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Leghemoglobins in the genus phaseolus and their significance for nitrogen fixation Lülsdorf, Monika Magdalena 1989

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LEGHEMOGLOBINS IN THE GENUS PHASEOLUS AND THEIR SIGNIFICANCE FOR NITROGEN FIXATION by Monika Magdalena L i i l s d o r f I n g . A g r a r . , U n i v e r s i t a t Bonn, Germany, F.R., 1982 M . S c , The U n i v e r s i t y of Ma n i t o b a , 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS. FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (DEPARTMENT OF PLANT SCIENCE) We a c c e p t t h i s t h e s i s as co n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA APRIL 1989 (c)Monika Magdalena L u l s d o r f , 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his 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 of The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT Leghemoglobins (Lbs) a r e i m p o r t a n t s o l u b l e p r o t e i n s i n the legume r o o t n o dule formed i n response t o i n f e c t i o n by s o i l R h i z o b i u m b a c t e r i a . W i t h the e x c e p t i o n o f P h a s e o l u s  v u l g a r i s , Lbs g e n e r a l l y show s t r u c t u r a l and f u n c t i o n a l h e t e r o g e n e i t y f o r t h e i r o x y g e n - b i n d i n g f u n c t i o n i n the n o d u l e . P_. v u l g a r i s seeds 'Contender' were mutagenized w i t h gamma-rays or e t h y l methane s u l f o n a t e . Among 1400 M2 o f f -s p r i n g d e r i v e d from the t r e a t e d p l a n t s s c r e e n e d f o r Lb v a -r i a b i l i t y , no m u t a t i o n i n Lb was d e t e c t e d . These o b s e r v a -t i o n s a r e c o n s i s t e n t w i t h a s i n g l e a c t i v e gene f o r Lb i n P h a s e o l u s . F i f t y a c c e s s i o n s from the genus P h a s e o l u s were s c r e e n e d f o r Lb v a r i a t i o n . P_. a c u t i f o l i u s s s p . c o n t a i n e d a s i n g l e Lb component which had a d i f f e r e n t e l e c t r o p h o r e t i c m o b i l i t y compared t o t h a t i n P_. v u l g a r i s . P_. f i l i f o r m i s showed two Lb bands, b o t h d i f f e r e n t from P_. v u l g a r i s , w h i l e P. l u n a t u s was found t o c o n t a i n two major and p r o b a b l y t h r e e minor Lb components. These d a t a r e p r e s e n t the f i r s t r e p o r t o f i n t r a - g e n e r i c Lb v a r i a b i l i t y i n P h a s e o l u s . U s i n g embryo r e s c u e t e c h n i q u e , h y b r i d s were produced from _P. v u l g a r i s x P. a c u t i f o l i u s s s p . and P. v u l g a r i s x P.. f i l i f o r m i s c r o s s e s . Rooted c u t t i n g s from each h y b r i d and i i i t s p a r e n t s were used t o e s t i m a t e the n i t r o g e n f i x a t i o n ( a c e t y l e n e r e d u c t i o n ) r a t e and t o d e t e r m i n e t h e t o t a l amount o f n i t r o g e n accumulated d u r i n g the growth p e r i o d . The P_. v u l g a r i s x P_. f i l i f o r m i s h y b r i d s had a s i g n i f i c a n t l y h i g h e r n i t r o g e n f i x a t i o n r a t e and accumulated more n i t r o g e n t h a n e i t h e r p a r e n t . The P_. f i l i f o r m i s p a r e n t p e r f o r m e d b e t t e r t h a n t h e P_. v u l g a r i s p a r e n t but o n l y i n t h e a c e t y l e n e r e d u c t i o n t e s t . One o f the P_. v u l g a r i s x P_. a c u t i f o l i u s h y b r i d s a l s o f i x e d n i t r o g e n a t a h i g h e r r a t e t h a n e i t h e r p a r e n t . Leghemoglobin components from a P_. f i l i f o r m i s h y b r i d and from P.. l u n a t u s 'Lima Hendersons* were i s o l a t e d by c e l l u l o s e ion-exchange chromatography. The N - t e r m i n a l amino a c i d sequence f o r peak I I o f t h e P. f i l i f o r m i s h y b r i d and peaks I and I I from P_. l u n a t u s n o d u l e s was d e t e r m i n e d . The f i r s t 37 amino a c i d s o f each component were i d e n t i c a l t o the p u b l i s h e d sequence f o r P_. v u l g a r i s . M o b i l i t y and/or f u n c t i o n a l d i f f e r e n c e s a r e l i k e l y t o be found i n the s e -quence n e a r e r the l o c a t i o n Of the heme c a v i t y . The d a t a . o f t h i s i n v e s t i g a t i o n s u p p o r t the c o n t e n t i o n t h a t Lb h e t e r o -g e n e i t y i s f u n c t i o n a l and c o n s t i t u t e s an a d a p t a t i o n f o r more e f f e c t i v e n i t r o g e n f i x a t i o n . TABLES OF CONTENTS ABSTRACT . . i i LIST OF TABLES v i LIST OF FIGURES v i i LIST OF ABBREVIATIONS v i i i GLOSSARY i x ACKNOWLEDGEMENTS x Chapter Page I . GENERAL INTRODUCTION 1 I I . GENERAL LITERATURE REVIEW 13 2.1. HEME 13 2.2. GLOBIN 15 2.3. BIOSYNTHESIS, ASSEMBLY AND DEGRADATION 17 2.4. INTRACELLULAR LOCATION 20 2.5. LEGHEMOGLOBIN FUNCTION 22 2.6. QUANTITATIVE RELATIONSHIP BETWEEN LEGHEMO-GLOBIN AND NITROGEN FIXATION 31 2.7. LEGHEMOGLOBIN HETEROGENEITY 3 4 2.8. EVOLUTION OF LEGHEMOGLOBIN GENES 40 I I I . INDUCED MUTAGENESIS FOR LEGHEMOGLOBIN VARIABILITY 50 3.1. INTRODUCTION 5 0 3.2. LITERATURE REVIEW 53 3.3. MATERIALS AND METHODS 6 0 3.4. RESULTS AND DISCUSSION 63 IV. LEGHEMOGLOBIN VARIABILITY IN THE GENUS PHASEOLUS 68 4.1. INTRODUCTION 68 4.2. LITERATURE REVIEW 7 0 iv 4.3. MATERIALS AND METHODS 76 4.3.1. PLANT MATERIAL 7 6 4.3.2. GROWING CONDITIONS 79 4.3.3. LEGHEMOGLOBIN TESTING 81 4.3.4. INTERSPECIFIC HYBRIDIZATION 82 4.3.5. EMBRYO RESCUE AND HYBRID GROWTH 8 3 4.3.6. NITROGEN FIXATION EXPERIMENT 85 4.3.7. PROTEIN SEQUENCING 8 7 4.4. RESULTS AND DISCUSSION 88 4.4.1. LEGHEMOGLOBIN VARIABILITY IN THE GENUS PHASEOLUS 88 4.4.2. INTERSPECIFIC HYBRIDIZATION 96 4.4.3. NITROGEN FIXATION EXPERIMENTS 98 4.4.4. PROTEIN SEQUENCING 112 V. GENERAL DICUSSION AND CONCLUSIONS 117 V I . SUMMARY AND CONCLUSIONS 124 LIST OF REFERENCES ' 125 .V LIST OF TABLES T a b l e Page I . P h a s e o l u s germplasm a c c e s s i o n s 77 I I . , G r o u p i n g o f ' P l a n t S p e c i e s ' f o r a n a l y s i s 99 I I I . A n a l y s i s of v a r i a n c e f o r ARA of P. v u l g a r i s , P_. a c u t i f o l i u s , P_. f i l i f o r a l i s and i n t e r -s p e c i f i c h y b r i d s . A d d i t i o n a l a n a l y s i s f o r the p a r t i t i o n e d sums o f s q u a r e s f o r ' P l a n t S p e c i e s ' i s a l s o shown 100 IV. O r t h o g o n a l c o n t r a s t s among groups f o r t h e ARA ex p e r i m e n t 101 i V. Mean a c e t y l e n e r e d u c t i o n (nm/plant/hour) f o r each ' P l a n t S p e c i e s ' and group measured i n th e ARA e x p e r i m e n t 102 V I . O r t h o g o n a l c o n t r a s t s w i t h i n groups f o r t h e ARA ex p e r i m e n t 103 V I I . A n a l y s i s o f v a r i a n c e f o r T o t a l N i n P_. v u l g a r i s , P. a c u t i f o l i u s , P. f i l i f o r m i s and i n t e r s p e c i f i c h y b r i d s . A d d i t i o n a l a n a l y s i s o f t h e p a r t i t i o n e d sums o f squ a r e s f o r ' P l a n t S p e c i e s ' i s a l s o shown 105 V I I I . O r t h o g o n a l c o n t r a s t s among groups f o r t h e T o t a l N e x p e r i m e n t 106 IX. Means (mg t o t a l n i t r o g e n ) f o r each ' P l a n t S p e c i e s ' and group o f the T o t a l N ex p e r i m e n t 107 X. O r t h o g o n a l c o n t r a s t s w i t h i n groups f o r t h e T o t a l N e x p e r i m e n t i n v o l v i n g P_. v u l g a r i s , P_. a c u t i f o l i u s , .P. f i l i f o r m i s and i n t e r -s p e c i f i c h y b r i d s 109 v i LIST OF FIGURES F i g u r e Page 1. CAE Lb p r o f i l e showing P_. v u l g a r i s (C) , P.. a c u t i f o l i u s N1602 (A^ ) , and P.. a c u t i f o l i u s ssp. l a t i f o l i u s (A|_) 89 2. CAE Lb p r o f i l e showing P_. v u l g a r i s w i l d (P^) , P_. f i l i f o r m i s (P p) , P. mi cr ocarpus (P^ ) and P. v u l g a r i s ' G a l l a t i n 1 (G) 91 3. CAE Lb p r o f i l e showing a P_. v u l g a r i s x P_. f i l i f o r m i s h y b r i d ( 1 ) , P. a c u t i f o l i u s ( 2 ) , P. v u l g a r i s x P_. a c u t i f o l i u s h y b r i d (3) and P_. v u l g a r i s (4) 92 4. CAE Lb p r o f i l e showing P_. v u l g a r i s (C) , P.. l u n a t u s 'Lima Hendersons' ( L ) , £. l u n a t u s 'Market Sample' (M) and P_. a c u t i f o l i u s ( A ) . . 93 5. E l u t i o n p r o f i l e f o r P. v u l g a r i s x P. f i l i f o r m i s h y b r i d Lb components on DE5 2 - c e l l u l o s e columns 113 6. E l u t i o n p r o f i l e f o r P_. l u n a t u s 'Lima Henderson' Lb components on D E 5 2 - c e l l u l o s e columns .... 115 vii LIST OF ABBREVIATIONS ADP aden o s i n e d i p h o s p h a t e ALA a m i n o l e v u l i n i c a c i d ARA a c e t y l e n e r e d u c t i o n a s s a y ATP ade n o s i n e t r i p h o s p h a t e CAE c e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s CDNA complementary DNA CI AT C e n t r o I n t e r n a c i o n a l de A g r i c u l t u r a T r o p i c a l e DE d i e t h y l a m i n o e t h y l -DF degrees o f freedom EMS e t h y l methane s u l f o n a t e Hb hemoglobin IAEA I n t e r n a t i o n a l A t o m i c Energy Agency IEF i s o e l e c t r i c f o c u s i n g Lb l e g h e m o g l o b i n Ml mutant g e n e r a t i o n one MS mean squ a r e s RCBD randomized complete b l o c k d e s i g n TDM t o t a l d r y m a t t e r T i tumor i n d u c i n g Tn t r a n s p o s o n .vi i i GLOSSARY Rhizobiu m i s a g e n e r i c name and r e f e r s t o t h e genus of b a c t e r i a known as Rhizobium. The p l u r a l R h i z o b i a r e f e r s t o more t h a n one c e l l o f R h i z o b i u m , and r h i z o b i a l i s an a d j e c t i v e d e s c r i b i n g some a t t r i b u t e o f R h i z o b i u m . B a c t e r o i d T h i s term i s used t o d e s c r i b e the n i t r o g e n f i x i n g form o f R h i z o b i a found i n r o o t n o d u l e s . P e r i b a c t e r o i d Membrane or membrane en v e l o p e r e f e r s t o the p l a n t membrane s u r r o u n d i n g each b a c t e r o i d or group o f b a c t e r o i d s i n n i t r o g e n f i x i n g r o o t n o d u l e s . P e r i b a c t e r o i d Space r e f e r s t o t h e space between the p e r i -b a c t e r o i d membrane and i t s c o n t a i n e d b a c t e r o i d s . P e r i p l a s m i c Space r e f e r s t o t h e space between t h e b a c t e r o i d i n n e r (plasma) and o u t e r ( c e l l w a l l ) membrane. Leghemoglobin (Lb) r e f e r s t o t h e o x y g e n - c a r r y i n g hemepro-t e i n found i n legume r o o t n o d u l e s . Oxy-Lb i s the oxygenated form, deoxy-Lb r e f e r s t o the deoxygenated form. Hemoglobin (Hb) i s a g e n e r a l term f o r hemeproteins r e g a r d l e s s o f a n i m a l or p l a n t o r i g i n . A c c e s s i o n r e f e r s t o a sample i n a gene bank; i t s number i s u n i q u e . C u l t i v a r r e f e r s t o a c u l t i v a t e d v a r i e t y . Ml g e n e r a t i o n r e f e r s t o t h e f i r s t mutated g e n e r a t i o n , i . e . p l a n t s grown from t h e t r e a t e d seeds. N i t r o g e n r e f e r s t o t h e gaseous element N2; s h o u l d be c o r r e c t l y termed ' d i n i t r o g e n ' . T o t a l N r e f e r s t o t h e t o t a l amount o f n i t r o g e n f i x e d . (References: Appleby 1984, Date 1978, Summerfield 1988.) .ix ACKNOWLEDGEMENT I would l i k e t o thank Dr. Galway from Cambridge U n i v e r s i t y , U.K., and Dr. M i c h a e l s from t h e U n i v e r s i t y of Guelph, Canada, f o r t h e p l a n t m a t e r i a l . F u r t h e r m o r e , I w i s h t o thank my t h e s i s s u p e r v i s o r Dr. F.B. H o l l f o r h i s gui d a n c e and s u p p o r t t h r o u g h o u t t h i s s t u d y . Thanks a r e extended t o my committee members, Dr. A . J . G r i f f i t h s , Dr. V.C. R u n e c k l e s , Dr. M. Upadhyaya, and my e x t e r n a l examiner Dr. D.P.S. Verma f o r t h e i r t i m e and e f f o r t f o r r e v i e w i n g t h i s r e s e a r c h . I a l s o w i s h t o thank Dr. G. Eaton f o r h i s a s s i s t a n c e w i t h the s t a t i s t i c a l a n a l y s i s of t h i s e x p e r i m e n t . S p e c i a l t h a n k s t o my husband, Claude L a p o i n t e , f o r h i s s u p p o r t and encouragement w i t h o u t which I might never have f i n i s h e d t h i s p r o j e c t . F i n a l l y , t hanks t o a l l t h o s e f r i e n d s who made me l a u g h and t h u s made t h e s i t u a t i o n a b i t more b e a r a b l e . x I. GENERAL INTRODUCTION The successful establishment of nitrogen f i x i n g no-dules r e l i e s on a complex sequence of phys i o l o g i c a l pro-cesses, many of which involve i n t e r a c t i o n between the as-sociated Rhizobium bacterium and i t s host plant. A cha-r a c t e r i s t i c of t h i s symbiotic process i s the presence of a red pigment which was f i r s t shown to be a hemeprotein by Kubo i n 1939. He proposed that the phys i o l o g i c a l s i g n i f i -cance of t h i s protein could relate to the transport and storage of oxygen similar to the monomeric myoglobin. These findings were la t e r confirmed by K e i l i n and Wang (1945) and Virtanen (1945). Virtanen and Laine (1946) suggested the name "leghemoglobin" for the root nodule hemoprotein. Leghemoglobins (Lbs) consist of a prosthetic group, the heme, which i s synthesized by the bacteroids, and a globin component which i s encoded by the host plant and synthesized on plant ribosomes (Bergersen 1977, E l l f o l k 1972, Verma and Bal 1976). Globins are widely d i s t r i b u t e d i n nature and occur i n many d i f f e r e n t forms including the tetrameric hemoglobins of higher vertebrates, the monomeric, dimeric, tetrameric 1 and higher polymeric forms of invertebrates, monomeric myoglobin, and dimeric hemoglobins found i n root nodules of nitrogen f i x i n g non-legumes (Appleby et a l . 198 3a, Goodman et a l . 1988, Hunt et a l . 1978, Magnum 1976, Te r w i l l i g e r 1980). Recently, hemoglobins have also been found in root nodules formed by" the a c t i n o r r h i z a l Frankia endophyte with genera as diverse as Alnus, Casuarina, Ceanothus, Comptonia, Eleagnus and Myrica (Roberts et a l . 1985, Tjepkema 1983). In addition, Landsmann et a l . (1986) re-ported that Parasponia Lb complementary DNA (cDNA) hybri-dizes to sequences i n Trema, a species which i s related to Parasponia but non-nodulating. Hattori and Johnson (1985) also detected cross-hybridizing DNA sequences i n Betula and Ceratonia, neither of which f i x nitrogen. Consequently, leghemoglobins or closely related proteins seem to be widespread i n the plant kingdom and may be present and functioning i n some as yet undiscovered capacity i n a l l plants (Roberts et a l . 1985). A l l nitrogen f i x i n g symbioses involving the bacterium Rhizobium require the presence of leghemoglobin since mu-tants d e f i c i e n t i n hemoglobin synthesis are i n e f f e c t i v e i n nitrogen f i x a t i o n (Fuller and Verma 1984, Lang-Unnasch et a l . 1985). The establishment of a symbiotic nitrogen f i x i n g re-2 l a t i o n s h i p as reviewed by Sprent (1979) and Bergersen (1982), begins with the release of plant compounds from the germinating root into the s o i l . These compounds at t r a c t the associated rhizobia bacteria and induce them to multi-ply and thus lead to a build-up of a b a c t e r i a l population within the rhizosphere. The f i r s t obvious symptom of plant i n f e c t i o n i s usually observed as a curling of the root hair due to the release of a b a c t e r i a l product (Yao and Vincent 1969). Sprent (1979) has suggested that c u r l i n g might delimit the area where e s s e n t i a l substances accumulate or that entry holes made i n the crook of a c u r l may be more e a s i l y plug-ged to prevent entry of pathogens. Once the bacteria are properly aligned and bound to the root hair, they enter by l o c a l i z e d digestion of the c e l l wall (Callaham and Torrey 1981) . The i n f e c t i o n pro-ceeds with the formation of i n f e c t i o n threads which consist of rows of d i v i d i n g bacteria enclosed by a host membrane derived from the plasmalemma. The plant root reacts to b a c t e r i a l synthesized auxins with c e l l enlargement and d i -v i s i o n which r e f l e c t s the i n i t i a t i o n of nodule formation. The thread continues to grow between and through the c o r t i c a l c e l l s u n t i l i t reaches the inner cortex (Sprent 1979, Bergersen 1982, Sprent and Minchin 1985). There, the bacteria are released into the plant c e l l cytoplasm but 3 they remain enclosed, singly or i n groups, within the host membrane, also c a l l e d the peribacteroid membrane (Bergersen and Goodchild 1973a). The next step i n a successful symbiosis involves the enlargement and d i f f e r e n t i a t i o n of the bacteria into bac-teroids. At t h i s l a t t e r stage, they are capable of f i x i n g atmospheric nitrogen into ammonia, which i s then released into the host cytoplasm and incorporated into plant pro-teins v i a the glutamine/glutamate pathway (Schubert 1986, Sprent 1979). The actual process of nitrogen f i x a t i o n i s catalyzed by the enzyme nitrogenase which consists of two complex subunits. The process of reducing nitrogen requires con-siderable amounts of energy i n the form of ATP, which i s produced by nodule r e s p i r a t i o n coupled to oxidative phos-phorylation with oxygen as the terminal electron acceptor (Appleby et a l . 1975). However, the occurrence of the ne-cessary amounts of free oxygen i n the c e l l would i r r e v e r -s i b l y inactivate the oxygen-sensitive nitrogenase enzyme complex. Hence, nitrogen f i x i n g plants have solved t h i s problem through the production of r e s t r i c t i v e structures which l i m i t oxygen d i f f u s i o n into the nodule (Minchin et a l . 1985, Sheehy et a l . 1985, Hunt et a l . 1988) and by the synthesis of leghemoglobins which function by f a c i l i t a t i n g oxygen transport and thus d i s t r i b u t i o n (Wittenberg et a l . 4 1974) . Consequently, a rapid flux of oxygen i s maintained throughout the nodule, s u f f i c i e n t for ATP production, but maintaining a concentration of free oxygen s u f f i c i e n t l y low that the nitrogenase complex i s not affected (Appleby et a l . 1975, Bergersen and Turner 1975a,b, Wittenberg et a l . 1974). Phaseolus vulgar is L. (kidney bean) has t r a d i t i o n a l l y been regarded as a poor nitrogen f i x e r i n comparison to other species such as V i c i a faba L. (broad bean) and Glycine max L. (Merr.) (soybean), e s p e c i a l l y during the important l a t e p o d - f i l l i n g stage, although this view may r e f l e c t the absence of s p e c i f i c s e l e c t i o n for t h i s t r a i t (Graham 1981, Rennie and Kemp 1983). Breeding for improved nitrogen f i x a t i o n has been focused on three targets: the bacteria, the host, or coincident s e l e c t i o n of both. Rhizobium str a i n s d i f f e r i n t r a i t s such as the time required for nodule development, cold and nitrogen f e r t i -l i z e r tolerance, nitrogenase a c t i v i t y , and the presence of an uptake hydrogenase system which recovers some of the energy l o s t due to hydrogen evolution (Attewell and B l i s s 1985, Barnes et a l . 1984, Hardarson et a l . 1984, Ligero et a l . 1987, Rennie and Kemp 1981) . Consequently, one or a combination of these t r a i t s could be used to select im-proved s t r a i n s . However, the major problem of t h i s s t r a t e -gy i s the d i f f i c u l t y i n establishing a p a r t i c u l a r Rhizobium 5 s t r a i n i n the s o i l where indigenous strains are already established (Johnson et a l . 1965, Roughley et a l . 1983, Weaver and Frederick 1974). In addition, even i f t h i s problem can be overcome successfully, the introduced s t r a i n may not necessarily make up the majority of the nodules (Caldwell and Vest 1970, Vest et a l . 1973). The same problems apply to coincident selection of host and microbe. Results have indeed shown that some c u l t i v a r s f i x considerably more nitrogen with one or few s t r a i n s (Heichel 1982, Rennie and Kemp 1983, Mytton and Livesey 1983). However, i n addition to d i f f i c u l t i e s i n est a b l i s h i n g s p e c i f i c s t r a i n s , Mytton et a l . (1984) ob-served that non-additive interactions can account for over 70% of the t o t a l v a r i a t i o n and thus make the phenotypic expression of nitrogen f i x a t i o n subject to substantial and unpredictable v a r i a t i o n when d i f f e r e n t Rhizobium genotypes nodulate a plant. Given these impediments, a promising target for im-proving nitrogen f i x a t i o n constitutes the host i t s e l f . Because of the large amounts of energy required for n i t r o -gen f i x a t i o n , i t has been proposed that the supply of car-bohydrates constitutes an important l i m i t i n g factor to no-dule function (Brunner and Zapata 1984, Smith et a l . 1980). Hence, s e l e c t i o n might focus for example on prolonging the preflowering period and/or on delaying leaf senescence 6 (Piha and Munns 1987, Rennie and Kemp 1981). Enhancement of photosynthetic rate has indeed been a target for selec-t i o n (Attewell and B l i s s 1985, Barnes et a l . 1984, Graham and Temple 1984, McFerson et a l . 1981). However, some authors have pointed out that nitrogen f i x a t i o n might also be oxygen-limited (Denison et a l . 1988, Suganuma et a l . 1987, Tajima et a l . 1986). Tajima et a l . (1986) and Denison et a l . (1988) proposed that oxygen sup-ply may be a more l i m i t i n g factor for nitrogen f i x a t i o n than the demand of energy. Furthermore, a c o r r e l a t i o n has been observed between the a b i l i t y of nodules to f i x n i t r o -gen and their Lb content, suggesting that t h i s hemeprotein plays a fundamental role i n nitrogen f i x a t i o n (Appleby 1984, Wittenberg et. a l 1974). The importance of leghemo-globin may also be r e f l e c t e d i n the observation that up to 40% of the t o t a l soluble protein of the nodule i s Lb (Nash and Schulman 1976). In addition, Lb genes are inherited codominantly and are stably expressed (Holl et a l . 1983) i n contrast to many quantita t i v e l y inherited nitrogen f i x i n g t r a i t s which often show low h e r i t a b i l i t y and large environmental interactions (Mytton et a l . 1984). Thus, leghemoglobin because of i t s importance i n the nodule would seem to be an obvious target for s e l e c t i o n i n breeding for enhanced nitrogen f i x a t i o n of P_. vulgar i s . 7 Many s t r u c t u r a l genes occur i n multiple copies i n the genome of higher eukaryotes. If such a group of genes descended from a common ancestral gene, then they c o n s t i -tute a gene family (Lewin 1985). The members of such a family may be clustered on the same or dispersed on d i f -ferent chromosomes, or a combination of both. If the co-pies are (almost) i d e n t i c a l , then they usually provide a source of increased (amplified) gene products. However, i f the genes have diverged during evolution, then gene fami-l i e s can provide s l i g h t l y d i f f e r e n t proteins for p a r t i c u l a r circumstances (Lewin 1985, Suzuki et a l . 1986). Hemoglobin, myoglobin and leghemoglobin constitute such a globin "super-family", derived from a common ances-t r a l gene (Lewin 1985). With the exception of myoglobin, which i s represented only by a single gene in the human genome, hemoglobins and leghemoglobins usually occur i n multiple copies (Lewin 1985, Suzuki et a l . 1986). An excellent example of a globin gene family i s the leghemoglobin genes i n soybean. Lee and Verma (1984) i d e n t i f i e d four l o c i containing four functional, two pseudo and two truncated leghemoglobin genes. Proteins coded by the four functional genes d i f f e r s l i g h t l y in their amino acid sequence, the homology being over 90%. The functional genes encode four d i s t i n c t products which can be separated by i s o e l e c t r i c focusing into four major (Lba, Lbc^, Lbc£, 8 Lbc3) and four minor components, the l a t t e r being post-t r a n s l a t i o n a l modifications of the major components (Fuchsman and Appleby 1979, Whittaker et a l . 1979). In contrast, P_. vulgaris was shown to contain four globin genes which might be arranged i n one locus but are not c l o s e l y linked (Lee and Verma 1984). However, i f leg-hemoglobin i s extracted from common bean nodules and sepa-rated by c e l l u l o s e acetate electrophoresis (CAE) or i s o -e l e c t r i c focusing (IEF), then only one major Lb component (Lba) can be detected (Lehtovaara and E l l f o l k 1975a). This observation raises the question of whether only a single gene i s expressed and the other three are s i l e n t , or whether a l l four genes encode a single component? There i s evidence to support expression of a single gene i n the s t r u c t u r a l d e t a i l s available; the complete amino acid sequence of P_. vulgaris Lba has 85% i d e n t i t y with G_. max Lbc^ and 79% i d e n t i t y with G_. max Lba i n d i -cating t h e i r close evolutionary r e l a t i o n s h i p (Lehtovaara and E l l f o l k 1975b, Lee and Verma 1984). In addition, the intragenic structure of the P_. vulgar i s globin gene shows the same intron/exon arrangement as that of the G_. max leghemoglobin genes, including extensive sequence homology i n both coding, and 5' and 3 ' non-coding regions (Lee and Verma 1984) . Experimental evidence suggests that the Lbc^gene of 9 soybean i s activated before the Lba gene and that Lbc^ i s activated after the Lbc3 gene (Fuchsman and Appleby 1979, Verma et a l . 1983). The amount of Lbc i n young nodules i s f i v e to eight times as great as the amount of Lba. This r a t i o changes when the nodules mature to about 1.5:1 (Fuchsman and Appleby 1979). The genes are apparently transcribed i n reverse order of their arrangement on the chromosome and their rate of synthesis i s regulated sepa-rately i n d i c a t i n g that leghemoglobin heterogeneity i s functional (Marcker et a l . 1984, Jensen et a l . 1988, Verma et a l . 1979) . Soybean Lba was found to have a higher oxygen-binding capacity than Lbc and i s , therefore, more e f f e c t i v e i n oxygen transport. The r e l a t i v e increase i n Lba content might be necessary for the regulation of oxygen concentra-t i o n when the nodule structure becomes more complex with age (Fuchsman and Appleby 197 9) . Fuchsman and Palmer (1985) have studied Lbs from a g e n e t i c a l l y diverse s e l e c t i o n of cultivated and wild soy-beans. They concluded that the conservation of both leg-hemoglobin heterogeneity and also a l l four major globin structures provided strong circumstantial evidence that t h i s heterogeneity i s functional. This view was further supported by the studies of Uheda and Syono (1982a,b) with peas (Pisum sativum L.) who concluded that "leghemoglobin 10 heterogeneity i s an evolutionary adaptation for more e f f e c t i v e nitrogen f i x a t i o n " . Leghemoglobin v a r i a b i l i t y i s widespread among nitrogen f i x i n g plant genera e.g. Glycine, Lathyrus, Lens, Lupinus, Medicago, Ornithopus, Pisum, Sesbania, and Tr ifolium (Cutting and Schulman 1971, Dilworth 1969, Holl et a l . 1983, Kortt et a l . 1987, Kuhse and Punier 1987, Richardson et a l . 1975). Thus, P. vulgaris and i t s r e l a t i v e P_. coccineus, which contain only a single globin component each, seem to be rare among the nitrogen f i x i n g species. I f , indeed, Lb heterogeneity i s an evolutionary adap-ta t i o n for more e f f e c t i v e nitrogen f i x a t i o n and t h i s capa-b i l i t y could be transferred to P_. vulgaris, one might an-t i c i p a t e the development of a more e f f i c i e n t nitrogen f i -xing bean species. Such a modified plant type might be expected to have a higher f e r t i l i z e r - u s e e f f i c i e n c y and/or to be more cost e f f i c i e n t i n nitrogen f e r t i l i z e r use. Therefore, t h i s study was conducted to assess the p o s s i b i l i t y of introducing leghemoglobin heterogeneity into Phaseolus vulgar i s and to evaluate such changes as a means to improve the nitrogen f i x a t i o n e f f i c i e n c y of t h i s species. The s p e c i f i c objectives of th i s investigation were: 11 To introduce or create v a r i a b i l i t y for leghemo-globins i n Phaseolus vulgar i s . To determine whether leghemoglobin v a r i a b i l i t y introduced into Phaseolus vulgar i s enhances the nitrogen f i x a t i o n c a p a b i l i t y of th i s crop species. 12 I I . GENERAL LITERATURE REVIEW 2.1. HEME The prosthetic group of bean leghemoglobin consists of protoheme IX (Lehtovaara and E l l f o l k 1975a). The iron atom i s i n a t r i v a l e n t state and coupled to the nitrogen atoms of the four pyrrole rings which l i e i n a plane around i t . The other two bonds of the iron are perpendicular to and on either side of the heme. The f i f t h ligand i s bound to an imidazole side chain of h i s t i d i n e of the globin chain and the s i x t h position i s available for binding oxygen '•' •T ( E l l f o l k 1972, White et a l . 1973). In the nodule, the iron i s maintained i n a physiolo-g i c a l l y active ferrous form due to the presence of reduc-tases which probably originate i n the periplasmic space between the bacteroid plasmamembrane and the plant c e l l wall (Appleby 1984, Rigaud 1983). The biosynthetic pathway for protoheme IX formation involves the aminolevulinic acid (ALA) synthetase pathway and was i n i t i a l l y postulated to take place i n the bacte-roids (Tait 1968), a proposal which was confirmed by O'Brian et a l . (1987). The authors obtained a transposon-induced Bradyrhizobium mutant, d e f i c i e n t i n protoporphyri-nogen oxidase a c t i v i t y which i s needed for the penultimate 13 step i n heme biosynthesis. This mutant induced nodules on soybeans which lacked leghemoglobin. However, experiments by Guerinot and Chelm (1986) with a heme mutation i n the f i r s t step of the ALA synthesis pathway showed that part or a l l of the heme synthesis can also be ca r r i e d out by the host plant and hence the authors proposed that there may be a coordinated e f f o r t of plant and bacteria to produce the necessary heme. Even though the bacteroids have now been i d e n t i f i e d as the major source of heme, the mechanism for the regulation of heme biosynthesis remains unclear. Avissar and Nadler (1978) suggested that reduced oxygen tension induces heme formation and excretion from Bradyrhizobium. In contrast, Dilworth and Appleby (1979) proposed a feed-back mechanism for heme regulation. Iron i s inserted into protoheme IX by a bacteroid ferrochelatase (Porra 1975). The heme moiety l i e s within a hydrophobic cavity created by the globin chain. Protoheme IX i s s t r u c t u r a l l y the same i n most plant or animals and thus, differences i n a f f i n i t y for oxygen and other charac-t e r i s t i c s of the hemoglobins are attributable to v a r i a t i o n i n the amino acid composition of the globin chain (White et a l . 1973). Such differences would be consistent with the suggestion that Lb heterogeneity may have functional s i g -nificance (Fuchsman and Palmer 1985) . 14 2.2. GLOBIN In addition to the heme group, leghemoglobins are comprised of a globin apoprotein. Phaseolus vulgar i s Lba has a molecular weight of 16,900 daltons and consists of one polypeptide chain with 145 amino acids (Lehtovaara and E l l f o l k 1975a). With the exception of pea, a l f a l f a , lupin, and broad bean, no other leghemoglobins have been found to include methionine ( E l l f o l k 1972, Dilworth and Appleby 1979). Cysteine has not been reported i n any leghemoglobin, ana-logous to mammalian myoglobins but not hemoglobins (Hunt et a l . 1978). Usually, myoglobins and hemoglobins have a gly-cine or valine residue as N-terminal amino acids; kidney bean Lb contains glycine (Lehtovaara and E l l f o l k 1975b). Like Glycine max, P_. vulgar i s contains two h i s t i d i n e s i n position 61 and 92. The h i s t i d i n e i n position 61 i s most l i k e l y the f i f t h ligand to heme (Appleby 1974). E l l f o l k (1972) pointed out that prolines are situated close to points where the globin chain i s folded and are thus important for the t e r t i a r y structure of the protein. Hence, the author proposed that leghemoglobins are folded s i m i l a r l y to vertebrate globin chains. Four out of f i v e prolines are i n the same p o s i t i o n i n P_. vulgaris and G. max. 15 O l l i s et a l . (1983) observed s i m i l a r structures for soybean Lba and other monomeric hemoglobins except that the D-helix i s missing i n leghemoglobins. Consequently, leg-hemoproteins consist only of seven h e l i c a l segments repre-senting 77% of the structure. The amino acids asparagine 55 to glutamine 75 constitute the E-helix which would com-prise an important part of the pocket surrounding the heme (Appleby 1974, E l l f o l k 1972, Lehtovaara and E l l f o l k 1975a,b). Appleby (1974) speculated that the E-helix might have some a b i l i t y to move i n a v e r t i c a l d i r e c t i o n and thus contribute to i t s high oxygen a f f i n i t y i n contrast to other hemeproteins. In addition, the hydrophobicity of the heme cavity influences the reversible binding of oxygen to a large extent ( E l l f o l k 1972). Lee and Verma (1984) determined that the intragenic structure of bean Lba showed three intervening sequences i n the same positions as those i n soybean, although they are, however, shorter. These authors also reported the presence of four hybridizing bands i n d i c a t i n g the presence of four leghemoglobin genes i n the Phaseolus genome. The bean Lb locus, l i k e soybean, contains a common sequence s p e c i f i c to the 5' and 3 1 end. Since only one copy of each sequence was found i n Phaseolus, the authors proposed that the four bean globin genes are arranged i n one locus. 1 6 2.3. BIOSYNTHESIS, ASSEMBLY AND DEGRADATION Leghemoglobins are only synthesized when plant and Rhizobium develop i n symbiosis. Recently, small amounts of Lb have been detected i n uninfected i n t e r s t i t i a l c e l l s of soybean root nodules (VandenBosch and Newcomb 1988). How-ever, the authors could not determine whether i t was the holoprotein or only the apoprotein of Lb. Derepression of the leghemoglobin genes appears to be under the control of the bacteroid or of nodule tissue which i s formed after the i n f e c t i o n . Verma et a l . (1979) demonstrated that the i n i t i a t i o n of the globin genes does not depend on the appearance of nitrogenase since leghemo-globin was detected six days after i n f e c t i o n by i n v i t r o t r a n s l a t i o n of nodule polysomes and thus, p r i o r to the ap-pearance of nitrogenase a c t i v i t y . Furthermore, a transposon Tn5-induced cytochrome-deficient mutant which could not ca-talyse the penultimate step of the heme biosynthesis path-way s t i l l contained apoleghemoglobin (O'Brian et a l . 1987). Thus, the authors concluded that heme i s not necessary for apoglobin synthesis. However, O'Brian et a l . (1987) did not rule out a regulatory role for a heme precursor. Marcker et a l . (1984) investigated the ac t i v a t i o n of the soybean leghemoglobin genes and concluded that leghe-moglobins contain two discrete a c t i v a t i o n s i t e s with which two possible d i f f e r e n t regulatory molecules interact i n a 17 cooperative manner. These re s u l t s were confirmed by Jensen et a l . (1988) who mapped two d i s t i n c t regions i n the 5' upstream region of the soybean Lbc^ gene which strongly bind a nodule s p e c i f i c factor. The i n i t i a l a c t i v a t i o n would then be due to the interaction at one s i t e and a se-cond i n t e r a c t i o n at the second s i t e would explain the ob-served large increase i n tr a n s c r i p t i o n of the Lbc^ gene after about 12 days. However, the o r i g i n of the i n i t i a l a c t i v a t i o n remains to be elucidated.. It has been suggested that the a c t i v a t i o n of leghemoglobin genes i s a response of the plant to the altered physiological environment created within the nodule due to the presence of the bacteria (Appleby 1984, Marcker et a l . 1984). Since the location of leghemoglobin i s s t i l l disputed (see chapter 2.4.), i t i s not ce r t a i n where the assembly of heme and globin takes place. Verma et a l . (1979) concluded that since the apoprotein does not have a t r a n s i t sequence, i t i s most l i k e l y that the globin remains i n the host cy-toplasm after t r a n s l a t i o n from plant ribosomes and that heme i s excreted into the cytoplasm by the bacteroids. In this scenario, assembly l i k e l y takes place i n the host no-dule cytoplasm. Once heme i s present, the polypeptide and the prosthetic group can combine spontaneously ( E l l f o l k and Sievers 1965). They are rapidly and strongly bound and 18 o n l y low l e v e l s of f r e e heme a r e found i n h e a l t h y n o d u l e s ( P o r r a 1975) . Legumes w i t h m e r i s t e m a t i c nodule t i s s u e l i k e f a b a beans and l u p i n s , a c t i v e l y s y n t h e s i z e l e g h e m o g l o b i n a t one end o f the nodule w h i l e i t i s b e i n g degraded a t the o t h e r end. Nodules l a c k i n g meristems l i k e common beans and soy-beans s y n t h e s i z e l e g h e m o g l o b i n o n l y over a b r i e f p e r i o d o f nodule f o r m a t i o n and th e c o n c e n t r a t i o n remains about the same u n t i l nodule senescence ( D i l w o r t h and App l e b y 1979, D i l w o r t h 1980) . Based on p u l s e - c h a s e e x p e r i m e n t s w i t h J - ^ c - l a b e l l e d p r o t e i n s , C o v e n t r y and D i l w o r t h (1976) e s t i m a t e d t h a t l u p i n Lb had an ap p a r e n t h a l f - l i f e o f about 18 days. I n con-t r a s t , B i s s e l i n g e t a l . (1980) d e t e r m i n e d t h a t pea Lb had an average t u r n o v e r - r a t e o f about two days. The a u t h o r s used two methods, k i n e t i c s o f 35s i n c o r p o r a t i o n i n t o p r o -t e i n and c h l o r a m p h e n i c o l i n h i b i t i o n o f b a c t e r o i d p r o t e i n s y n t h e s i s , which gave s i m i l a r e s t i m a t e s . A t h i r d method, p u l s e - c h a s e e x p e r i m e n t s w i t h 35grj 2-f w a s found u n s u i t a b l e s i n c e t h i s p r o c e d u r e tended t o o v e r e s t i m a t e h a l f - l i f e o f p r o t e i n s due t o r e - u t i l i z a t i o n o f r e l e a s e d amino a c i d s de-r i v e d from p r o t e i n c a t a b o l i s m . Thus, l e g h e m o g l o b i n and b a c t e r i a l p r o t e i n s seem t o have t h e same t u r n o v e r r a t e s ( B i s s e l i n g e t a l . 1980) . I n g e n e r a l , d e g r a d a t i o n o f l e g h e m o g l o b i n s t a r t s w i t h 19 flowering and i t s decrease i s concomitant with but not correlated to the decrease i n the nitrogenase a c t i v i t y (Klucas 1974, Virtanen and Miettinen 1963). Pladys et a l . (1988) reported that senescence (and n i t r a t e treatment) induced a decrease i n the i n t r a c e l l u l a r pH of bean nodules. This decline i n nodule pH was found to stimulate Lb hydro-l y s i s and oxidation which contributed to depression of nitrogen f i x a t i o n i n ageing nodules of t h i s species. 2.4. INTRACELLULAR LOCATION Bacteroids are located s i n g l y or in groups within membrane-bound v e s i c l e s (Bergersen and Briggs 1958, Goodchild 1977) which a r i s e from the plasmalemma following endocytotic processes during nodule development (Dixon 1967). In 1949, Smith determined that, i n soybean, leghemo-globin was located i n the bacteroid-containing c e l l s of the nodule tissue. However, the exact i n t r a c e l l u l a r l o c ation of the hemeprotein remains a controversial issue. One group of researchers has reported that the hemo-globin i s located e x c l u s i v e l y i n the plant cytoplasm of d i f f e r e n t legumes using various methods. For example, Verma and Bal (1976) and Verma et a l . (1978) isolated i n -tact membrane envelopes which contained no material immu-noreactive to leghemoglobin antibodies. The authors con-20 eluded that Lb was l o c a l i z e d i n the host c e l l cytoplasm. S i m i l a r l y , when Robertson et a l . (1978) i s o l a t e d complete membrane-enclosed bacteroids they could f i n d only traces of leghemoglobin within the membranes; they also concluded that the protein must be located i n the host cytoplasm. In contrast, i t has also been proposed that leghemo-globin i s exclusively located i n the peribacteroid space. Dilworth and Kidby (1968) concluded from electron micros-copic autoradiography studies using 59pe-labelled Serradella nodules that leghemoglobin was located i n the peribacteroid membrane and thus i n d i r e c t contact with the bacteroids. Bergersen and Goodchild (1973a) used oxidized diaminobenzidine staining to v i s u a l i z e soybean leghemoglo-bin for electron microscopy. They confirmed that the heme protein was present i n the membrane envelopes whereas the cytoplasm was not stained. Bergersen*and Appleby (1981) using antiserum and Gourret and Fernandez-Arias (1974) using the leghemoglobin reaction with diaminobenzidine ob-tained the same r e s u l t s . Recently, Robertson et a l . (1984) demonstrated that leghemoglobin can be detected i n the plant cytoplasm and the host nucleus but not i n the peribacteroid spaces, using immuno-gold staining. Since leghemoglobin i s not synthe-sized as a precursor molecule for transport across a mem-brane (Verma et a l . 1979) and since the nodule cytosol 21 contains enzymes both for protection of the heme from o x i -dation and for leghemoglobin reduction (Puppo et a l . 1982, Saari and Klucas 1984), i t seems ce r t a i n that the hemepro-t e i n i s indeed located i n the cytoplasm. A simulation study by Sheehy and Bergersen (1986), based on the experiments by Appleby (1969c), determined that when leghemoglobin was absent from the cytoplasm, n i -trogenase a c t i v i t y would be greatly reduced, whereas the presence of the globin exclusively i n the peribacteroid space had no e f f e c t on enzyme a c t i v i t y l e v e l s . However, since the authors observed a discrepancy between the l e v e l of oxygenation required and that observed experimentally by Appleby (1969c), they suggested that leghemoglobin i s l i k e l y present i n both cytoplasm and peribacteroid space. 2.5. LEGHEMOGLOBIN FUNCTION K e i l i n and Wang (1945) investigated leghemoglobin spectroscopically and observed that the red pigments i n nodules have 'an a f f i n i t y for oxygen'. Further spectro-photometry studies by Appleby (1962) indicated that the function of leghemoglobin could be the transport of oxygen. Measurement of the oxygen binding curves for p u r i f i e d soybean leghemoglobin were plotted as rectangular hyper-bolae; such curves would be expected for monomeric hemo-globins without heme-heme in t e r a c t i o n (Behlke et a l . 1971, 22 Imamura et a l . 1972). In 1973, Bergersen and coworkers added leghemoglobin to a bacteroid suspension and found that stimulation of nitrogenase a c t i v i t y was much greater than stimulation of oxygen uptake, suggesting that there may be a more energy-efficient ATP producing pathway. However, i t was Wittenberg et a l . (1974) who demons-trated i n i n v i t r o experiments that leghemoglobin func-tions by transporting oxygen through a solution and thereby making oxygen available for bacteroid respiration. Adding oxygenated leghemoglobin to bacteroid suspensions enhanced the rate of oxygen consumption, the amount of ATP formed, the ATP/ADP r a t i o , the reduction of acetylene to ethylene (a measure of the nitrogenase a c t i v i t y ) , and the incorpo-rati o n of l a b e l l e d into ammonium. These authors con-cluded from e q u i l i b r i a and k i n e t i c measurements that oxygen was responsible for the increase i n bacteroid phosphoryla-tion and thus nitrogenase a c t i v i t y . In addition, Wittenberg et a l . (1974) also reported that other oxygen c a r r i e r s such as vertebrate and invertebrate myoglobins and hemoglobins, hemerythrin and hemocyanin could substitute for leghemoglobin i n the enhancement of acetylene reduction although proteins with high molecular weight, low a f f i n i t y or slow oxygen dissociation-rates were less e f f i c i e n t . Leghemoglobin shows an unusually high oxygen a f f i n i t y 23 due to a combination of a fa s t oxygen association-rate constant and a r e l a t i v e l y slow dissociation-rate constant (Imamura et a l . 1972, Wittenberg et a l . 1972). Thus, t h i s protein f a c i l i t a t e s the transport of oxygen to the bacte-roids at a rate which i s s u f f i c i e n t to allow bacteroid phosphorylation, as r e f l e c t e d i n enhanced leve l s of ATP, ATP/ADP r a t i o and nitrogenase a c t i v i t y (Appleby et a l . 1975, Wittenberg et a l . 1972), but low enough to avoid damage to nitrogenase (Bergersen and Turner 1975a,b). However, the inter p r e t a t i o n of i n v i t r o experiments l i k e those by Wittenberg et a l . (1974) and Appleby et a l . (1975) i s lim i t e d because dissolved oxygen concentrations and f r a c t i o n a l oxygenation could not be measured and be-cause leghemoglobin could have been f a c i l i t a t i n g the d i s -t r i b u t i o n of oxygen within the shaken l i q u i d which might have no relevance to i t s i n vivo function (Bergersen 1978, 1982). Thus, Bergersen and Turner (1975a,b) developed a no-gas phase assay i n which deoxygenation of Lb was spec-trophotometrically monitored. The authors showed that i n contrast to previous r e s u l t s , Lb increased oxygen uptake with increasing oxygen concentration (Bergersen and Turner 1975a). Furthermore, the authors reported that maximum nitrogenase rates occurred i n a range of oxygen concentra-tions which supported no a c t i v i t y i n the absence of leghe-moglobin. Nitrogenase a c t i v i t y was enhanced because of 24 higher ATP/ADP r a t i o s due to a more e f f i c i e n t respiratory pathway. The dependence of nitrogen f i x a t i o n on the presence of molecular oxygen (Bergersen and Turner 1975a,b) and studies with the oxidative phosphorylation i n h i b i t o r carbonyl cyanid m-chlorophenylhydrazone (Appleby et a l . 1975) confirmed that ATP generation i s linked to electron transport and not to substrate l e v e l phosphorylation. In the absence of leghemoglobin, bacteroid r e s p i r a t i o n i s r e s t r i c t e d due to li m i t e d d i f f u s i o n of free oxygen. The high concentration of leghemoglobin i n the nodule buffers the bacteroids against fluctuations i n oxygen concentration which may occur through external perturbation or as a con-sequence of fluctuations i n the supply of energy y i e l d i n g substrates (Bergersen 1978). Stokes (1975) also recognized the considerable storage and buffering capacity, and thus the s t a b i l i z a t i o n of delivered oxygen tension, to be an important point of the leghemoglobin function. The abundance of oxygenated leghemoglobin i n the cy-toplasm constitutes a large store of oxygen which would ensure a uniform supply for bacteroid r e s p i r a t i o n . In ad-d i t i o n , the concentration of free oxygen i n the cytoplasm w i l l be kept low because of the extraordinary a f f i n i t y of leghemogobin for oxygen, and thus damage to the nitrogenase protein may be avoided (Wittenberg 1980). Oxygen enters the nodules v i a l e n t i c e l s on their sur-25 face (Pankhurst and Sprent 1975). From i t s point of entry, the oxygen diff u s e s through the nodule v i a i n t r a c e l l u l a r spaces (Bergersen and Goodchild 1973b) with a possible d i f f u s i o n barrier i n the cortex (Tjepkema and Yocum 1973). Hunt et a l . (1988) developed a model which predicted that a d i f f u s i o n barrier i n form of water plugs was present i n the i n t r a c e l l u l a r spaces of a layer of c e l l s between the inner and outer cortex. The presence of oxygen d i f f u s i o n bar-r i e r s together with high respiratory oxygen consumption keeps the oxygen concentration i n infected c e l l s low (Hunt et a l . 1988, Minchin et a l . 1985, Tjepkema and Yocum 1973, 1974). Sheehy et a l . (1985) concluded from th e i r mathema-t i c a l model that, as a consequence of the primary need for nitrogenase protection, nodules function under a degree of oxygen l i m i t a t i o n . Membrane envelopes are thought to be impermeable to leghemoglobin but not to free dissolved oxygen (Bergersen 1982). The r e s u l t s of Bergersen (1980) suggested that a small amount of leghemoglobin present i n the envelope space would ensure an even d i s t r i b u t i o n of oxygen to a l l bacte-roids . The author concluded that i n the absence of leghe-moglobin i n the envelope space, oxygen would be d e f i c i e n t through much of the space and optimum bacteroid r e s p i r a t i o n could not be maintained. In contrast, other researchers have advocated that 26 there i s no leghemoglobin present i n the envelope space (see chapter 2.4.). In the l a t t e r case, the question a r i -ses of how oxygen i s delivered to the bacteroid terminal oxidases. The experiment by Bergersen and Turner (1975b) con-firmed the presence of two d i f f e r e n t nodule terminal o x i -dase systems; a high a f f i n i t y system, sensitive to i n h i b i -t i o n by N-phenylimidazole and a low a f f i n i t y , i n h i b i t o r -i n s e n s i t i v e system. When oxygen was supplied at a s t a b i -l i z e d low concentration, the high a f f i n i t y pathway produced up to f i v e times greater bacteroid ATP concentrations than the low a f f i n i t y oxidase pathway. The authors proposed that cytochrome P450 was involved i n the high a f f i n i t y system. However, Inozemtseva et a l . (1979) proposed that cytochrome P450 does not d i r e c t l y receive oxygen from le g -hemoglobin. Findings reported by Melik-Sarkisan et a l . (1982) showed that i n order to f i x nitrogen, bacteroids require the presence of the peribacteroid membrane. In contrast, removal of the peribacteroid membrane did not a f f e c t the rate of oxygen consumption by bacteroids and thus must not be involved i n bacteroid r e s p i r a t i o n . They concluded that the reduction of oxygen i s accomplished by electron trans-porters present i n the peribacteroid space and not by bacteroid oxidases. 27 It i s d i f f i c u l t to comprehend how oxygen i s d e l i -vered i n a regulated manner to the bacteroids. Wittenberg (1980) suggested that i f the Brownian movement of the bacteroids i s f a s t , then th e i r surfaces might be in transient contact with the inner face of the membrane envelope for a long enough period of time to allow oxygen transfer. Recently, VandenBosch et a l . (1989) reported that bacteroids are indeed i n close contact with the peribacte-roid membrane. Many researchers have attempted to elucidate the st r u c t u r a l features which confer upon leghemoglobin i t s , unusual combination of rapid combination rate with oxygen and a moderately slow d i s s o c i a t i o n rate and thus i t s very high oxygen a f f i n i t y (Appleby 1962, Imamura et a l . 1972). One c h a r a c t e r i s t i c of leghemoglobin i s i t s a b i l i t y to bind bulky ligands such as n i c o t i n i c acid, reversibly (Appleby et a l . 1973). Peive et a l . (1972) suggested that such binding i s due to a greater degree of a c c e s s i b i l i t y of the heme pocket. X-ray analysis by Vainshtein (1978) confirmed that the size of the heme pocket i n leghemoglobin i s larger on the d i s t a l side of h i s t i d i n e than i n other globins. S i m i l a r l y , O l l i s et a l . (1983) observed an exceptionally large cavity on the d i s t a l side of the heme i n soybean leghemoglobin. When n i c o t i n i c acid i s bound, h i s t i d i n e i s displaced towards the edge of the porphyrin ring concomi-28 tant with a displacement of the iron atom from the plane of the heme group (Vainshtein 1978) . This displacement may-account for the high oxygen a f f i n i t y of leghemoglobin (Appleby et a l . 1980). O l l i s et a l . (1983) found that the heme group i n soy-bean Lba i s closer to the G and H helices and further from the B and C he l i c e s than i n myoglobin. This s h i f t also causes an increase i n the size of the heme pocket on the d i s t a l side. The authors also proposed that there may be two routes by which ligands have access to the heme. The f i r s t pathway l i e s between the G helix and the junction of the B and C helic e s (Gin 101=G3 and Lys 36=B16); the second l i e s between the d i s t a l h i s t i d i n e 61 (E7) and leucine 65 ( E l l ) . However, Armstrong et a l . (1980) argued that heme d i s t o r t i o n i s not the primary factor leading to rapid re-action with oxygen. They suggested a strong ligand f i e l d could lower the energy required for spin p a i r i n g i n the t r a n s i t i o n from the high spin deoxy to the low spin oxy-leghemoglobin and/or s t a b i l i z e the complex by decreasing the d i s s o c i a t i o n rate constant and thus f a c i l i t a t e the re-action of leghemoglobin with oxygen. Appleby et a l . ,(1980) also considered the perturbation of the heme e l e c t r o n i c structure i n conformational changes of the protein as a l i k e l y cause of the high oxygen a f f i n i t y . 29 Laser f l a s h photolysis analysis by Stetzkowski et a l . (1985) showed that the faster association rate i n leghemo-globin compared to myoglobin i s due to faster bond forma-t i o n . These authors observed that the migration of oxygen from the solvent to the heme pocket was much faster i n leghemoglobin. Rapid transfer was f a c i l i t a t e d by the pre-sence of solvent molecules i n the heme pocket which gained access v i a an open channel to the pocket. However, the question s t i l l remains as to the expla-nation for the large differences observed i n the of f - r a t e constants for leghemoglobin i n comparison to hemoglobin or myoglobin. Appleby et a l . (1983a) suggested that the high a f f i n i t y of leghemoglobin could be due to protonation of the imidazole of the d i s t a l h i s t i d i n e (his 61) which would lead to formation of a hydrogen bond with bound oxygen and thus a f f e c t the e l e c t r o n i c configuration of the heme. This conformational change would cause a decrease i n the rate of oxygen d i s s o c i a t i o n but would have no e f f e c t on the oxygen combination rate. The importance of oxygen, and thus leghemoglobin, for optimum nitrogen f i x a t i o n a c t i v i t y has been emphasized by the findings of Suganuma et a l . (1987). The res u l t s of their study implied that even i n the presence of oxygenated leghemoglobin, oxygen-limiting conditions p r e v a i l i n the nodule. S i m i l a r l y , Tajima et a l . (1986) and Denison et a l . 30 (1988) recognized that oxygen supply may be a more impor-tant factor than the demand of energy for nitrogen f i x a t i o n . 2.6. QUANTITATIVE RELATIONSHIP BETWEEN LEGHEMOGLOBIN AND NITROGEN FIXATION The discussion i n the previous chapter (2.5.) des-cribed the fundamental role of leghemoglobin i n nitrogen f i x a t i o n . However, does t h i s role imply a quantitative r e l a t i o n s h i p between the amount of leghemoglobin i n the nodule and the amount of nitrogen fixed by bacteroids? Virtanen et a l . (1947a,b) and other researchers (Graham and Parker 1961, LaRue and Child 1979) reported such a c o r r e l a t i o n between leghemoglobin content and acetylene reduction rate. S i m i l a r l y , Becana et a l . (1986) observed a close r e l a t i o n s h i p between the hemeprotein content and nitrogen f i x a t i o n a c t i v i t y i n a l f a l f a nodules during the vegetative growth period which followed an exponential curve. These authors suggested that the-functional state of leghemoglobin could have an e f f e c t on the decline of nitrogenase a c t i v i t y i n ageing nodules. P a t i l and Rasal (1985) reported a good c o r r e l a t i o n between leghemoglobin content and nitrogen f i x e d at a l l stages of crop development i n chickpea. In contrast, Koundal et a l . (1987) could not confirm t h i s r e l a t i o n s h i p 31 for the chickpea c u l t i v a r i n their study. They suggested that the nitrogenase a c t i v i t y was limi t e d by the sugar content, and hence the energy supply, of the nodule. In v i t r o experiments by Wittenberg et a l . (1974) showed an increase i n both oxygen uptake and acetylene re-duction by bacteroid suspensions with increasing concen-t r a t i o n of leghemoglobin, to a maximum value between 1 and 2 mM. In contrast, Bergersen and Turner (1975a) found the optimum leghemoglobin concentration to be about 50 )iM. The authors attributed the differences i n Lb concentration to li m i t a t i o n s of in v i t r o experiments (as discussed i n chap-ter 2.5.). Increasing Lb concentration above 50 .UM had no ef f e c t on nitrogenase a c t i v i t y at a given oxygen concen-t r a t i o n (Bergersen and Turner 1975a). Troitskaya et a l . (1979) measured the content of the hemoglobin i n d i f f e r e n t species during the budding and flowering period. The amount varied from 0.5 mg/g of fresh nodule mass for chickpea to a maximum of 4.0 mg/g for soy-bean. Kidney beans contained 2.3 to 3.6 mg of leghemoglo-bin per gram of fresh nodule mass. The amount of leghemo-globin also varied depending on the developmental phase (e.g. vegetative, flowering, f r u i t formation) of each species. The same authors reported that maximum nitrogen f i x i n g capacity of the nodules was not correlated with the maximum leghemoglobin content of the d i f f e r e n t crops. 32 Species which accumulated the largest amount of hemeprotein did not achieve the highest l e v e l of nitrogen f i x a t i o n and vice versa. Furthermore, the time of maximum nitrogen f i x -ation rate and maximum leghemoglobin content showed a lack of coincidence with the stage of plant development and was d i f f e r e n t for d i f f e r e n t species. For example, i n fababeans maximum leghemoglobin content preceded, whereas i n soybeans i t lagged behind, the period of maximum nitrogen f i x a t i o n a c t i v i t y . Kidney beans were the poorest nitrogen f i x e r with only 1 to 1.5% of the a c t i v i t y of soybean nodules although the former species contained the second highest amount of leghemoglobin (Troitskaya et a l . 1979). Nash and Schulman (1976) showed that leghemoglobin content i n soybean was correlated wiflh nodule size such that larger nodules contained more hemeprotein. Bergersen and Goodchild (1973a) suggested that large nodules increase their leghemoglobin content to compensate for the increased oxygen d i f f u s i o n pathway. Nash and Schulman (1976), how-ever, pointed out that such compensation i s not f u l l y e f f e c t i v e and that other factors necessary for maintenance of nitrogenase a c t i v i t y become more l i m i t i n g . In summary, v a r i a b i l i t y for leghemoglobin content exists between and within d i f f e r e n t species. However, the co r r e l a t i o n between Lb content and nitrogen f i x a t i o n i s not simple or l i n e a r . One might speculate that at le a s t part of 3 3 the e f f e c t which confounds t h i s r e l a t i o n s h i p could be at-tributed to differences i n oxygen a f f i n i t y of Lb components from d i f f e r e n t species, e.g. a species containing highly e f f e c t i v e Lb components might not need as much Lb as an-other species with less e f f e c t i v e components. 2.7. LEGHEMOGLOBIN HETEROGENEITY Leghemoglobin v a r i a b i l i t y i s widespread among nitrogen f i x i n g species with very few exceptions (Appleby 1974, 1984). For example, a l f a l f a (Medicago sativa L.) and Lathyrus contain four and Lupinus polyphyllus produces three leghemoglobin components (Holl et a l . 1983). Tr i f o l i u m subterraneum was separated by ion exchange chro-matography into four d i s t i n c t components (Holl et a l . 1983, Thulborn et a l . 1979). The most frequently studied l o c i i n soybean show sim i l a r heterogeneity for the leghemoglobin gene family. The four soybean leghemoglobin genes encode four d i s t i n c t gene products which can be separated by i s o e l e c t r i c focu-sing into four major components (Lba, Lbc^ , Lbc^ , Lbcj ) and four minor components (Lbb, Lbd^ , Lbd^ , Lbd^ ) (Fuchsman and Appleby 1979, Lee and Verma 1984). The minor components ari s e from the major components by cleavage of the N-ter-minal v a l y l residue and subsequent acetylation of the ex-34 posed alanyl residue and are thus po s t - t r a n s l a t i o n a l modi-f i c a t i o n s of the major isomers (Whittaker et a l . 1979). Experimental evidence suggests that the Lbc^ gene i s activated before the Lbc^ gene and that Lbc2 i s activated at about the same time as the other two Lbc genes (Marcker et a l . 1984). Thus, during the very early stages of nodule development only Lbc type mRNAs are synthesized. After a few more days, the Lba gene i s activated. Consequently, soybean leghemoglobin genes are activated i n reverse order of their chromosomal arrangement (Marcker et a l . 1984). The amount of Lbc i n young nodules i s more than seven times as great as the amount of Lba. This r a t i o changes when the nodules mature, to le s s than two for three-week old nodules due to the increase i n biosynthesis of Lba (Fuchsman and Appleby 1979, Fuchsman et a l . 1976, Verma et a l . 1979, 1983). The rate of synthesis of each Lb compo-nent i s regulated separately from the others, i n d i c a t i n g that leghemoglobin heterogeneity i s functional (Marcker et a l . 1984, Fuchsman and Appleby 1979). Lba was found to have a higher oxygen binding capacity than Lbc and i s , therefore, more e f f e c t i v e i n enhancing nitrogen f i x a t i o n and oxygen consumption. The r e l a t i v e increase i n Lba content might be required for the regula-t i o n of oxygen concentration when the nodule structure becomes more complex with age (Appleby 1962, Fuchsman and 35 Appleby 1979, Uheda and Syono 1982 b). Fuchsman and Palmer (198 5) studied leghemoglobins from a g e n e t i c a l l y diverse s e l e c t i o n of c u l t i v a t e d and wild soybeans by i s o e l e c t r i c focusing. They concluded that the conservation of both leghemoglobin heterogeneity and also a l l four major leghemoglobin structures i n an otherwise diverse population provides strong circumstantial evidence that leghemoglobin v a r i a b i l i t y i s functional and that t h i s heterogeneity i s an evolutionary adaptation for more ef-f e c t i v e nitrogen f i x a t i o n . Uheda and SyOno (1982a) detected two major (Lbl and LblV) and three minor ( L b l l , L b l l l , and LbV) leghemoglobin components i n pea using disc gel electrophoresis. In young nodules, Lbl i s synthesised at a higher rate than LblV whereas i n older nodules, LblV i s predominantly produced. Thus, the r a t i o of the two major components (I/IV) de-creased with increasing age. Similar to soybeans, the component which i s synthesized at a higher rate i n older nodules (LblV) also has a higher oxygen binding a f f i n i t y . Hence, since low oxygen tension seems to p r e v a i l i n older nodules, the higher oxygen binding capacity of LblV would r e s u l t i n a better capacity for oxygen transport. The authors suggested that changes i n the r e l a t i v e amounts of the leghemoglobin components during nodule development contribute to more e f f e c t i v e nitrogen f i x a t i o n which i s 36 mediated by changes i n the capacities for oxygen transport within the nodule (Uheda and Syono 1982b). Ho l l et a l . (1983) described a l i n e of Tr ifolium  subterranean which contained only three leghemoglobin com-ponents; L b l , a major component of a l l other T. subterranean c u l t i v a r s , was missing. The amount of L b l l was approximately twice that usually found i n nodules, suggesting a compensating response for the lack of Lbl or a r e f l e c t i o n of mutation i n the Lbl component which caused i t to coelute with L b l l i n chromatographic and electrophoretic separations. Although differences i n nitrogen f i x a t i o n were observed, the widely d i f f e r e n t phenological patterns of the three- and four-banded l i n e s precluded any d e f i n i t e comparisons. Lupins contain two major (Lbl and L b l l ) and one minor ( L b l l l ) component, the l a t t e r being a posttranslational modification of Lbl (Szybiak-Strozycka et a l . 1987). In early nodule development, Lbl was predominant whereas in later stages L b l l prevailed (Sikorski et a l . 1986). The oxygen a f f i n i t y for each component i s not known; however, the authors proposed that leghemoglobin v a r i a b i l i t y has a p h y s i o l o g i c a l r o l e . Sesbania rostrata i s a nitrogen f i x i n g legume with both stem and root nodules. Stem nodules contain photo-synthesizing chloroplasts with p o t e n t i a l for oxygen evolu-37 t i o n adjacent to bacteroid-containing tissue (Dreyfus and Dommergues 1981). Anion exchange chromatography demons-trated that there are seven major leghemoglobin components i n both root and stem nodules although i n d i f f e r e n t pro-portions. At l e a s t six of these components are separate gene products (Bogusz et a l . 1987). Twelve days after inoculation, Sesbania stem nodules contain about equal amounts of Lb2, Lb3, and Lb5 while Lb4 predominates and Lbl i s barely detectable. This r e l a t i o n -ship changes i n mature nodules such that Lb2 and Lb5 are predominant, Lb3 and Lb4 are moderately abundant and Lbl i s a minor component (Lajudie and Huguet 1986). Bogusz et a l . (1987) found that the amount of Lb2 was highest i n both stem and root nodules about 20 to 30 days after inocula-tion. Decreasing r e l a t i v e amounts were detected for Lb2, Lb7, Lb5, Lb6, Lb4, Lbl and Lb3 i n stem nodules and for Lb2, Lb7, Lb3, Lb5, Lb4, Lbl and Lb6 i n root nodules. The predominant Sesbania Lb2 component had the highest oxygen a f f i n i t y yet recorded for any leghemoglobin as de-termined by Wittenberg et a l . (1985). The concentration of free oxygen i n the nodule was extremely low. The associa-t i o n of the high oxygen a f f i n i t y with the predominant com-ponent i s consistent with observations for soybean and pea leghemoglobins. Kortt et a l . (1987) compared the amino acid sequence 38 of Sesbania Lb2 to soybean, kidney bean, broad bean and pea leghemoglobin and found them to be very s i m i l a r indicating that very small changes must be responsible for the higher oxygen a f f i n i t y . Furthermore, Fleming et a l . (1987) also reported he-moglobin heterogeneity for Casuarina glauca, a non-legume which forms root nodules i n association with the actino-mycete Frankia. I s o e l e c t r i c focusing separated the hemo-globin into three major and multiple minor components, re-f l e c t i n g a multigene family. Molecular weights were simi-l a r to other plant globins and since these hemproteins can bind oxygen re v e r s i b l y and have a high oxygen a f f i n i t y , i t i s l i k e l y that hemoglobin i n Casuarina i s f u n c t i o n a l l y active i n nitrogen f i x a t i o n . An exception to leghemoglobin heterogeneity i s Parasponia, a member of the Ulmaceae which forms nodules with Rhizobium. However, i t contains an unusual dimeric hemoglobin, the only one known i n plants (Appleby et a l . 1983b, Landsmann et a l . 1986). K i n e t i c measurements re-vealed s u f f i c i e n t l y f a s t oxygen association and d i s s o c i a -t i o n rate constants and extremely high oxygen a f f i n i t y to allow Parasponia hemoglobin to function s i m i l a r l y to known leghemoglobins. In conclusion, the l i t e r a t u r e has shown considerable v a r i a t i o n for leghemoglobin i n almost a l l species examined. 39 Hence, i t i s surprising and noteworthy that _P_. vulgar i s , which i s so c l o s e l y related to G_. max, contains only one component. 2.8. EVOLUTION OF THE GLOBIN GENES The o r i g i n of globin genes i n nitrogen f i x i n g plants i s disputed. Three possible sources have been suggested from which leghemoglobins might have evolved: a transfer from Rhizobium, a v i r a l vector, or the plant i t s e l f . Appleby (1969a) reported that Bradyrhizobium contain a pigment with the c h a r a c t e r i s t i c s of a hemoglobin. Thus, Broughton and Dilworth (1973) proposed that a p r i m i t i v e -hemoglobin was transferred from Rhizobium to the host plant s i m i l a r to the natural Agrobacterium T i plasmid system which then evolved into an e f f i c i e n t oxygen c a r r i e r . However, leghemoglobin genes contain introns (Lee and Verma 1984, Hyldig-Nielson et a l . 1982) whereas prokaryotic genes do not (Lewin 1985) and thus, a b a c t e r i a l o r i g i n for the globin gene i s u n l i k e l y . Furthermore, Appleby (1969b) pointed out that the bacteroid hemeprotein he discovered was unrelated to leghemoglobin. The second theory by J e f f r e y s (cited i n Lewin 1981) suggests that globin genes were transferred by an insect-borne plant pathogenic virus to an ancestral legume. Nucleotide s u b s t i t u t i o n experiments by Brown et a l . (1984) 40 and X-ray crystallographic studies by Vainshtein et a l . (1975) estimate the time of divergence between plant and animal globin genes to about 900 and 1400 m i l l i o n years. Goodman et a l . (1975, 1988) placed the duplication of the globin gene after the emergence of the vertebrates about 680 m i l l i o n years ago. Since i t seems u n l i k e l y that the globin genes of a donor would have diverged p r i o r to t h i s , time, t h i s would exclude the p o s s i b i l i t y of transfer from an insect donor. Further evidence against such a horizontal transfer comes from the f a c t that animal globin genes contain only two introns unlike leghemoglobins which possess three (Go 1981). Verma et a l . (1983) observed that exon fusion must have occurred before the duplication of the globin genes, about 500 m i l l i o n years ago. Thus, the authors reasoned that a vectoral transfer occurring long before the emer-gence of legumes (about 140 m i l l i o n years ago) seems un-l i k e l y . In addition, Lee et a l . (1983) argue that i f such a h o r i z o n t a l gene transfer has taken place, then leghemoglo-bins should show a close c o r r e l a t i o n to one p a r t i c u l a r do-nor. However, analysis of 245 globin genes by Goodman et al.(1988) using the parsimony p r i n c i p l e indicate that there i s no such close c o r r e l a t i o n . Lee et a l . (1983) explain that a more rapid divergence 41 of globin genes i n contrast to animal hemoglobin could ac-count for the equally close c o r r e l a t i o n of leghemoglobin to a l l animal globin genes. However, Goodman et a l . (1975) reported that i n the beginning, mutations which improved hemoglobin function also took place at an accelerated rate i n animals and only decelerated when a l l functional oppor-t u n i t i e s had been exploited. The t h i r d theory invokes a plant o r i g i n for leghemo-globins either by d_e novo development or due to a primitive globin common to a l l hemoglobin carrying organisms. Appleby (1974) advocated a de novo evolution of l e g -hemoglobin from a duplicated plant hemoprotein l i k e cyto-chrome due to the need to improve an oxygen-limited inef-f e c t i v e symbiosis. However, Broughton and Dilworth (1973) argue that, since leghemoglobins are s i g n i f i c a n t l y related to myoglobins and primitive hemoglobins and since the he-terogeneity i n leghemoglobins follows the pattern of legume evolution, i t does not seem l i k e l y that they evolved de  novo. Consequently, i t i s possible that a l l hemoproteins arose from a common o r i g i n a l globin gene before the sepa-ra t i o n of the animal, fungi and plant kingdom. Brown et a l . (1984) estimated that the separation of the three eukaryotic groups dates back about 1.4 to 1.5 b i l l i o n years. Ramshaw et a l . (1972) came to similar con-clusions from th e i r studies with cytochrome c. However, 42 Verma et a l . (1983) determined that legumes appeared only about 140 m i l l i o n years ago. Hence, Appleby (1974) argues that "an apparently useless gene would not have survived for over 1 b i l l i o n years before i t s f i r s t expression". Estimation of time scales are often only educated guesses (Goodman et a l . 1975). Axelrod (1958) and Heywood (1971) reported finding f o s s i l i z e d legumes i n tertiary-formations (around 50 m i l l i o n years ago). Since legumes consist of s o f t material, they might not h.ave been well preserved and thus their age could have been s u b s t a n t i a l l y underestimated (Dilworth and Appleby 1979) sim i l a r to other angiosperms. Ramshaw et a l . (1972) reported that * angiosperms originated at l e a s t several geological periods before the Cretaceous, which i s the e a r l i e s t period i n geological record i n which their f o s s i l s have been found. Another strong argument for a plant o r i g i n comes from the occurrence of globin genes outside the legume family. Southern b l o t experiments by Hattori and Johnson (1985) and Roberts et a l . (1985) showed that non-nodulating legumes and even non-legumes have sequences which cross-hybridize to soybean leghemoglobin probes. Globin genes occur i n such d i s t a n t l y related f a m i l i e s as Rosiflorae (Casuarinaceae, Betulaceae, Myricaceae), Malviflorae (Ulmaceae, Elaegnaceae), Rutiflorae (Coriariaceae), and Fabiflorae (Leguminosae) according to Dahlgren (1980) and 43 Landsmann et a l . (1986). Hattori and Johnson (1985) also suggested that globin genes might be more widespread i n the plant kingdom than expected and were at some time, or s t i l l are present, i n a l l plants. Further support for t h i s theory comes from western blot analyses by Fleming et a l . (1987) who detected strong r e c i p r o c a l cross-reactions between hemoglobins from Casuarina and Parasponia, two nitrogen f i x i n g non-legumes which belong to widely separated plant orders. Parasponia i s a plant which fi x e s nitrogen i n association with Rhizobium. Its leghemoglobin cDNA hybridizes to genes i n the genome of Casuarina, a genus which forms a nitrogen f i x i n g association with Frank i a . Because of the high degree of hemoglobin homology between these two species and their d i f f e r e n t symbiotic associations, Landsmann et a l . -(1986) suggested that a separate evolution of plant leg-hemoglobin and nitrogen f i x i n g symbioses might have taken place. A l l these findings support the theory that hemepro-teins have an ancient o r i g i n and that globin sequences be-came silenced i n plants which have not aquired a nitrogen f i x i n g c a p a b i l i t y or do not form a c t i n o r h i z a l associations (Appleby et a l . 1988, Lee and Verma 1984). Many researchers have used amino acid sequences to construct phylogenetic trees (e.g. Barker and Dayhoff 1972, 44 Goodman et a l . 1988). However, there are very few amino acids i n the hemeprotein which are preserved i n a l l globins ( E l l f o l k 1972, Guy 1985, Lee and Verma 1984, O l l i s et a l . 198 3). H u r r e l l et a l . (1979) investigated 55 hemoglobins. Only two amino acid residues out of 145 were found to be invariant i n a l l species. In contrast, s i z e , helix content and d i s p o s i t i o n and thus, the o v e r a l l geometry of the he-moglobin molecule, were remarkably si m i l a r for molecules from such diverse or i g i n s as mammals and plants. Guy (1985) compared c r y s t a l structure and sequences of vertebrate hemoglobins, myoglobins, insect larva globins and plant leghemoglobins to determine th e i r evolutionary r e l a t i o n s h i p . Analysis of the globin sequences showed that very few residues i n homologous positions were i d e n t i c a l and thus were not very useful for evaluating s t r u c t u r a l relationships. Usually, globin chains consist of eight h e l i c a l rods with the exception of leghemoglobins where the short D-helix i s missing (Hurrell et a l . 1979). During evolu-t i o n , changes are more l i k e l y to occur at the interface of the molecule with i t s environment than i n the i n t e r i o r which incorporates the heme group since changes i n the heme environment are more l i k e l y to be disruptive and thus cause loss of functional a c t i v i t y . When changes occurred, they 45 were usually conformationally conservative, i . e . amino acids were substituted by residues with s i m i l a r properties (Hurrell et a l . 1979). In addition, Lehtovaara et a l . (1980) demonstrated that r e l a t i v e l y few mutations have i n -volved, the amino terminal (A-helix) and carboxy terminal (H-helix) portions of the globin chain, probably because these ends are important for the preservation of the cor-rect conformation of the molecule. However, Appleby (1974) argued that homology i n the heme cavity and i n the terminal regions of d i f f e r e n t hemo-globins does not imply that these globins have the same evolutionary o r i g i n . The author points out that according to Margoliash et a l . (1968) two requirements have to be f u l l f i l l e d to show ancestral relationship; i t has to be proven that sequences are g e n e t i c a l l y related to a greater degree than can be expected by chance, and i t must be shown that s i m i l a r i t i e s are greater i n extent than necessitated by the functional requirements of the hemeproteins. Thus, Dilworth and Appleby (1979) concluded that the greater than expected homologies found between myoglobin, hemoglobin and leghemoglobin are due to convergence of unrelated proteins and not due to a descent from a common ancestor. It seems very d i f f i c u l t to d i s t i n g u i s h between con-vergence of unrelated proteins during evolution (analogy) and descent from a common ancestor (homology) (Ramshaw et 46 a l . 1972). However, i n view of common regulatory sequences at the 5' end of animal and plant globin genes, the idea of an analogy to globin proteins cannot be upheld. Thus, Brisson and Verma (1982) reported that i n addition to the t y p i c a l eukaryotic 'CAAT1 and 1TATAA1 boxes, the 5' region of leghemoglobin genes contain an imperfect, tandemly re-peated sequence which seems necessary for a l l globin func-tion (Verma et a l . 1983). In addition, Brown et a l . (1984) showed that t h i s region also contains a sequence comple-mentary to the 3' end of 18S rRNA. Yamaguchi et a l . (1982) proposed that such a region might enhance the rate of i n i -t i a t i o n of complex formation i n protein synthesis. Hence, Verma et a l . (1983) concluded on the basis of the conser-vation of globin regulatory sequences that a l l globin genes have a common o r i g i n . The coding regions of leghemoglobins are interrupted by three introns. S i m i l a r l y , Parasponia (Landsmann et a l . 1986) and Trema (Bogusz et a l . 1988), the l a t t e r i s a non-nodulating species, contain hemoglobins with three introns in i d e n t i c a l positions to those i n leghemoglobins. In con-t r a s t , a l l other hemoglobins contain only two intervening sequences. The two common introns of g-globin and leghe-moglobin genes are i n simi l a r positions. The t h i r d intron of leghemoglobin divides the cen t r a l exon which encodes the proximal and d i s t a l heme contacts (Go 1981). 47 Blake (1978) argued that i f exons correspond to a folded protein fragment, then mutational changes would be much more l i k e l y to y i e l d stable globular conformations. Thus, Go (1981) proposed that the central intron was ex-cised from an ancient ancestral hemoglobin gene. Nishioka et a l . (1980) reported the presence of an a, -globin-1 ike pseudogene i n mice which lacks the two introns that are normally present i n hemoglobins and thus a mechanism for s p l i c i n g out introns must e x i s t i n eukaryotes. Based on these findings, Appleby et a l . (1988) suggested that a l l globin genes have evolved from a primitive globin-carrying proto-organism. Furthermore, Appleby et a l . (1988) speculated that the expression of hemoglobin i n Trema and i n un-nodulated Parasponia and Casuarina roots ensured i t s persistence du-rin g evolution. The authors suggested that the small amount of hemoglobin i n the un-nodulated roots may be at-tributed to "the sensing of anaerobiosis and the i n i t i a t i o n of anaerobic response mechanisms, rather than f a c i l i t a t i o n of oxygen d i f f u s i o n " . In summary, the close evolutionary relationships of leghemoglobins with other hemoglobins as well as the simi-l a r i t i e s i n intron p o s i t i o n and regulatory sequences and the presence of globin sequences i n non-nitrogen f i x i n g species provide strong evidence that a l l hemoglobins have 48 indeed evolved from a common ancestral gene. 49 I I I . INDUCED MUTAGENESIS FOR LB VARIABILITY 3.1. INTRODUCTION Breeding implies s e l e c t i o n of variants which either arose spontaneously or were induced by mutagenic agents. In general, a plant breeder would f i r s t screen c u l t i v a t e d or wild accessions to determine i f a desired character i s present within the species or genus. If v a r i a t i o n i n the character e x i s t s , then hybridization and backcrossing w i l l e s t a b l i s h the t r a i t i n a crop. I f , however, a variant cannot be found or i f a l l hybridization attempts f a i l , then inducing v a r i a b i l i t y by means of mutagenic agents i s often the only solution (Simmonds 1981) . A mutation i s defined as a "heritable change i n the genotype of an organism" (Lewin 1985). Accordingly, a mutagen may be any agent which increases the rate of muta-ti o n to a higher than spontaneous l e v e l (Lewin 1985). Spontaneous mutations and subsequent recombination are the basis for ex i s t i n g v a r i a b i l i t y i n plant evolution. Most mutations are recessive i n nature and natural muta-tions generally occur at low frequencies (10*6 per genera-t i o n ) . Such changes are l i k e l y a response to u l t r a - v i o l e t and background ra d i a t i o n , errors i n DNA r e p l i c a t i o n , ageing of the DNA and transposable genetic elements (Simmonds 50 1981). For breeding purposes, the o v e r a l l rate of spontaneous mutation i s often too low to be of p r a c t i c a l use; further-more, mutations of the desired type may be p a r t i c u l a r l y rare. Increasing mutation rates with the help of mutagenic agents l i k e radiation or chemical mutagens increases the p r o b a b i l i t y of creating and enhances the potential for finding the desired mutant. The foundations of induced mutagenesis were l a i d by De Vries (1909, 1910) at the beginning of t h i s century while working with Oenothera lamarckiana. Schliemann (1912) was the f i r s t to investigate the e f f e c t s of chemical mutagens on Aspergillus niger. The experiments of Muller (1927) and Stadler (1928) showed that X-rays d i r e c t l y increased the frequency of mutations. Since then, the induction of mu-tations has often been suggested as a tool to enlarge v a r i a t i o n i n a crop (Gottschalk and Wolff 1983). However, since most mutagenic agents are non-selec-t i v e , any treatment w i l l produce a random array of muta-tions from which one has to sort out those of in t e r e s t . Adequate s e l e c t i o n procedures among the mutagenized popu-l a t i o n are an e s s e n t i a l component of a successful mutation breeding program. The choice of a method usually depends on the f a c i l i t i e s available. In P_. vulgaris, r a d i a t i o n with gamma-rays and chemical treatment with a l k y l a t i n g 5 1 agents such as ethyl methane sulfonate (EMS) have been more commonly used for mutation induction (Crocomo et a l . 1973, Delgado de l a Flor 1978, Hussein and Disouki 1976, Motto et a l . 1975). 52 3.2. LITERATURE REVIEW A l l radiation releases energy as i t passes through matter. The energy i s absorbed i n the atoms of the target and i s released i n form of f a s t charged p a r t i c l e s . These p a r t i c l e s can inter a c t with the electrons of the atom by expelling electrons out of o r b i t . The atom thus becomes ionized which can a f f e c t the whole molecule i n such a way to i n i t i a t e a change i n configuration. Such changes may resu l t i n the loss of the functional a c t i v i t y of the molecule (Timofe"ef f-Ressovsky 1940) . Timofe"eff-Ressovsky et a l . (1935) developed the theory that only one " h i t " would be necessary for a gene mutation to be induced. Consequently, the number of mutations i n -creases l i n e a r l y at low to medium doses. Early theories were developed i n the absence of knowledge about the DNA. Since then, th e i r correctness has been confirmed (IAEA 1977). Hence, at low doses, the chances of chromosome breakage are small because breakage requires a "two-hit" event; during the time required for a second h i t to occur, the f i r s t break may have been repaired. Hence, the f r e -quency of chromosome breakage does not show a simple l i n e a r increase; the response curve r e f l e c t s an increase i n breakage approximately r e f l e c t i n g the square of the dose. It seems that the l e t h a l e f f e c t s of radiation are often due to double strand breaks. Single h i t events lead to dele-53 t i o n s , gene mutation, and s i s t e r chromatid unions (IAEA 1977, M u l l e r 1954a). In g e n e r a l , gamma-rays have a s h o r t e r wavelength and t h e r e f o r e possess more energy per photon than X-rays. In a d d i t i o n , X-rays are always l e s s homogeneous and hence, gamma-rays (obtained from Cobalt-60 or Caesium-137) are more f r e q u e n t l y used f o r the i n d u c t i o n of mutations (IAEA 1977). Beside the d i r e c t e f f e c t s on g e n e t i c m a t e r i a l , the experiments by Stone e t a l . (1947) and Wyss e t a l . (1947, 1948, 1950) showed t h a t a t l e a s t p a r t of the mutations produced are the r e s u l t of an i n d i r e c t chemical e f f e c t of r a d i a t i o n . The authors found t h a t i r r a d i a t i n g the media with U V - l i g h t , on which Staphylococcus aureus was subse-q u e n t l y grown, caused mutations. T h i s r a d i o c h e m i c a l e f f e c t i n v o l v e s the formation of hydrogen p e r o x i d e and f r e e r e a c -t i v e chemical i n t e r m e d i a t e s from water i n the presence of molecular oxygen. Beside the p o l a r i z a t i o n of water molecules, water i s a l s o important as a d i f f u s i o n medium f o r f r e e r a d i c a l s and oxygen (Weiss 1944, M u l l e r 1954b). Consequently, the p r e -sence or absence of water and/or oxygen has a profound i n -f l u e n c e on the e f f e c t i v e n e s s of r a d i a t i o n . Konzak e t a l . (1970) poi n t e d out t h a t r a d i o c h e m i c a l damage can be p r e -vented by u s i n g presoaked seeds f o r gamma r a d i a t i o n t r e a t -54 ment. However, soaking seeds i n water also induces germi-nation and growing tissue has been shown to be more a f f e c -ted by radiati o n than inactive tissue (Savin et a l . 1968). There are numerous factors beside oxygen and water which modify the e f f e c t s of radiation. Temperature i s an important secondary factor which influences the extent of r a d i c a l movement and thus interaction. For example, pre-and p o s t - i r r a d i a t i o n heat treatments have been shown to reduce radiation-induced damage i n seeds while increasing the mutation frequency (Ehrenberg 1955, IAEA 1977). In addition, i t seems that the e f f e c t of radiati o n also depends on the t r a i t studied. For example, Crocomo-et a l . (1973) demonstrated that a dosage of 6 - 7 Krad was optimal for mutagenesis for increasing the t o t a l amount of bean protein, whereas at l e a s t 7 Krad was necessary for s p e c i f i c a l l y increasing the methionine l e v e l . Thus, com-parisons between d i f f e r e n t experiments of even the same species are extremely d i f f i c u l t due to the number of variables a f f e c t i n g an experiment. In 1943, Oehlkers demonstrated the mutagenic ef f e c t s of chemicals by treating Oenothera stems with a urethane solution and by observing subsequent chromosome transloca-tions during meiosis. Since then, many mutagenic chemicals have been des-cribed (IAEA 1977). The p r i n c i p a l categories of chemical 55 mutagens are a l k y l a t i n g agents, base analogues, i n t e r c a l a -t i n g agents and deaminating agents. Al k y l a t i n g agents are by far the most important group of mutagenic chemicals with ethyl methane sulfonate (EMS) being the most frequently used, since methylating agents are more toxic (IAEA 1977). If the electron density i s s u f f i c i e n t l y high, a l k y l a -ting agents such as EMS can transfer their reactive a l k y l group(s) either to the phosphate backbone, r e s u l t i n g i n an unstable phosphate t r i e s t e r which might lead to chromosome breakage, or to the nitrogeneous bases which i n turn might cause point mutations due to pair i n g errors (IAEA 1977, Suzuki et a l . 1986). The e f f e c t i v e concentration of the treatment solution and the duration of the treatment depends on the properties of the agent, on the solvent medium and on the b i o l o g i c a l system (IAEA 1977). The uptake of a l k y l a t i n g agents de-pends on simple d i f f u s i o n laws and requires that the con-centration of the chemical i n solution i s greater than that in the plant c e l l . In addition, the e f f e c t s of treatment are also i n f l u -enced by temperature. EMS hydrolyses and has a h a l f - l i f e of 93 hours at 20° C but only 26 hours at 30° C (IAEA 1977). Hydrolysis of EMS leads to methane sulphuric acid and ethyl alcohol which are damaging to plants without apparent mutagenic a b i l i t y (Froese-Gertzen et a l . 1964). 56 Consequently, extensive post-treatment washing i s recom-mended to remove the p o t e n t i a l e f f e c t s of the EMS hydro-l y s i s products. Konzak et a l . (1970) concluded that wa-shing as a means of stopping mutagen treatment does not achieve' complete removal of the agent because the mutagenic chemicals are too t i g h t l y bound to l i p i d s within the t i s s u e . Pre-treatment of seeds by soaking them i n water seems to f a c i l i t a t e the d i f f u s i o n of mutagenic substances through membranes and to achieve better infusion of the chemicals into the tissue (Konzak et a l . 1970). However, Mikaelsen et a l . (1967) reported that the uptake of EMS was indepen-dent of presoaking time. Other factors influencing the e f f e c t s of a l k y l a t i n g agents include post-mutagenic treatments such as reported by Mikaelsen et a l . (1967) who found that chlorophyll mutations and chromosome aberrations increased when seeds were dried and stored after the mutagenic treatment. These ef f e c t s depended on storage time, temperature and also on the i n i t i a l l e v e l of damage. Gaul (196 2) proposed that EMS i s the most powerful chemical for the induction of chlorophyll mutations i n plants. In 1966, Gaul and coworkers compared the use of X-rays and EMS for mutagenic treatment of barley seeds. EMS induced a larger genetic variance and was less l e t h a l 57 than X-rays under s i m i l a r experimental conditions. However, the authors also found that the e f f e c t of EMS showed a large dependence on pre v a i l i n g environmental con-d i t i o n s and thus often precluded s a t i s f a c t o r y experimental reproducability. An experiment on the e f f e c t s of EMS (0.1% and 0.2% w/v) and gamma rays (0-100 Gray) on f i v e P_. vulgar i s v a r i e t i e s was performed by Hussein and Disouki (1976). They reported v a r i e t a l differences i n mutagen-sensivity, with the vari e t y 'Contender' being somewhat more.resistant to either treatment. About 30% of the 'Contender' seeds treated with either 100 Gray of gamma rays or 0.1% (w/v), of EMS survived i n the Ml generation. However, EMS induced a higher degree of s t e r i l i t y than the gamma-ray treatment, which the authors attributed to the phy s i o l o g i c a l damage produced by EMS and i t s hydrolysis products. They suggested that P_. vulgar i s , l i k e other legumes, should be treated with 0.1% (w/v) or less EMS i n order to obtain a reasonable number of Ml survivors. However, the authors recommended the use of EMS and gamma rays as "the most e f f i c i e n t p h y s i c a l , respectively chemical agents available nowadays" for the induction of mutations (Hussein and Disouki 1976). Chemical as well as radiatio n treatments are usually applied to seeds, which i s e s s e n t i a l l y a treatment of the 58 embryo meristems. Since the meristematic regions of an embryo usually consist of several i n i t i a l meristematic c e l l s , any mutation induced w i l l only a f f e c t the part of the plant which arises from t h i s mutated c e l l . Hence, mutagenic treatments often give r i s e to chimeras (Gottschalk and Wolff 1983, IAEA 1977). The experiment by Motto et a l . (1975) showed chimeric Ml P_. vulgaris plants with often only a small mutated sector. Based on the segregation r a t i o s obtained, the average number of i n i t i a l c e l l s present i n the a p i c a l meristem was estimated to be eight. However, i n heavily damaged plants, the average number of i n i t i a l c e l l s de- * creased to three. In one p a r t i c u l a r mutant, a single meristematic i n i t i a l c e l l generated the whole plant. Delgado de l a Flor (1978) reported that EMS tended to i n -duce a larger size of chimera than gamma-rays. 59 3.3. MATERIALS AND METHODS Approximately 4000 seeds of P_. vulgar i s L. c u l t i v a r •Contender' were used. Kidney beans are d i p l o i d and are normally s e l f - p o l l i n a t e d . 'Contender* i s an early bush bean variety with buff to brown seeds. The average thousand seed weight i s about 387g. Seed protein content ranges from 20-22 % on a dry matter basis (Adams et a l . 1985). 'Contender' was released from the USDA Southeast Vegetable Breeding Laboratory i n 1949. The c u l t i v a r was derived from crosses involving Commodore x Streamliner x US#5 Refugee. 'Contender' i s re s i s t a n t to common bean mosaic and powdery mildew (J.L. Morris, personal communication). The experiment was car r i e d out i n the greenhouses of the Department of Plant Science, University of B r i t i s h Columbia i n 1986 and 1987. Well developed seeds of about the same seed size were selected for the experiment. For either mutation treatment, the Ml s u r v i v a l rate was tar-geted at 20%-30%. For the gamma-ray treatment, seeds were soaked over-night i n d i s t i l l e d water and treated i n a Gammacell 220 i r r a d i a t i o n f a c i l i t y using Cobalt-60 i n the Faculty of A g r i c u l t u r a l Sciences, University of B r i t i s h Columbia. The doses used were 100, 150 and 200 Gray. For the EMS treatment, seeds were soaked overnight in EMS solutions at 24° C t2. The concentrations of the 60 treatment solutions were 0.08% (w/v), 0.1% (w/v), 0.2% (w/v) and 0.4% (w/v). After an incubation period l a s t i n g 18 hours, seeds were washed thoroughly under running water for one hour. A l l seeds from both treatments were immediately seeded i n 15 cm pots (one plant per pot) containing a soil:peat' moss mixture (4:1 v/v) and Osmocote®, a 14-14-14 slow re-lease f e r t i l i z e r . A t o t a l of 708 Ml and 571 Ml plants were obtained from the gamma-ray treatment and the EMS treat-ment, respectively. At l e a s t one seed from every Ml plant (and thus the M2 generation) was seeded i n a styrofoam cup containing Turface® (Montmorillonite c l a y ) . The M2 plants were inocu-lated with a commercial Rhizobium leguminosarum biovar phaseoli inoculant and f e r t i l i z e d twice weekly with a n i -trogen-free nutrient solution (Holl 1975). After three to four weeks, nodules from each M2 plant were excised, crushed i n 0.1 M sodium phosphate buffer pH 7.4 and c e n t r i -fuged at 12,400 x g i n an Eppendorf microcentrifuge for 2 minutes. The supernatant containing leghemoglobin was used for c e l l u l o s e acetate electrophoresis (CAE). The CAE was run for 15 minutes at 420 volt s (Holl et a l . 1983). After separation, the Sepraphore® s t r i p s were stained for the presence of protein with Ponceau S and counterstained for the presence of heme using the method of Owen et a l . (1958) 61 as described i n H o l l et a l . (1983) . 62 3.4. RESULTS AND DISCUSSION Plants of the Ml generation showed a mutagenic spec-trum similar to other reported experiments, which included decreased s u r v i v a l rates, chlorophyll mutations, changes in leaf size and shape, a l t e r a t i o n s i n growth habit from de-terminate to indeterminate and varying degrees of s t e r i l i t y (Hussein and Disouki 1976, Motto et a l . 1975, Rubaihayo 1975). On average, plants of the Ml generation obtained from the gamma ray treatment produced 5.28 seeds per plant, the va r i a t i o n ranging from 0-23 seeds, whereas EMS-induced Ml mutants produced 4.98 seeds per plant on average, the va-r i a t i o n ranging from 0-10 seeds. About 780 M2 seeds derived from the gamma-ray t r e a t -ment and 620 M2 seeds derived from the EMS treatment were sown for leghemoglobin t e s t i n g . Approximately 55 M2 seeds did not germinate, while 20 M2-gamma and 36 M2-EMS plants either did not develop any nodules or developed i n e f f e c t i v e nodules without leghemoglobin. Other nitrogen f i x i n g mu-tants were also detected. For example, variations were observed i n nodule number and size compared to seed-derived control plants. Thus, both mutagens proved to be e f f e c t i v e in inducing mutations i n P_. vulgaris. Nevertheless, although 1400 plants were screened for v a r i a b i l i t y i n leghemoglobin, no heterogeneity for t h i s 63 t r a i t could be detected. This observation i s not incon-s i s t e n t with previous reports of genetic conservation for t h i s protein (Holl et a l . 1983, Fuchsman and Palmer 1985). There are a number of possible explanations for the f a i l u r e to induce any mutational changes i n Lb pattern. F i r s t l y , c e l l u l o s e acetate electrophoresis separates proteins according to their e l e c t r i c charge and i s not the most discriminating method. I s o e l e c t r i c focusing (IEF) can d i s t i n g u i s h proteins d i f f e r i n g by only 0.01 pH unit i n their i s o e l e c t r i c point (Fuchsman 1983). However, unlike CAE, IEF i s a more co s t l y , time-consuming procedure which cannot be routinely used as a rapid screening technique for large nodule numbers. Thus, i f mutations had been induced which effected only a s l i g h t change i n the charge of the Lb molecule, they would not l i k e l y have been detected with the CAE procedure. Secondly, the number of plants screened may have been too small a population to permit detection of a rare muta-genic event. T h e o r e t i c a l l y , any gene can mutate, but such a mutational event i s a matter of chance and the expected rate of mutation at any s p e c i f i c locus i s d i f f i c u l t to es-timate (IAEA 1977). Micke (1984) estimated that the muta-tio n rate i n a p a r t i c u l a r single gene occurs with a f r e -quency of 10*4 to 1 0 p e r generation. Given that t h i s frequency range i s a reasonable estimate, a population of 6 4 10,000 - 100,000 plants would be required to detect muta-t i o n i n a single Lb gene, a much larger sample than was available for t h i s experiment. However, t h i s estimate i s based on the occurrence of a single active gene. Lee and Verma (1984) proposed that there might be four active genes present i n the Phaseolus genome, which would imply that a fo u r - f o l d decrease i n the population s i z e should be adequate. Consequently, the pr o b a b i l i t y of finding the desired mutant i n the population studied, might not have been as small as suggested. Furthermore, increasing the mutation rate by increa-sing the dosage provides no obvious solution, since a l l mutagenic treatment i s damaging and higher doses are also l i k e l y to produce too many s t e r i l e plants and/or too high a l e v e l of mortality. In the l i t e r a t u r e , treatment of P_. vulgar i s with gamma-rays has been reported using 30 - 400 Gray, and treatment with EMS using from 0.02 - 0.2 M (Crocomo et a l . 1973, Hussein and Disouki 1976, Motto et a l . 1975, Romero and Garcia 1981) . The dosage for che-mical treatments also depends on the exposure time, which has been varied for EMS treatments from 6 - 1 2 hours (Delgado de l a Flor 1970, 1978, Moh 1969). As noted i n the introduction to t h i s section, i n ad-d i t i o n to the absolute dose of mutagen, mutation rates are influenced by many other factors, including the c u l t i v a r 65 (Hussein and Disouki 1976) , temperature (IAEA 1977) , water and oxygen content (Weiss 1944, Muller 1954b), pre- and post-treatment conditions (Conger et a l . 1969, Delgado dela Flor 1970, Konzak et a l . 1970) as well as the number of i n i t i a l meristematic c e l l s i n the growing point of the seed (Gottschalk and Wolff 1983, Motto et a l . 1975). In t h i s experiment, usually only one seed per plant was tested. However, an average of f i v e seeds per Ml plant was harvested. According to Motto et a l . (1975) , i t i s l i k e l y that the mutagenic treatment of P_. vulgaris induced chimeras since the number of i n i t i a l c e l l s present i n the a p i c a l meristem of th i s species was estimated to be eight. If the production of chimeras for Lb v a r i a b i l i t y i s consi-dered a p o s s i b i l i t y , then every single nodule would have to be screened. However, since over 4000 seeds were harves-ted, the l o g i s t i c s of such an analysis were beyond t h i s research. The r e s u l t s of th i s mutagenesis work again rais e the question of whether a l l four leghemoglobin genes i n P_. vulgaris encode a single i d e n t i c a l component or i f only one gene i s active and the other three are s i l e n t (Lee and Verma 1984). About 1400 seeds were grown and screened for leghemoglobin v a r i a b i l i t y . If there are four active genes, one might speculate that i t should have been possible to detect a mutant. However, the fa c t that 56 mutants did not 66 form any nodules or were d e f i c i e n t i n leghemoglobin pro-duction gives some in d i c a t i o n that only one gene might be activ e . Thus, one might further speculate that i n case only a single gene i s active, i t i s quite possible that inducing a mutation i n the Lb gene i s an extremely rare event and/or i f i t occurs, i t has most l i k e l y a l e t h a l e f f e c t since any mutation w i l l a f f e c t the structure as well as the function of the leghemoglobin molecule. Probably only very few mutations are acceptable which w i l l not d i s -rupt i t s function, and even fewer of those w i l l improve i t s oxygen-carrying c a p a b i l i t y . In conclusion, many plant breeders and g e n e t i c i s t s are s i m i l a r l y s c e p t i c a l with regard to the effectiveness of mutagenesis for plant improvement (Stadler 1930, Micke 1984, Simmonds 1981). Micke (1984) pointed out that many pos i t i v e r e s u l t s obtained so far with mutation breeding are due to e a s i l y i d e n t i f i a b l e , predominantly morphological mutations which are often monogenetically inherited and thus can be screened for on a large scale. 67 IV. LEGHEMOGLOBIN VARIABILITY IN THE GENUS PHASEOLUS 4.1. INTRODUCTION P_. vulgar i s L. i s commonly known as French, kidney, bush, snap, navy, haricot, or common bean (Evans 1980) . Gentry (1969) considers Central and probably South America to have been the centre of o r i g i n and from there beans spread to Europe and A f r i c a during the sixteenth century. Archeological findings date the domestication of beans between about 7680 and 10,000 BC (Kaplan et a l . 1973). The genus Phaseolus belongs to the Leguminosae (Fabaceae) family, subfamily Papilionoideae and t r i b e Phaseoleae. P_. vulgar i s i s a. d i p l o i d species with 2n=22 chromosomes. In view of their common morphological cha-racters, _P_. coccineus L. (scarlet runner bean) and _P. a c u t i f o l i u s Gray (tepary bean) are close r e l a t i v e s . P_. lunatus L. (lima bean) i s more d i s t a n t l y related as re-f l e c t e d i n the f a i l u r e of i n t e r s p e c i f i c crosses with _P. vulgar i s . Beans are autogamous with the exception of P_. coccineus which shows predominant c r o s s - f e r t i l i t y ( B l i s s 1980, Brucher 1977, Evans 1980, Kloz 1971). Analysis of leghemoglobins from a range of legume species has revealed a consistent pattern of heterogeneity within a species (Dilworth and Appleby 1979). Preliminary 68 disc electrophoretic studies by Cutting and Schulman (1971) indicated that P_. lunatus 'Prizetaker' had two leghemoglo-bin components. However, i t should be noted that, accor-ding to their r e s u l t s , P_. vulgar i s also showed two compo-nents. This has not been confirmed by our results using c e l l u l o s e acetate electrophoresis (CAE) or by the use of the more sophisticated technique of i s o e l e c t r i c focusing (IEF) (Holl and Lulsdorf, unpublished data). These l a t t e r methods demonstrated that nodules of bush beans contain only one major leghemoglobin component and a minor compo-nent which i s l i k e l y a deamination product of the major one, as suggested by Lehtovaara and E l l f o l k (1975a). Since leghemoglobins i n the genus Phaseolus have never been investigated systematically, t h i s project was conduc-ted to evaluate representatives of the genus and to deter-mine i f globin heterogeneity i s present. If v a r i a t i o n for leghemoglobin e x i s t s between species, then t h i s plant ma-t e r i a l could be used to transfer the variant leghemoglobins into P_. vulgaris i n order to determine the impact of Lb heterogeneity on the f i x a t i o n e f f i c i e n c y of the altered bean. Furthermore, knowledge of the arrangement of leghemo-globin genes i n the genus Phaseolus and the amino acid se-quences of the proteins could greatly enhance our under-standing of the evolution of the globin genes. 69 4.2. LITERATURE REVIEW Immunological studies by Kloz (1971) and cluster ana-l y s i s by S u l l i v a n and Freytag (1986) determined that P_. vulgar i s i s c l o s e l y related to P_. coccineus (runner bean) , less c l o s e l y to P_. a c u t i f o l i u s (tepary bean) and only d i s -t a n t l y to P_. lunatus (lima bean) . P_. f i l i f o r m i s can be grouped between tepary and lima beans i n t h i s geneology. A genus i s an excellent source of genetic v a r i a b i l i t y for transfer of characters not otherwise available within a crop species. Mendel (1865) accomplished the f i r s t suc-cessful i n t e r s p e c i f i c hybridization between P_. vulgaris and P_. coccineus. Since then, many crosses have been performed between these two species because of the r e l a t i v e ease with which hybrids can be obtained. For example, Lamprecht (1941) reported extensive hy-b r i d i z a t i o n studies between P_. vulgar i s and P. coccineus (multiflorus) c a r r i e d out during the nineteen t h i r t i e s . Since f e r t i l i t y of the hybrids was reduced, the author proposed that hybridization b a r r i e r s due to cytoplasmic factors caused the high gamete s t e r i l i t y i n the F l genera-ti o n . From further studies, Lamprecht (1957) concluded that the larger pollen of runner beans car r i e d over a small but s u f f i c i e n t quantity of cytoplasm to permit restoration of f e r t i l i t y whereas the re c i p r o c a l cross using P_. vulgaris 70 pollen remained i n f e r t i l e due to the smaller cytoplasmic content of p. vulgar i s pollen grains. In contrast, i n t e r s p e c i f i c hybridization with other species i n the Phaseolus genus has been more d i f f i c u l t , and embryo rescue methods are generally required. Honma (1956) crossed P_. vulgar i s ('Great Northern') with _P_. a c u t i f o l i u s in order to transfer common bl i g h t resistance. Three to twenty-four days after p o l l i n a t i o n , tissue culture of the aborting embryos was required. However, the r e s u l t i n g hybrids were s e l f - f e r t i l e and produced viable seeds. A l - Y a s i r i and Coyne (1964) reported that applying naphthaleneacetamide and potassium gibberellate to the base of p o l l i n a t e d flowers improved seed set i n i n t e r s p e c i f i c crosses. Pod growth was stimulated and embryo abortion delayed, but embryo rescue was s t i l l necessary for plant s u r v i v a l . S i m i l a r l y , Smartt (1970) obtained hybrids of common and tepary beans and their reciprocals, although the l e v e l of success was low. A few ind i v i d u a l s were obtained without the help of tissue culture. The author concluded that the lower v i a b i l i t y of hybrids i n an a c u t i f o l i u s plasmon was due to cytoplasmic e f f e c t s . Mok et a l . (1978) also produced immature embryos from re c i p r o c a l crosses of P_. vulgaris ('Great Northern' and 'Ga l l a t i n 50') and P.. a c u t i f o l i u s . Cotyledon development of the hybrids was uneven and the rate of growth as well as 71 the f i n a l s i z e of the hybrid embryos were found to be dependent on the genotype of both parents. Further studies by Rabakoarihanta et a l . (1979) re-vealed that f e r t i l i z a t i o n i n i n t e r s p e c i f i c crosses might be completed but incompatibility arose during endosperm f o r -mation; development was e s p e c i a l l y delayed i f tepary bean was the female parent. Cytological observations indicated bivalent and univalent chromosomes during Metaphase I. In addition, F l pollen f e r t i l i t y was found to be low and s e l -f i n g of the hybrids was not possible (Rabakoarihanta et a l . 1980). Pratt et a l . (1985) reported that using heterozygous female P_. vulgar i s parents almost t r i p l e d the frequency at which i n t e r s p e c i f i c hybrids could be produced. However, a l l F l plants were s t e r i l e and no viable F2 offspring were obtained, due to meiotic abnormalities. Furthermore, the authors pointed out that the genotype of the female parent was the most important factor determining r e l a t i v e success of the hybridization procedure. These somewhat contradictory findings by d i f f e r e n t authors were c l a r i f i e d by the experiment of Parker and Michaels (1986) i n which an examination of nine parental P_. vulgar i s l i n e s indicated that common beans carry an a l l e l e for i n t e r s p e c i f i c incompatibility which interacts with a nuclear factor i n the P. a c u t i f o l i u s genome. Thus, some 72 c u l t i v a r s l i k e 'lea pij a o ' and 'Sacramento Light Red Kidney' do not contain t h i s a l l e l e and are able to produce f e r t i l e hybrids. In contrast to P_. acu t i f o l i u s , h ybridization between P_. vulgar i s and P_. lunatus has not achieved such a degree of success. For example, Honma and Heeckt (1959) reported that crosses between kidney and lima beans usually f a i l e d within seven days of p o l l i n a t i o n . A further attempt using an int r a s p e c i f i c heterozygous P_. vulgar i s hybrid as a f e -male parent yielded two pods with mature seeds. The F2 plants showed intermediate morphological characters and a segregant with the desired green seed coat and green coty-ledon was obtained. However, Smartt (1979) expressed doubt / that these plants were true i n t e r s p e c i f i c hybrids because he was unable to reproduce these re s u l t s i n si m i l a r expe-riments. The author suggested that the progeny were pro-ducts of either s e l f - f e r t i l i z a t i o n or apomixis. In order to examine these contradictory reports, Leonard et a l . (1987) investigated the e f f e c t of maternal heterozygosity on the subsequent development and growth of i n t e r s p e c i f i c hybrids. They concluded that the maternal P_. vulgaris genotype affected the number as well as the size of the embryos produced. However, i n contrast to the f i n -dings by Honma and Heeckt (1959) , i n t r a s p e c i f i c maternal hybrids resulted i n s i g n i f i c a n t l y smaller embryos than pure 73 maternal l i n e s . In addition, since no r e c i p r o c a l effects were observed, a cytoplasmic influence was ruled out. Savova and Zagorska (1987) confirmed i n their experi-ment that the success of i n t e r s p e c i f i c hybridization de-pends on the heterozygosity of the female parent. Mok et a l . (1978) rescued P_. vulgaris x P_. lunatus hybrid embryos i n the pre-heart-shape stage. Reciprocal crosses did not succeed. These authors also observed that hybrid embryo development depended on the parental geno-types. S i m i l a r l y to P_. a c u t i f o l i u s crosses, f e r t i l i z a t i o n occurs but endosperm d i v i s i o n i s either delayed or does not take place. I t appears that these hybrids cannot complete d i f f e r e n t i a t i o n (Rabakoarihanta et a l . 1979). A few viable p l a n t l e t s were obtained from these experiments and trans-ferred to s o i l . However, the p l a n t l e t s did not continue to grow beyond the four t r i f o l i a t e l eaf stage (Mok et a l . 1986) suggesting an incompatibility b a r r i e r . Cytoembryological studies by Molkhova and Tsoneva (1986) indicated that the incompatibility of kidney and lima bean crosses i s r e f l e c t e d i n abnormal development of the embryo and suspensor haustorium and i n hypertrophy of the tapetum of the integument. Thus an e f f e c t i v e incompa-t i b i l i t y b arrier exists between the two species. F i n a l l y , the l i t e r a t u r e on hybridization between P_. vulgar i s and P_. f i l i f o r m i s i s scarce. Tau et a l . (1986) 74 produced viable F l p l a n t l e t s from t h i s cross using embryo rescue techniques. Reciprocal crosses were not successful. The F l hybrids were completely s t e r i l e due to non-viable pollen. Backcrossing also f a i l e d . Treatment of the F l hy-brids with c o l c h i c i n e doubled the chromosome number and some viable seeds were produced. 75 4.3. MATERIALS AND METHODS 4.3.1. PLANT MATERIAL The plant material used i n t h i s study consisted of a s e l e c t i o n of Phaseolus germplasm obtained from Dr. N. Galway of the Cambridge University, U.K., two P. a c u t i f o l i u s accessions from the Centro Internacional de Agricultura T r o p i c a l (CIAT) , and the P_. vulgaris c u l t i v a r 'lea p i j a o ' from the University of Guelph, Canada. Furth-ermore, one of the s c a r l e t runner bean (P_. coccineus) c u l -t i v a r s , the c u l t i v a r s 'Contender', 'G a l l a t i n 50' (P_. vulgaris) , and 'Lima Hendersons' (P_. lunatus) were obtained from Buckerfield's Ltd., Vancouver, B.C.. Table 1 l i s t s a l l accessions and their geographical source. Beans are d i p l o i d and are normally s e l f - p o l l i n a t e d with the exception of P_. coccineus (Adams et a l . 1985, Brucher 1977). The average thousand seed weight ranged from about 8 g for P_. f i l i f o r m i s to about 1150 g for P_. lunatus (Market Sample). Seed coat color varied from white, through beige, brown, purple to black and variegated types. A l l plants with the exception of 'Contender' and ' G a l l a t i n 50' were pole beans. Some of the wild accessions with hard seed coats nee-ded s c a r i f i c a t i o n and/or soaking i n 0.5 mM g i b b e r e l l i c acid 76 Table I. Phaseolus germplasm accessions SPECIES VARIETY CULTIVAR or GEOGRAPHICAL PLANT INTRODUCTION SOURCE P_. vulgar i s V P_. vulgar i s 2851 840 841 868 869 871 872 2249 2832 2860 2895 2960 3701 3782 3798 3807 Europe Eur ope Europe Eur ope Europe Eur ope Europe Central Central Central Central Central Central Central Central Central Amer ic a Ame r i c a America America America America America America America P. vulgar i s P. vulgar i s wild N 1695 -wild N 1735 -wild N 11002 -wild N 11042 -wild N 11089 -wild N 11091 — wild N 1404 Mexico wild N 1406 A Mexico wild N 1407 Mexico wild N 1544 Mexico wild N 1575 Mexico wild N 1578 Mexico wild N 1580 Mexico wild N 1581 Mexico wild N 1848 Mexico aborigineus N 1190 — abor igineus N 1573 — aborigineus N 1622 — abor igineus N 1627 — 77 Table I. continued. SPECIES VARIETY CULTIVAR or PLANT INTRODUCTION GEOGRAPHICAL SOURCE P. vulgar i s P. coccineus P. a c u t i f o l i u s l a t i f o l i u s P_. lunatus P_. f i l i f o r m i s P_. microcarpus 'Contender' USA 'Gallatin 50' USA 1 lea pijao' 'Hammond's Dwarf* NC44 'Crusader' C196 'Exhibition Scarlet Prizewinner 1 C184 'Scarlet Runner' CIAT 79069 Mali CIAT 40107 Mexico N 1775 N 1602 Market Sample 'Lima Hendersons' N N 1600 1708 78 to induce germination. Following the res u l t s and recommendations by Parker and Michaels (1986), the P_. vulgaris c u l t i v a r 'lea p i j a o 1 was used as a female parent for a l l P_. vulgar i s x P_. a c u t i f o l i u s i n t e r s p e c i f i c crosses. A l l other crosses were attempted with *Ica p i j a o * , 'Gallatin 50', or 'Contender' as the female parent. The nitrogen f i x a t i o n experiment was conducted only with hybrids derived from crosses involving 1 lea p i j a o ' . 4.3.2. GROWING CONDITIONS The experiments were conducted i n the greenhouses and growth chambers of the Department of Plant Science, University of B r i t i s h Columbia from 1986 to 1988. Plants were grown i n i t i a l l y i n growth cabinets with day and night temperatures of 22°C and 18°C, respectively. A l l P_. a c u t i f o l i u s accessions, except P. a c u t i f o l i u s Mali, are short-day plants and thus required a maximum day-length of 10 hours for flowering; a l l other accessions were grown under a 16 hour day/ 8 hour night regime. Depending on the season and the siz e of the plants, the accessions and the hybrids were transferred to the greenhouse and supplemented with fluorescent l i g h t during the f a l l and winter months according to their day-length requirements. Spider mites and white f l i e s were controlled 79 when necessary. For the hybridization experiments and hybrid multi-p l i c a t i o n , each plant was grown i n a .15 cm diameter f l e x i -ble p l a s t i c pot with a s o i l : peat moss (4:1 w/w) mix con-taining Osmocote®, a 14-14-14 slow release f e r t i l i z e r . After about two months of growth, the plants were f e r t i -l i z e d twice weekly with Peters®water soluble f e r t i l i z e r (20-20-20). Throughout the growth period, a l l plants were watered with tap water as required. According to the recommendations by Mok et a l . (1986) for hybridizations with _P_. lunatus, the female P_. vulgar i s parent was grown i n hydroponic culture i n a medium supple-mented with 10 >uM benzyladenine. Nutrient solutions were prepared according to Murashige and Skoog (1962) at one-h a l f strength; one l i t e r per plant. Aeration was provided from a compressed a i r l i n e , at about one l i t e r per minute. A l l female parent plants were maintained i n growth chambers at 22°C with a 16 hour photoperiod. For the CAE and IEF experiments, at least one seed from every accession or one well-rooted cutting from every hybrid was planted i n a styrofoam cup containing Turface® and inoculated with a commercial Rhizobium leguminosarum biovar phaseoli inoculant (Nitragin Company Ltd) . Since P_. lunatus accessions nodulated only poorly with t h i s inocu-lum, t h i s species was inoculated with a R. leguminosarum 80 bv. phaseoli s t r a i n TAL 22, s p e c i f i c for lima beans, ob-tained from the University of Hawaii, NifTAL Project. A l l plants were f e r t i l i z e d twice weekly with a nitrogen-free nutrient solution. For the nitrogen f i x a t i o n experiment, well-rooted cuttings from each hybrid were grown i n 8 cm diameter p l a s t i c pots f i l l e d with Turface®, inoculated and f e r t i -l i z e d s i m i l a r to the CAE and IEF procedure. 4.3.3. LEGHEMOGLOBIN TESTING Three to f i v e weeks after inoculation, nodules from each accession were excised, crushed i n 0.1 M sodium phosphate buffer, pH 7.4 and centrifuged at 12,400 x g i n an Eppendorf microcentrifuge for 2 minutes. The superna-tant containing leghemoglobin was either used d i r e c t l y for CAE and IEF or further p u r i f i e d according to methods des-cribed by Holl et a l . (1983) via ammonium sulfate p r e c i p i -t a t i o n , treatment with potassium ferricyanide and d i a l y s i s . Samples were concentrated using Centricon® microconcen-tr a t o r s . The CAE was run for 15 minutes at 420 v o l t s (Holl et a l . 1983) . After separation, the Sepraphore®strips were stained for protein with Ponceau S and counterstained for heme using the method of Owen et al.(1958) as described i n Ho l l et a l . (1983) . 81 IEF was performed i n an automated PhastSystem® (Pharmacia Ltd.) using precast Phast Gels® with a pH 4-6.5 ampholyte gradient. Two gels loaded with the same samples were run simultaneously. A protein standard (Pharmacia C a l i b r a t i o n Kit®, pH 2.5-6.5) was used for c a l i b r a t i o n . The t o t a l running time including prefocusing was 500 Vo l t -hours at 15°C and lasted about 25 minutes. One gel was stained with Coomassie Blue for protein according to the recommendations i n the manual for the Phast Gel® IEF system (Phast Development Technique F i l e No. 200); the other gel was stained for heme using the method by Owen et a l . (1958) as described by Holl et a l . (1983). 4.3.4. INTERSPECIFIC HYBRIDIZATION Flowers of P_. vulgar i s were emasculated one day before opening ( B l i s s 1980) and p o l l i n a t e d immediately using the hooking method described by Buishand (1956). To prevent dessication, p o l l i n a t e d buds were enclosed i n cellophane tape as recommended by B l i s s (1980) . Only one flower per raceme was po l l i n a t e d ; a l l other flowers were removed to decrease competitive e f f e c t s . The number of i n t e r s p e c i f i c pods growing at the same time on one plant was limi t e d to f i v e . Pods showed v i s u a l l y d i s c e r n i b l e growth about three to four days afte r p o l l i n a t i o n . 82 4.3.5. EMBRYO RESCUE AND HYBRID GROWTH Developing pods for embryo rescue were removed from the P_. a c u t i f o l i u s hybrids 23 days after p o l l i n a t i o n (Parker and Michaels 1986), and 12 to 15 days after anthe-s i s from crosses involving P_. lunatus (Mok et a l . 1978, Leonard et a l . 1987). P_. f i l i f o r m i s hybrids were rescued 15 to 20 days after p o l l i n a t i o n . Pods were rinsed with 95% ethanol for one minute, and surface s t e r i l i z e d for 15 minutes by immersion i n an aqueous solut i o n of commercial bleach (20% v/v) containing two drops of Tween-20®. A l l subsequent procedures were car r i e d out i n a laminar flow hood. The s t e r i l i z e d pods were rinsed twice with s t e r i l e d i s t i l l e d water. Ovules were removed from pods and embryos dissected under a binocular dissecting microscope. The excised P_. a c u t i f o l i u s embryos were placed in culture v i a l s containing 15 ml of B5 medium (Gamborg et a l . 1968) supplemented with 3% (w/v) sucrose and 0.8% (w/v) agar. Following the procedure of Parker and Michaels (1986), the t e s t tubes were placed i n the dark at 25°C un-t i l hypocotyl elongation took place. Then, embryos were transferred to a growth chamber with a 16 hour photoperiod. After 12 to 15 days, vigorously growing p l a n t l e t s were transferred to 8 x 8 cm square clear p l a s t i c containers f i l l e d with 80 ml of the same B5 medium. 83 About one month l a t e r , well-developed p l a n t l e t s with roots were transferred to 8 x 8 cm square f l e x i b l e p l a s t i c pots containing a s o i l : sand : peat moss (3:1:1, w/w/w) mixture with Osmocote® f e r t i l i z e r , a 14-14-14 slow release f e r t i l i z e r . The plants were placed on culture trays i n closed aquariums f i l l e d with about 3 cm of water (plants were not i n contact with water) to maintain high r e l a t i v e humidity. Later, the aquariums were p a r t i a l l y opened u n t i l the plants were adapted to ambient growing conditions; the adjustment required about two to three weeks. P_. f i l i f o r m i s hybrids were treated s i m i l a r l y to P_. a c u t i f o l i u s embryos except for the tissue culture medium. Excised P_. f i l i f o r m i s and P. lunatus i n t e r s p e c i f i c embryos were placed i n culture v i a l s containing 15 ml of a three quarter strength Murashige and Skoog (1962) embryo rescue medium prepared according to Mok et a l . (1978) , with 0.125 /iM benzyladenine, 0.7 mM glutamine, 0.8% (w/v) agar, and 3% (w/v) sucrose. P_. lunatus i n t e r s p e c i f i c embryos were placed i n growth chambers with continuous l i g h t at 28°C for f i v e weeks (Leonard et a l . 1987) and then maintained with a 16 hours photoperiod and a temperature of 26°C. 84 4.3.6. NITROGEN FIXATION EXPERIMENT The nitrogen f i x a t i o n experiment was conducted with cuttings obtained from P_. vulgar i s x P. a c u t i f o l i u s and P. vulgaris x P_. f i l i f o r m i s i n t e r s p e c i f i c hybrids ( a l l had 'lea pijao* as female parent) and from each of the parents. Cuttings were taken from healthy plants, including at l e a s t one growing point, and dipped i n Stim-Root® No.l, a rooting hormone powder containing 0.1% indole butyric acid, and planted i n 8x8 cm square pots containing a s o i l : sand mixture (4:1, v/v). The p l a n t l e t s were placed i n aquariums as described i n chapter 4.3.5. and grown under fluorescent l i g h t s (16 hour photoperiod) u n t i l they were well rooted^ (about two weeks). Three-week old rooted cuttings of about the same size were selected for the experiments. The s o i l mixture was c a r e f u l l y washed o f f the roots, and cuttings planted i n 8cm diameter p l a s t i c pots f i l l e d with Turface®, inoculated with Rhizobium leguminosarum biovar phaseoli and watered twice weekly with nitrogen-free nutrient s o l u t i o n . The nitrogen f i x a t i o n tests were analysed as rando-mized complete block designs (RCBD) with seven r e p l i c a t e s for each of the acetylene reduction assay (ARA) and Total N experiments. For each ARA, two samples were analysed. Plants were grown for eight weeks i n two growth cham-bers at 22°C/ 18°C and a 16 hour photoperiod. Plant height 85 was measured at the beginning and the end of the exper iment. After eight weeks, the acetylene reduction assay was ca r r i e d out using a closed system and analysis by flame io n i z a t i o n gas chromatography as described by H o l l et a l . (1983) . This method i s based on the a b i l i t y of the n i t r o -genase to reduce acetylene to ethylene (Dilworth 1966). The ARA i s a k i n e t i c measurement of a c t i v i t y which deter-mines the nitrogen f i x i n g rate of a plant at the time of measurement (Boddey 1987, Upchurch 1987). D i r e c t l y a f t e r the ARA, nodule number and nodule fresh weight were recorded, the plants were dried, weighed for total.dry matter (TDM), and ground for micro-Kjeldahl ana-l y s i s to determine t o t a l nitrogen (Lepo and Ferrenbach 1987). T o t a l nitrogen gives an estimate of the cumulative nitrogen f i x a t i o n a c t i v i t y over the period of plant growth. The determination of t o t a l nitrogen was conducted i n the Department of S o i l Science, University of B r i t i s h Columbia. S t a t i s t i c a l analysis of the nitrogen f i x a t i o n experi-ment was c a r r i e d out according to Cochran and Cox (1960). A l l other s t a t i s t i c a l procedures were as described i n Steel and Torrie (1980). 86 4.3.7. PROTEIN SEQUENCING Leghemoglobins from P_. lunatus 'Lima Hendersons' and from an i n t e r s p e c i f i c P_. f i l i f o r m i s hybrid were extracted and p u r i f i e d as described i n Chapter 4.3.3. After p u r i f i -cation, Lbs were applied to a 1.5 x 26 cm column of DE-52 (Whatman®) for c e l l u l o s e ion-exchange chromatography (Holl et a l . 1983). Leghemoglobins from the P_. f i l i f o r m i s hybrid and from P_. lunatus were eluted with a l i n e a r gradient of 5 - 150 mM sodium acetate (pH 5.4) i n a 250 ml volume. Sam-ples were c o l l e c t e d and the absorbance monitored at 405 nm to determine the e l u t i o n of Lb components. The co l l e c t e d fractions were concentrated i n Centricon® microconcentra-tors using an IEC® c l i n i c a l centrifuge. Salts were removed by desalting with the same microconcentrators. Apoproteins for the Lb components were prepared by the acid-acetone procedure ( E l l f o l k 1961). Precipitated pro-teins were re-dissolved i n d i l u t e sodium bicarbonate solu-t i o n (50 mg/ml). Three samples were prepared for sequen-cing: peak I and II from P_. lunatus 'Lima Hendersons' and peak II from a P_. f i l i f o r m i s hybrid. Sequencing of the N-terminal end of the proteins was c a r r i e d out by the Protein Sequencing Laboratory of the University of V i c t o r i a using an automated Edman degradation. 87 4 . 4 . RESULTS AND DISCUSSION 4.4.1. LB VARIABILITY IN THE GENUS PHASEOLUS A l l Phaseolus accessions (see Table I, page 77) were screened for Lb v a r i a b i l i t y using CAE and IEF, three to f i v e weeks after inoculation. The r e s u l t s of both methods were i d e n t i c a l i n regard to numbers of heme-stained Lb bands. In addition, the only difference between p u r i f i e d and unpurified Lb extracts was that the bands were sharper with the former samples. I s o e l e c t r i c points were estimated from IEF gels using protein standards. A l l P. vulgaris accessions (including P_. vulgaris wild and P. vulgar i s aborigineus) as well as P_. coccineus ac-cessions and P.. microcarpus showed only one Lb band i n the same position as P.. vulgar i s controls. Occasionally, a second band was v i s i b l e which has been i d e n t i f i e d as a de-amidation product of the major component (Lehtovaara and E l l f o l k 1975a). Lehtovaara and E l l f o l k (1975a) reported i s o e l e c t r i c points of 4.7 for Lba and 4.55 for Lbb from P. vulgaris. The i s o e l e c t r i c points estimated from the pre-sent i s o e l e c t r i c focusing study confirmed those r e s u l t s . A l l P_. a c u t i f o l i u s accessions also contained one major component; however, th i s band migrated more slowly i n the electrophoretic systems i n comparison to P_. vulgaris 88 Fig. 1. CAE Lb p r o f i l e showing P_. vulgar i s (C) , P_. acuti f o l iu s N1602 (A ^ ) , and _P. a c u t i f o l i u s ssp. l a t i f o l i u s (A j_) • 89 (Fig. 1). The basic p r i n c i p l e of CAE, as reviewed by Chin (1970), i s the movement of charged proteins through a sup-port medium toward the electrode with the opposite charge. The speed of migration of a protein depends, beside many other factors, on i t s net charge. Consequently, the d i f -ferent migration pattern r e s u l t from changes i n one or more amino acids. I s o e l e c t r i c focusing re s u l t s gave an estimate of 4.8 for the P_. a c u t i f o l i u s Lb i s o e l e c t r i c point. In contrast, both P. f i l i f o r m i s and P_. lunatus acces-sions contain more than one Lb component. P_. f i l i f o r m i s (Fig. 2) shows two bands when separated by CAE. Both com-ponents migrate more slowly than that of P. vulgar i s . One (the faster of the two) migrated to a p o s i t i o n s i m i l a r to that of P_. a c u t i f o l i u s Lb (Fig. 3) . The i s o e l e c t i c points of the two f i l i f o r m i s components were estimated to be about 4.8 and 4.9. The most int e r e s t i n g species examined i n the survey was P_. lunatus (Fig. 4 ) , since t h i s species produced two major and probably three minor components. The major com-ponents migrated faster than P_. vulgar i s and their i s o -e l e c t r i c points have been estimated as 4.5 and 4 . 4 . One minor component seems to be i n a similar position as the P_. a c u t i f o l i u s band, the other i s i n a p o s i t i o n s i m i l a r to the P.. vulgar i s band; the t h i r d one has an i s o e l e c t r i c point of approximately 4 . 4 . 90 F i g . 2. CAE Lb p r o f i l e s h o wing P. v u l g a r i s w i l d ( P w ) , P. f i l i f o r m i s (P p) , P. m i c r o c a r p u s (P M ) and P. v u l g a r i s ' G a l l a t i n ' ( G). 91 Fig. 3. CAE Lb p r o f i l e showing P_. vulgar i s x P. f i l i f o r m i s hybrid (1), P. a c u t i f o l i u s (2), P. vulgar i s x P.. a c u t i f o l i u s hybrid (3) and p_. vulgaris (4). 92 9 3 The only report of i n t r a - s p e c i f i c leghemoglobin v a r i -a b i l i t y has been described by Holl et a l . (1983) who d i s -covered a Tr ifolium subterranean l i n e with a major compo-nent missing. In contrast, Fuchsman and Palmer (1985) who studied leghemoglobins from a g e n e t i c a l l y diverse s e l e c t i o n of c u l t i v a t e d (G_. max) and wild soybeans (G_. soja) could not detect any differences even though they used IEF, a more se n s i t i v e method. This present study i s the only one to screen a genus i n which differences between species for leghemoglobin genes have been discovered. S u l l i v a n and Freytag (1986) investigated seed protein patterns i n the genus Phaseolus and s i m i l a r l y found very l i t t l e v a r i a t i o n i n protein pro-f i l e s within most species, while considerable v a r i a t i o n was evident among species. However, the same authors reported considerable v a r i a t i o n within the P. a c u t i f o l i u s species which did not have a single e a s i l y recognizable protein p r o f i l e . In contrast, the present study did not detect any heterogeneity for Lb between P_. a c u t i f o l i u s subspecies. This investigation studied only a small number of species within the Phaseolus genus which comprises 31 (Mare"chal et a l . 1978) to 150 (Briicher 1977) species. It would be i n t e r e s t i n g to determine i f further v a r i a t i o n e x i s t s i n t h i s genus since, i n general, i t would appear 94 that the more d i s t a n t l y related the species, the more d i f -ferent are their Lb p r o f i l e s . The only exception to t h i s generalization was P_. microcarpus (Fig. 2) which had only a single Lb isomer, similar to P_. vulgaris. P_. microcarpus has been shown to be more c l o s e l y related to P. lunatus than to P_. vulgar i s (Sullivan and Freytag 1986) . Leghemoglobin components from many species have been shown to d i f f e r i n their oxygen a f f i n i t y and thus i n their enhancement of bacteroid oxygen consumption and support of bacteroid nitrogen f i x a t i o n (Fuchsman and Appleby 1979, Uheda and Syono 1982a,b, Wittenberg et a l . 1985). Often, only very small changes i n the amino acid composition were found to be responsible for t h i s higher oxygen a f f i n i t y (Kortt et a l . 1987). Unfortunately, we could not determine oxygen association and d i s s o c i a t i o n rates of the d i f f e r e n t leghemoglobins i n Phaseolus but i f differences i n oxygen a f f i n i t y could be confirmed i n t h i s genus, then those f i n -dings would further support the theory that Lb heteroge-neity i s functional and that i t i s an adaptation for more e f f e c t i v e nitrogen f i x a t i o n as suggested by Fuchsman et a l . (1976) and Uheda and Syono (1982a,b). 95 4.4.2. INTERSPECIFIC HYBRIDIZATION Based on the re s u l t s obtained from CAE and IEF, P_. a c u t i f o l i u s , V. f i l i f o r m i s , and _P. lunatus were chosen as parents for transfer of Lb v a r i a b i l i t y into P_. vulgar i s . A t o t a l of about 500 crosses was performed, from which about 450 developed into pods. However, many of those pods con-tained seeds with aborted embryos, e s p e c i a l l y when P_. lunatus was one of the parents. This observation i s in agreement with that by Alvarez et a l . (1981) and Mok et a l . (1978). Hybrid embryos from P_. lunatus crosses only deve-loped to an oval-rod-shape stage s i m i l a r to reports by Mok et a l . (1978) . Furthermore, growth of the i n t e r s p e c i f i c p l a n t l e t s i n tissue culture was slow. Due to a growth chamber malfunction which destroyed the i n t e r s p e c i f i c hy-brids obtained from th i s cross, t h i s part of the research project was discontinued. In contrast, embryos from i n t e r s p e c i f i c hybridizations involving P. a c u t i f o l i u s ssp. (ssp. a c u t i f o l i u s , ssp. l a t i f o l i u s , ssp. a c u t i f o l i u s Mexico and ssp. a c u t i f o l i u s Mali) developed to the cotyledon stage. As reported by Mok et a l . (1978), these embryos often showed c h a r a c t e r i s t i c asymmetric development of the two cotelydons and t y p i c a l retarded endosperm development (Rabakoarihanta et a l . 1979). Hybrids grew well i n tissue culture and transfer to s o i l and adaptation to ambient growing conditions resulted 96 i n very few losses. However, even though a l l i n t e r s p e c i f i c plants from t h i s cross flowered, the hybrids were generally s t e r i l e , with l i t t l e pollen developed i n the anthers. The exceptions to t h i s s t e r i l i t y were two P_. vulgaris x P_. a c u t i f o l i u s Mexico plants which were s e l f - f e r t i l e and de-veloped seeds. S i m i l a r l y , Haghighi (1987) reported ob-taining some se l f e d seeds from t h i s combination. Hybrid Lb phenotypes were intermediate between the two parents. Results from CAE and IEF showed two Lb bands for the hy-brids (Fig. 3) and thus, Lbs are inherited codominantly. Few of the _P_. vulgar i s x P_. f i l i f o r m i s hybrid pods contained seeds with embryos s u f f i c i e n t l y large for e x c i -sion. Both P_. vulgar i s c u l t i v a r s 'Contender' and 'lea pijao ' were successfully used as female parents, and hy-brids from both crosses grew well i n tissue culture and s o i l . Leaf shape, size and texture of the hybrids were intermediate between the two parents. Plants flowered profusely but again, a l l hybrids were completely s t e r i l e . Tau et a l . (1986) also reported obtaining s t e r i l e hybrids from th i s cross, due to formation of nonviable pollen^ These authors used colch i c i n e to produce tetr a p l o i d s i n order to restore p a r t i a l f e r t i l i t y and to induce seed de-velopment. I n t e r s p e c i f i c hybrids also showed codominant inheritance of Lb components when separated by CAE and IEF (Fig. 3). 97 4.4.3. NITROGEN FIXATION EXPERIMENTS Treatment of 'Plant Species' and grouping of parents and hybrids for analysis are summarized i n Table I I . In the analysis of variance shown i n Table III highly s i g n i f i c a n t differences (P< 0.01) were detected for 'Plant Species' i n the ARA experiment, indicating that species d i f f e r e d i n the i r nitrogen f i x a t i o n rates. Further p a r t i -tioning of the sums of squares for 'Plant Species' showed highly s i g n i f i c a n t differences among the f i v e groups and within group V (involving P_. f i l i f o r m i s ) . C a l c u l a t i o n of sin g l e degree contrasts among groups (Table IV) revealed that groups I to IV (involving P_. a c u t i f o l i u s ssp.) d i f - , fered s i g n i f i c a n t l y (P< 0.01) from group V (involving _P. f i l i f o r m i s ) , indicating that group V had a higher nitrogen f i x a t i o n rate than any of the other groups (Table V). Furthermore, contrasts calculated within groups of the ARA experiment (Table VI) showed that i n group I, both parents (P_. vulgaris (1) and P_. a c u t i f o l i u s N1602 (6) ) d i f f e r e d s i g n i f i c a n t l y from their associated hybrid (11). Thus, the hybrid mean (Table V) i s s i g n i f i c a n t l y higher than either parent. Whether t h i s difference i s due to heterosis or a consequence of parent II (P_. a c u t i f o l i u s N1602) remains a matter of speculation, since the difference between parent I and II was not s i g n i f i c a n t (Table VI). 98 Table I I . Grouping of 'Plant Species' for analysis. Group 1 Parent I Parent II Hybrid I P.v.2 (1) P.a. N1602 (6) P.v. x p . 3.N1602 (11) II P. v. (2) P.a. Mexico (7) P.v. x p. a.Mexico (12) III P.v. (3) P.a. Mali (8) P.v. X p. a.Mali (13) IV P.v. (4) P.a. l a t i f . (9) P.v. X p. a . l a t i f . (14) V P.v. (5) P. f i l i f . (10) P.v. X p . f i l i f . (15) x For analysis, 'Plant Species' were coded from 1 to 15 (numbers i n parentheses) and grouped into f i v e groups, each consisting of parent I, parent II and the respective hybrid. 2 Abbreviations: P. vulgaris = P.v.; P. a c u t i f o l i u s = P.a.; l a t i f o l i u s = l a t i f . ; P. f i l i f o r m i s = P. f i l i f . 99 Table I I I . Analysis of variance for ARA of P. vulgaris, P_. a c u t i f o l i u s , P. f i l i f o r m i s and i n t e r s p e c i f i c hybrids. Additional analysis for the pa r t i t i o n e d sums of squares for 'Plant Species' i s also shown. Source of Variation DF MS Replicates Plant Species Error 6 14 84 558,778.4 1,662,522.5 646,859.2 0.864 2.570** Among Groups Within Group I Within Group II Within Group III Within Group IV Within Group V 2 2 2 2 2 2,492,596.8 1,710,954.0 1,015,737.8 97,770.3 193,964.3 3,634,037.8 3.853** 2, 1, 645 570 0.151 0.230 5.618** *,** S i g n i f i c a n t at the 0.05 and 0.01 l e v e l of p r o b a b i l i t y . 100 Table IV. Orthogonal contrasts among groups for the ARA exper iment. Compar ison of Groups r l c 2 i MS(Q) F I,II,III,IV vs. V 21 (20) 6,594,641. 8 10 .195** II,III,IV vs. I 21(12) 2,094,909. 7 3 .239 I,III,IV vs. II 21 (12) 202,296. 0 0 .313 I,II,IV v s . I l l 21(12) 249,026. 8 0 .385 .** S i g n i f i c a n t at the 0.01 l e v e l of p r o b a b i l i t y . 101 Table V. Mean acetylene reduction (nm/plant/hour) for each 'Plant Species' and group measured i n the ARA experiment. Group Group Parent 1 Parent 2 Hybrid Means I 414.04 773.47 1,391.48 859.66 II 404.78 501.16 1,107.45 671.13 III 360.71 524.60 590.16 491.83 IV 308.20 494.81 162.73 321.91 V 386.76 1,538.68 1,712.55 1,212.66 102 Table VI. Orthogonal contrasts within groups for the ARA experiment. Comparison within group between species r E o! MS(Q) F I 1, 6 and 11 7(6) 2,969,736.1 4.591* 1 and 6 7(2) 452,171.9 0.699 II 2, 7 and 12 7(6) 1,998,960.8 3.090 2 and 7 7(2) 32,514.8 0.050 III 3, 8 and 13 7(6) 101,534.1 0.157 3 and 8 7(2) 94,006.5 0.145 IV 4, 9 and 14 7(6) 266,047.1 0.411 4 and 9 7(2) 121,881.5 0.188 V 5, 10 and 15 7(6) 2,623,845.1 4.056* 5 and 10 7(2) 4,644,230.4 7.180** *,** S i g n i f i c a n t at the 0.05 and 0.01 l e v e l of p r o b a b i l i t y . 103 The most in t e r e s t i n g r e s u l t s occurred within group V. Both parents (P_. vulgar i s (5) and P_. f i l i f o r m i s (10) ) d i f -fered s i g n i f i c a n t l y from their associated hybrid (15) (Table VI). Their respective means (Table V) showed that their hybrid had a higher nitrogen f i x i n g rate than either parent. In addition, parent I (P_. vulgar is) d i f f e r e d s i g -n i f i c a n t l y (P< 0.01) from parent II (P. f i l i f o r m i s ) (Table VI). The mean rate of nitrogen f i x a t i o n (Table V) of the P. f i l i f o r m i s parent i s about four times higher than that of the P.. vulgar i s parent i n d i c a t i n g that the hybrid de-rived i t s higher nitrogen f i x i n g a b i l i t y from the P_. f i l i f o r m i s parent. The analysis of variance (Table VII) also detected highly s i g n i f i c a n t differences among 'Plant Species' i n the Total N experiment, in d i c a t i n g that some species accumu-lated more nitrogen during their growth period than others. P a r t i t i o n i n g the DF and sums of squares for 'Plant Species' (Table VII) revealed that there were s i g n i f i c a n t d i f f e r e n -ces among groups and within group V for the t o t a l amount of nitrogen accumulated over the growing period. To determine the source of the differences, orthogonal contrasts were car r i e d out among and within groups. Table VIII shows that groups I to IV (involving _P. a c u t i f o l i u s ssp.) d i f f e r e d s i g n i f i c a n t l y (P< 0.01) from the P. f i l i f o r m i s (V) group. 104 Table VTI. Analysis of variance for Total N i n P_. vulgar i s , P.. a c u t i f o l i u s , P_. f i l i f o r m i s and i n t e r s p e c i f i c hybrids. Additional analysis of the p a r t i t i o n e d sums of squares for 'Plant Species' i s also shown. Source of Vari a t i o n DF MS Replicates Plant Species Error 6 14 84 451.4 2,507.4 527.9 0.855 4.749** Among Groups 6,827.2 12.933** Within Group I Within Group II Within Group III Within Group IV Within Group V 2 2 2 2 2 891.3 188.5 345.0 204.8 2,273.0 1.688 0.357 0.654 0.388 4.306* *,** S i g n i f i c a n t at the 0.05 and 0.01 l e v e l of p r o b a b i l i t y . 105 Table VIII. Orthogonal contrasts among groups for the Total N experiment. Compar ison of groups r l c| MS(Q) F I,II,III,IV vs. V 21 (20) 20,092. 9 38.06** II,III,IV vs. I 21(12) 2,753. 5 5.22* I,III,IV vs. II 21 (12) 1,125. 6 2.13 I,II,IV v s . I l l 21(12) 1,868. 5 3.54 *,** S i g n i f i c a n t at the 0.05 and 0.01 l e v e l of p r o b a b i l i t y . 106 Table IX. Means (mg t o t a l nitrogen) for each 'Plant Species' and group of the Total N experiment. Group Group Parent 1 Parent 2 Hybrid Means I 27.5 23.6 44.8 32.0 II 21.1 10.8 15.3 15.7 III 22.3 32.6 35.7 30.2 IV 16.5 7.4 6.9 10.3 V 38.1 57.8 74.1 56.7 107 Similar to the ARA experiment, the means of the f i v e groups (Table IX) show that the P_. f i l i f o r m i s group accumulated more nitrogen during the growth period than either of the P_. a c u t i f o l i u s groups. Furthermore, group I (involving P_. a c u t i f o l i u s N 1 6 0 2 ) also d i f f e r e d s i g n i f i c a n t l y (P< 0 . 0 5 ) from groups I I , III and IV (Table V I I I ) . F i n a l l y , con-t r a s t s calculated within groups (Table X) showed s i g n i -f i c a n t differences only i n group V between the parents (P. vulgar i s (5) and P_. f i l i f o r m i s ( 1 0 ) ) and their associated hybrid ( 1 5 ). Their respective means (Table IX) confirmed that the hybrid accumulated more nitrogen than either parent. Whether t h i s r e s u l t i s attributable to the P_. f i l i f o r m i s parent as i n the ARA experiment i s not clear since the means of the parents do not d i f f e r s t a t i s t i c a l l y . However, i t i s tempting to speculate that the higher rate of ARA i n P_. f i l i f o r m i s may contribute to greater N accumulation i n the hybrid. In summary, 'Plant Species' d i f f e r e d i n their nitrogen f i x a t i o n rate as well as the t o t a l amount of nitrogen fixed over the growing period. Results from both methods showed that group V (involving P_. f i l i f o r m i s ) outperformed a l l other groups. Notably, the hybrid P_. vulgaris x P_. f i l i f o r m i s had a higher acetylene reduction rate and thus a higher nitrogen accumulation rate than a l l other 'species'. However, only i n the ARA experiment could t h i s be a t t r i b u -1 0 8 Table X. Orthogonal contrasts within groups for the Total N experiment involving P_. vulgaris, P. a c u t i f o l i u s . P_. f i l i f o r m i s and i n t e r s p e c i f i c hybrids. Compar ison within group between species 2 MS(Q) F I 1, 6 and 11 7(6) 1,7 29.3 3.276 1 and 6 7(2) 53.2 0.101 II 2, 7 and 12 7(6) 1.9 0.004 2 and 7 7(2) 369.3 0.699 III 3, 8 and 13 7(6) 318.7 0.604 3 and 8 7(2) 371.3 0.703 IV 4, 9 and 14 7(6) 119.7 0.227 4 and 9 7(2) 289.8 0.549 V 5, 10 and 15 7(6) 3,187.7 6.038* 5 and 10 7(2) 1,358.3 2.573 * S i g n i f i c a n t at the 0.05 l e v e l of p r o b a b i l i t y . 109 ted to the JP. f i l i f o r m i s parent. In addition, the P_. a c u t i f o l i u s N1602 hybrid also had a higher nitrogen f i x i n g rate at the time of measurement, although the t o t a l amount of nitrogen f i x e d did not d i f f e r s i g n i f i c a n t l y . A c o r r e l a t i o n analysis conducted between the A R A and Total N method, gave a c o r r e l a t i o n c o e f f i c i e n t of 0.40, ind i c a t i n g that the acetylene reduction assay i s a poor predictor of the amount of nitrogen f i x e d . Nitrogen f i x a -t i o n rates vary during the growth period as well as during the day (Rennie and Kemp 1981, Goh et a l . 1978). Rennie and Kemp (1981) found no s i g n i f i c a n t c o r r e l a t i o n between A R A and Tot a l N i n their experiments either. In contrast, Hungria and Neves (1987) reported a highly s i g n i f i c a n t c o r r e l a t i o n between these two parameters when A R A was mea-sured from flowering to mid p o d - f i l l stage of d i f f e r e n t bean c u l t i v a r s . From further studies with _P_. vulgaris, Rennie and Kemp (1984) concluded that A R A severely under-estimates nitrogen f i x a t i o n i n t h i s species. S i m i l a r l y , Pacovsky et a l . (1984) concluded that the amount of a s s i -milated nitrogen i s a better measure of symbiotic e f f i -ciency than the reduction of acetylene. Recent studies of nitrogen metabolism i n kidney bean suggest that the r e l a -tionship between acetylene reduction a c t i v i t y measurements and nitrogen accumulation are l i k e l y to be strongly i n f l u -enced by the timing of the assay during the growing period 110 (P. Singleton, NifTAL Project, Hawaii, personal communication). One factor which could not be taken into account i n t h i s experiment and thus might have contributed to the poor c o r r e l a t i o n between ARA and Total N, was the evolution of hydrogen i n the acetylene reduction assay. Pacovsky et a l . (1984) pointed out that the results of the ARA should be corrected for t h i s energy l o s s . Piha and Munns (1987) re-ported hydrogen evolution rates of 17-27 ymol/pot/hour for P_. vulgar i s , 15-16 jjmol for P_. a c u t i f o l ius, 21-25 ymol for the P_. vulgaris x P. a c u t i f o l i u s hybrid, and 7 jimol for P_. f i l i f o r m i s . However, the authors also estimated the n i -trogen f i x a t i o n rate for P_. f i l i f o r m i s as 18 jjmol/pot/h, which was much lower than either P_. vulgar i s , P_. a c u t i f o l i u s and their hybrid. In addition, these estimates were based on measurements of one pot with two plants. In the present experiment, estimates of the seven r e p l i c a t e s varied considerably. However, the mean of P_. f i l i f o r m i s a c t i v i t y highly s i g n i f i c a n t l y (P< 0.01) exceeded P_. vulgar i s using either method. The present study used cuttings instead of plants de-rived from seeds. When plants r e l y on nitrogen f i x a t i o n for t h e i r sole N supply, they have to overcome an i n i t i a l lag period, i n which they function under nitrogen stress. However, plants grown from seeds have some nitrogen re-I l l sources available i n the endosperm. In contrast, cuttings have to r e l y e n t i r e l y on the nitrogen which can be remobi-l i z e d from leaves, stem or roots u n t i l nitrogen f i x a t i o n sets i n . This puts cuttings under a more severe nitrogen stress and hence, r e s t r i c t s their growth. P_. f i l i f o r m i s and i t s hybrids seemed to overcome t h i s l i m i t a t i o n much faster than either P_. vulgaris or V_. a c u t i f o l i u s . Another confounding e f f e c t which could not be taken into account i n t h i s experiment, resulted from differences between the species i n respects other than nitrogen f i x a -t i o n . In order to eliminate t h i s source of error, one would have to use isogenic l i n e s which d i f f e r only i n Lb ',t components. Thus, one cannot conclude d e c i s i v e l y from these experiments that the higher acetylene reduction rates and the higher amounts of nitrogen fixed by P_. f i l i f o r m i s and i t s hybrids are due to more e f f i c i e n t function of Lb components i n those materials. However, these r e s u l t s suggest that i t might be worthwhile to explore t h i s hypothesis further. 4 . 4 . 4 . Protein Sequencing Cellulose ion exchange chromatography resolved Lbs from a P_. f i l i f o r m i s hybrid into three peaks (Fig. 5 ) . Peak II was chosen for the N-terminal analysis. This peak corresponds to the second Lb band i n CAE (Fig. 2 )» i t mi-1 1 2 X I E n o oo u o i FRACTION NUMBER F i g . 5. Elution profi le for P. vulgar is x P. f i l i f o r m i s hybrid Lb components on DE52-cellulose columns. Experimental conditions are described in section 4 . 3 . 7 . , page 87. 113 grated to a p o s i t i o n s i m i l a r to the band from Phaseolus  a c u t i f o l i u s . The same chromatographic method separated a Lb sample from P_. lunatus 'Lima Hendersons' into three peaks (Fig.6). Peaks I and II were chosen for the N-terminal analysis. Both peaks corresponded to the major components determined in CAE (Fig. 4 ) . The r e s u l t s of the protein sequencing showed that the f i r s t 37 amino acids of the N-terminal matched the known amino acid sequence of P_. vulgaris (Lehtovaara and E l l f o l k 1975b). Bean Lbs consist of 145 amino acids and show a close homology to both soybean Lba and Lbc3. The f i r s t Lb exon consists of 32 amino acids i n both species. Four of those i n Lbc3 and 5 of those i n Lba d i f f e r from the bean sequence (Lee and Verma 1984). The major changes were found i n the t h i r d exon. Consequently, i t i s possible that the changes which caused the d i f f e r e n t Lb p r o f i l e s with CAE from P. a c u t i f o l i u s , P. f i l i f o r m i s and P_. lunatus also af-fected mainly the heme cavity rather than the amino termi-nal end. Migration patterns i n CAE can d i f f e r due to a single amino acid change s i m i l a r to human hemoglobin (Chin 1970). Since only small differences can r e s u l t i n changes i n the oxygen a f f i n i t y and these changes would most l i k e l y take place i n the heme cavity, i t i s not surprising that 114 20 FRACTION NUMBER F ig . 6. Elut ion p r o f i l e for P_. lunatus 'Lima Hendersons' Lb components on DE52-cenulose columns. Experimental condit ions are described in sect ion 4 . 3 . 7 . , page 87. 1 1 5 the N-terminal sequences of a l l Lb components investigated in t h i s study are s i m i l a r . 116 V. GENERAL DISCUSSION AND CONCLUSIONS Beans have generally been considered to be poor n i -trogen f i x e r s i n comparison to species such as soybean. Among the explanations for t h i s apparent phenomenon i s the fact that kidney beans contain only one nodule Lb component which might be less e f f e c t i v e i n supporting the nitrogen f i x i n g bacteroids. In contrast, more e f f i c i e n t nitrogen f i x i n g crops l i k e soybeans or peas contain several Lb com-ponents which d i f f e r i n their oxygen a f f i n i t y and i n th e i r e f f i c i e n c y i n supporting nitrogen f i x a t i o n (Fuchsman et a l . 1976, Uheda and Syono 1982a,b). On the basis of their work with soybeans (Fuchsman et a l . 1976) and peas (Uheda and Syono 1982a,b), these authors have concluded that Lb he-terogeneity i s functional and that i t constitutes an adap-ta t i o n for more e f f e c t i v e nitrogen f i x a t i o n . Consequently, the present study was conducted to determine whether such Lb heterogeneity can be achieved i n P_. vulgaris and i f t h i s v a r i a b i l i t y indeed enhanced the nitrogen f i x i n g c a p a b i l i t y of t h i s crop. Leghemoglobin heterogeneity i n P_. vulgaris was ad-dressed using a dual strategy. On the one hand, beans seeds were treated with mutagenic agents (gamma rays and EMS) i n order to induce mutations i n the e x i s t i n g Lb gene(s). From the i r study with kidney beans, Lee and Verma 117 (1984) have concluded that there are four Lb genes present i n the Phaseolus genome. However, the authors could not determine i f only one gene was active, or i f a l l four genes produced an i d e n t i c a l component. About 1 , 4 0 0 M2 plants were tested i n the present study for Lb v a r i a b i l i t y using CAE. No mutation which affected the migration pattern of the Lb protein was detected. If there are indeed four ac-ti v e genes present i n the bean genome, i t should have been possible to detect mutation i n the sample tested. The ab-sence of detectable mutation suggests that only one gene i n the bean genome may be active and the other three are s i -lent or, that the gene i s not highly mutable. It i s l i k e l y that the number of plants screened was too small to detect a low frequency variant, and i f the method (CAE) was not sensitive enough, then mutation induction i s too laborious to consider as an alt e r n a t i v e method for achieving hetero-geneity for plant breeding of t h i s species. On the other hand, plant breeders often screen a genus for a desired t r a i t and use i n t r a - and i n t e r s p e c i f i c hy-b r i d i z a t i o n to transfer such characters into an acceptable crop background. The Phaseolus genus was screened for the presence of ex i s t i n g Lb v a r i a b i l i t y and considerable v a r i -ation for t h i s t r a i t was revealed. P_. a c u t i f o l i u s nodules also contained a single Lb component which migrated to a d i f f e r e n t p o s i t i o n i n comparison to the P_. vulgaris con-118 t r o l , suggesting differences i n the amino acid sequence. Furthermore, nodule extracts of P_. f i l i f o r m i s , a desert species, contained two Lb components, both migrating d i f -f e r e n t l y from P_. vulgar i s . _P. lunatus apparently produced two major and probably three minor Lb components, also showing a d i f f e r e n t electrophoretic p r o f i l e from P_. vulgaris. Thus, the present study constitutes the f i r s t report of Lb heterogeneity occurring within the genus. A further study was i n i t i a t e d to determine i f Lb va-r i a b i l i t y enhanced nitrogen f i x a t i o n i n i n t e r s p e c i f i c crosses. I n t e r s p e c i f i c hybrids were obtained from P.. vulgar i s x P.. a c u t i f o l ius and from P_. vulgar i s x P_. f i l i f o r m i s . Parents and their respective hybrids were ve-get a t i v e l y propagated for evaluation of nitrogen f i x a t i o n c a p a b i l i t y . Two nitrogen f i x a t i o n tests were used to assess the performance of the plants; ARA which gives an estimate of the nitrogen f i x i n g rate at the time of mea-surement and Total N which r e f l e c t s the t o t a l amount of nitrogen fixed over the growth period i n an N-free environ-ment. P_. vulgar i s x P. f i l i f o r m i s hybrids had a higher nitrogen f i x a t i o n rate as well as accumulated s i g n i f i c a n t l y more nitrogen over the growing period than either parent. However, only i n the ARA experiment could t h i s be c l e a r l y attributed to the P_. f i l i f o r m i s parent. In addition, the P_. vulgaris x P_. a c u t i f o l i u s N1602 hybrid performed better 119 than i t s parents i n the ARA experiment but data for the Total N experiment showed no s i g n i f i c a n t difference between parents and the hybrid. Interpretation of the data from the nitrogen f i x a t i o n tests should be made with caution. Comparisons i n t h i s experiment involved d i f f e r e n t species; differences i n per-formance may be confounded by the d i f f e r e n t genetic back-ground of the species involved i n a p a r t i c u l a r hybrid. De-f i n i t i v e tests of the contribution of Lb heterogeneity to nitrogen f i x a t i o n e f f i c i e n c y require the use of isogenic l i n e s , d i f f e r i n g only i n the genes defining the Lb compo-nents. Nevertheless, the within-group comparison of the P. vulgar is x P_. f i l i f o r m i s hybrids and the respective pa-rents, shows transgressive segregation for both ARA and Total N i n the hybrid. It i s tempting to speculate that at least part of the improved f i x a t i o n l e v e l i s a consequence of the heterogeneous Lb pattern i n the hybrid. N-terminal sequencing of one of the P. f i l i f o r m i s components (peak II) and two of the P_. lunatus 'Lima Hendersons' Lbs (peak I and II) showed no differences be-tween the f i r s t 37 amino acids of the three proteins and those of P_. vulgaris. However, since only one amino acid need vary i n order to a f f e c t the migration pattern i n CAE (Chin 1970) , these data are not unexpected and are consis-tent with the other observations of conserved sequences i n 120 Lb (Appleby 1984). Furthermore, i t i s l i k e l y that changes leading to an improvement i n Lb e f f i c i e n c y w i l l be located in the sequences associated with the heme cavity rather than at the N-terminal end. This study has i d e n t i f i e d d i f f e r e n t Lb components i n the genus Phaseolus. However, no tests were made to eval-uate the components for differences i n oxygen a f f i n i t y or to determine whether the molecules are regulated indepen-dently. Independent regulation would be i n accord with most other species studied so far and thus, t h i s point c l e a r l y needs further investigation. Furthermore, funding l i m i t a t i o n s precluded sequence analysis of the three Lb components beyond the 35-40 N-terminal amino acids. Hence, sequencing of a l l Lb mole-cules detected i n t h i s study should be c a r r i e d out to fu r -ther our understanding of sequence changes that may cause d i f f e r e n t oxygen a f f i n i t i e s . The d i f f i c u l t i e s i n obtaining f e r t i l e i n t e r s p e c i f i c hybrids from P_. vulgaris x P. lunatus c l e a r l y l i m i t s the potential transfer of genetic t r a i t s between those species. Even though p l a n t l e t s were obtained from t h i s cross, the plants did not develop beyond the formation of four t r i f o -l i a t e leaves. Leghemoglobin genes have been transferred from soybean to Lotus corniculatus using an Agrobacterium system; the chimeric gene was found to be expressed i n root 121 nodules formed on the transformed Lotus (Jensen et a l . 1986). Hence, t h i s l a t t e r method could be used to transfer Lb genes from lima to kidney beans i f the P_. lunatus Lbs are i d e n t i f i e d to be superior i n their oxygen a f f i n i t y . This procedure would also have the advantage of producing isogenic l i n e s without further backcrossing. Testing Lbs from a single nodule i s also an easy method to determine i f hybrids obtained from i n t e r s p e c i f i c crossing are, indeed, hybrid. Thus, simple electrophoretic analysis could have resolved the doubts expressed by Smartt (1979) about plants obtained by i n t e r s p e c i f i c h y b r idization of P_. vulgar i s x P. lunatus i n experiments by Honma and Heckt (1959) . An inte r e s t i n g question i s , how Lb v a r i a b i l i t y arose i n the genus Phaseolus from an evolutionary or geneological perspective. Did P_. vulgar is and P_. coccineus lose their Lb v a r i a b i l i t y after the i n i t i a l speciation events, or did other Phaseolus species only develop Lb heterogeneity f o l -lowing speciation? In the l a t t e r case, Lb heterogeneity must have been a r e l a t i v e recent evolutionary event. In t h i s context, i t i s also i n t e r e s t i n g to note that P_. microcarpus which has been described as more cl o s e l y r e l a -ted to P_. lunatus than P. vulgar i s (Sullivan and Freytag 1986) contains only one Lb component which migrates to the same position i n CAE as P. vulgar i s . Based on the s i m i l a -122 r i t i e s of their Lb p r o f i l e s , one might speculate that P_. microcarpus i s more clos e l y related to P_. vulgaris and P.  coccineus rather than _P. lunatus. However, since crossing data are not available, t h i s apparent contradiction cannot be resolved at present. Only r e l a t i v e l y few species i n the genus Phaseolus have been screened for Lb heterogeneity i n t h i s study. A further analysis of the species i n the genus might reveal more v a r i a t i o n and thus further our understanding of the evolution of leghemoglobins genes. In addition, t h i s i n -v e s t i g a t i o n might also resolve the controversy about the phylogenetic r e l a t i o n s h i p of some species which cannot be c l e a r l y attributed to either the genus Phaseolus or the genus Vigna on the basis of their morphological t r a i t s (Briicher 1977) . In conclusion, although Lb heterogeneity i s not e v i -dent i n common c u l t i v a r s of P_. vulgar i s , i t does occur in the genus Phaseolus and thus, these findings are consistent with the general theory that Lb heterogeneity i s functional and that i t constitutes an adaptation for more e f f e c t i v e nitrogen f i x a t i o n . If the Lb components found i n the genus are shown to have d i f f e r e n t oxygen a f f i n i t i e s which are r e f l e c t e d i n more e f f e c t i v e nitrogen f i x a t i o n , then any P_. vulgaris breeding program should u t i l i z e i n t e r s p e c i f i c hy-b r i d i z a t i o n or gene transfer and s e l e c t i o n for t h i s t r a i t . 123 VI. SUMMARY AND CONCLUSIONS Phaseolus vulgar i s seeds were mutagenized using gamma rays and ethyl methane sulfonate. Among 1400 M2 offsprings derived from the treated plants, no mutation i n the legemo-globin (Lb) gene was detected. The i n a b i l i t y to induce a mutation i n t h i s locus suggests that leghemoglobin c o n s t i -tutes a strongly conserved protein. F i f t y accessions were screened for the presence of exis t i n g Lb v a r i a b i l i t y i n the genus Phaseolus. Heteroe-neity was evident i n some of the species investigated. Therefore, the single Lb component found i n P. vulgaris constitutes an exception among the nitrogen f i x i n g species. I n t e r s p e c i f i c hybrids were produced i n order to trans-fer the ex i s t i n g Lb v a r i a b i l i t y into P. vulgar i s and to de-termine i f t h i s heterogeneity indeed enhances nitrogen f i x a t i o n . S i g n i f i c a n t differences i n the nitrogen f i x i n g c a p a b i l i t i e s of the d i f f e r e n t hybrids were determined. 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