h e s i s , but does not i n i t i a t e enzyme synthesis. The frequent express ion of AEM among bar ley c u l t i v a r makes i t necessary to reassess the ro le of the embryo in c o n t r o l l i n g endosperm mob i l i za t i on . Cu l t i vars exh ib i t ing th i s phenomenon must be i den t i f i ed as pre-harvest sprouting resu l ts in large crop losses af fect ing several i m p o r t a n t i n d u s t r i e s ( b r e w i n g , b r e a d - m a k i n g ) . However , the i den t i f i ca t i on of AEM is often d i f f i c u l t as i t s expression is var iab le under d i f ferent incubation condi t ions. I n c u b a t i o n o f w i l d oat endosperm t i s s u e i n s o l u t i o n s of pre-emergence herbicides affected GA^-induced reducing sugar release to va r y i ng ex ten ts when app l i ed at f i e l d a p p l i c a t i o n l e v e l s . Only t r i a l l a t e (25% reduct ion) and t r i f l u r a l i n (22% reduct ion) prevented sugar re lease (only at 3x10 M); no i n h i b i t i o n was seen fo l low ing incubation in EPTC (5x l0~ 5 , 5 x l 0 " 6 M), metribuzin (5x l0~ 5 , l x l O " 6 M) and - 5 - 6 oryza l in (3x10 , 3x10 M). Although reduction of starch hydrolys is is not a major target s i t e for these herb ic ides, the i nh ib i t i on caused by t r i a l l a t e and t r i f l u r a l i n may a f fec t i ve l y reduce the vigor of the seeds. 84 LITERATURE CITED ALEX, J . F . 1966. Survey of weeds of cu l t i va ted land in the p ra i r i e provinces, pp.2-3. Can. Dept. Agr . , Regina, Sask. AKAZAWA, T. and S. MIYATA. 1982. B i o s y n t h e s i s and. s e c r e t i o n of a-amylase and other hydrolases in germinating cereal seeds. Essays in Biochem. 18:41-78. ANONYMOUS. 1990. F ie ld crop guide to weed, d isease, insect , b i r d , and rodent control for commercial growers, p.34-35. Min is t ry of Agr icu l ture and F isher ies , Province of B r i t i s h Columbia. ASHFORD, A .E . and F. GUBLER. 1984. Mob i l i za t ion of polysacchar ide reserves from endosperm. pp.117-162. _In D. Murray (ed). Seed Physiology, Vo l . 2. Academic Press. N.Y. ASHTON, F.M. and A . S . CRAFTS. 1981. 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Regression analysis of data (p>0.05) Fig Treatment Model a b l b2 r2 P 1.3 cysteine y=a+bjX -0.05 0.00 - 1.00 0.00 cystine y=a+bjX -0.03 0.00 - 0.95 0.00 serine 2 y=a+bjX+b2x 0.05 0.00 0.00 0.92 0.00 tryptophan 2 y=a+bjX+b2x 0.06 0.00 0.00 0.73 0.00 tyrosine 2 y=a+b1x+b2x 0.04 0.00 0.00 0.68 0.00 1.4 none y=a+bjX 0.07 3.17 - 0.94 0.00 0.2 mM cys y=a+bjX 0.26 3.14 - 0.93 0.00 0.4 mM cys y=a+bjX 0.61 2.74 - 0.89 0.00 0.2 mM ser y=a+b1x 0.23 3.07 - 0.95 0.00 0.4 mM ser y=a+bjX 0.28 3.46 - 0.98 0.00 2.3 Klages y=a+bjX 1140.01 -129.57 • - - - - - 0.90 0.00 Virden 2 y=a+bjX+b2x -666.77 230.54 -17.62 0.37 0.06 2.5 Kl ages 2 y=a+bjX+b2x 8.12 4.39 0.19 0.88 0.00 Vi rden y=a+bjX 35.69 3.26 - 0.88 0.00 2.6 Klages 2 y=a+bjX+b2x 522.85 -105.82 7.64 0.71 0.00 Virden y=a+b1x 39.14 0.13 0.00 0.741 92 2 Fig Treatment Model a r 3.1 control 3xl0"5 M 3xl0"6"M y=a+bjX y=a+bjX y=a+bjX+b2x': •97.95 21.42 no variation 56.75 -18.13 0.97 0.00 1.48 0.99 0.00 3.2 control 5x10 J M 5xl0"6 M y=a+bjX y=a+bjX y=a+bjX •97.95 -60.35 -97.15 21.42 13.11 20.28 0.97 0.94 0.96 0.00 0.00 0.00 3.3 control y=a+bjX 5x10 u M •6 1x10 y=a+bjX+b2x' M y=a+b2X+b2x' -97.95 •119.33 36.90 21.42 36.74 -7.05 •1.64 1.05 0.97 0.98 0.96 0.00 0.00 0.00 3.4 control 3x10 J M 3xl0"u M y=a+bjX y=a+b1x y=a+b1x -97.95 21.42 no variation -98.20 20.75 0.97 0.97 0.00 0.00 3.5 control 3xl0"5 M 3xl0"6 M y=a+b1x y^a+bjX y=a+b1x -97.95 21.42 no variation -93.90 20.25 0.97 0.00 0.96 0.00 93 2 Fig Treatment Model a b j b^ r control y=a+b1x -79, .45 20.13 - 0 .96 0 .00 3xl0"5 M y=a+bjX no variation lxlO" 5 M y=a+bjX . no variation 3xl0"6 M 2 y=a+bjX+b2x 38, .30 -14.12 1.29 0, .99 0, .00 lxlO" 6 M 2 y=a+b,x+b?x 50, ,60 -2.24 0.89 0, .97 0, .00 3.11 control y=a+bTX -79.45 20.13 - 0.92 0.00 3xl0"5 M y=a+b1x no variation lxlO" 5 M y=a+bjX -75.70 18.98 0.95 0, ,00 3xl0"6 M y=a+bjX -65.00 18.26 0.90 0, .00 lxlO" 6 M y=a+b,x -56.05 19.35 0.98 0, .00 

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