Leghemoglobins in the Genus Phaseolus and Their Significance for Nitrogen Fixation

LEGHEMOGLOBINS IN THE GENUS PHASEOLUS AND THEIR SIGNIFICANCE FOR NITROGEN FIXATION

Monika Magdalena Liilsdorf

Ing. Agrar., Universitat Bonn, Germany, F.R., 1982 M.Sc, The University of Manitoba, 1985

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF

THE REQUIREMENTS. FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

THE FACULTY OF GRADUATE STUDIES

(DEPARTMENT OF PLANT SCIENCE)

We accept this thesis as conforming to the required standard

THE UNIVERSITY OF BRITISH COLUMBIA

APRIL 1989

(c)Monika Magdalena Lulsdorf, 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) are important soluble proteins in the legume root nodule formed in response to infection by soil Rhizobium bacteria. With the exception of Phaseolus vulgar is, Lbs generally show structural and functional heterogeneity for their oxygen-binding function in the nodule.

P_. vulgar is seeds 'Contender' were mutagenized with gamma-rays or ethyl methane sulfonate. Among 1400 M2 off• spring derived from the treated plants screened for Lb va• riability, no mutation in Lb was detected. These observa• tions are consistent with a single active gene for Lb in

Phaseolus.

Fifty accessions from the genus Phaseolus were screened for Lb variation. P_. acutifolius ssp. contained a single Lb component which had a different electrophoretic mobility compared to that in P_. vulgar is. P_. f ilif ormis showed two Lb bands, both different from P_. vulgaris, while

P. lunatus was found to contain two major and probably three minor Lb components. These data represent the first report of intra-generic Lb variability in Phaseolus.

Using embryo rescue technique, hybrids were produced from _P. vulgar is x P. acutif ol ius ssp. and P. vulgar is x P.. filiformis crosses. Rooted cuttings from each hybrid and

i i its parents were used to estimate the nitrogen fixation

(acetylene reduction) rate and to determine the total amount of nitrogen accumulated during the growth period.

The P_. vulgaris x P_. filiformis hybrids had a significantly higher nitrogen fixation rate and accumulated more nitrogen than either parent. The P_. filiformis parent performed better than the P_. vulgar is parent but only in the acetylene reduction test. One of the P_. vulgaris x P_. acutifolius hybrids also fixed nitrogen at a higher rate than either parent.

Leghemoglobin components from a P_. filiformis hybrid and from P.. lunatus 'Lima Hendersons* were isolated by cellulose ion-exchange chromatography. The N-terminal amino acid sequence for peak II of the P. filiformis hybrid and peaks I and II from P_. lunatus nodules was determined. The first 37 amino acids of each component were identical to the published sequence for P_. vulgaris. Mobility and/or functional differences are likely to be found in the se• quence nearer the location Of the heme cavity. The data.of this investigation support the contention that Lb hetero• geneity is functional and constitutes an adaptation for more effective nitrogen fixation. TABLES OF CONTENTS

ABSTRACT . . ii

LIST OF TABLES vi

LIST OF FIGURES vii

LIST OF ABBREVIATIONS viii

GLOSSARY ix

ACKNOWLEDGEMENTS x

Chapter Page

I. GENERAL INTRODUCTION 1

II. 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

III. 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

VI. SUMMARY AND CONCLUSIONS 124

LIST OF REFERENCES ' 125

.V LIST OF TABLES

Table Page

I. Phaseolus germplasm accessions 77

II. , Grouping of 'Plant Species' for analysis 99

III. Analysis of variance for ARA of P. vulgaris, P_. acutifolius, P_. filiforalis and inter• specific hybrids. Additional analysis for the partitioned sums of squares for 'Plant Species' is also shown 100

IV. Orthogonal contrasts among groups for the ARA experiment 101 i V. Mean acetylene reduction (nm/plant/hour) for each 'Plant Species' and group measured in the ARA experiment 102

VI. Orthogonal contrasts within groups for the ARA experiment 103

VII. Analysis of variance for Total N in P_. vulgaris, P. acutifolius, P. filiformis and interspecific hybrids. Additional analysis of the partitioned sums of squares for 'Plant Species' is also shown 105

VIII. Orthogonal contrasts among groups for the Total N experiment 106

IX. Means (mg total nitrogen) for each 'Plant Species' and group of the Total N experiment 107

X. Orthogonal contrasts within groups for the Total N experiment involving P_. vulgar is, P_. acutifolius, .P. filiformis and inter• specific hybrids 109

vi LIST OF FIGURES

Figure Page

1. CAE Lb profile showing P_. vulgaris (C) , P.. acutif olius N1602 (A^ ) , and P.. acutifol ius ssp. latif olius (A|_) 89

2. CAE Lb profile showing P_. vulgar is wild (P^) , P_. f iliformis (P p) , P. mi cr ocarpus (P^ ) and P. vulgar is 'Gallatin1 (G) 91

3. CAE Lb profile showing a P_. vulgaris x P_. filiformis hybrid (1), P. acutifolius (2), P. vulgaris x P_. acutifolius hybrid (3) and P_. vulgaris (4) 92

4. CAE Lb profile showing P_. vulgaris (C) , P.. lunatus 'Lima Hendersons' (L), £. lunatus 'Market Sample' (M) and P_. acutifolius (A).. 93

5. Elution profile for P. vulgar is x P. filiformis hybrid Lb components on DE5 2-cellulose columns 113

6. Elution profile for P_. lunatus 'Lima Henderson' Lb components on DE52-cellulose columns .... 115

vii LIST OF ABBREVIATIONS

ADP adenosine diphosphate ALA aminolevulinic acid ARA acetylene reduction assay ATP adenosine triphosphate

CAE cellulose acetate electrophoresis CDNA complementary DNA CI AT Centro Internacional de Agricultura Tropicale

DE diethylaminoethyl- DF degrees of freedom

EMS ethyl methane sulfonate

Hb hemoglobin

IAEA International Atomic Energy Agency IEF isoelectric focusing

Lb leghemoglobin

Ml mutant generation one MS mean squares

RCBD randomized complete block design

TDM total dry matter Ti tumor inducing Tn transposon

.vi i i GLOSSARY

Rhizobium is a generic name and refers to the genus of bacteria known as Rhizobium. The plural Rhizobia refers to more than one cell of Rhizobium, and rhizobial is an adjective describing some attribute of Rhizobium.

Bacteroid This term is used to describe the nitrogen fixing form of Rhizobia found in root nodules.

Peribacteroid Membrane or membrane envelope refers to the plant membrane surrounding each bacteroid or group of bacteroids in nitrogen fixing root nodules.

Peribacteroid Space refers to the space between the peribacteroid membrane and its contained bacteroids.

Periplasmic Space refers to the space between the bacteroid inner (plasma) and outer (cell wall) membrane.

Leghemoglobin (Lb) refers to the oxygen-carrying hemeprotein found in legume root nodules. Oxy-Lb is the oxygenated form, deoxy-Lb refers to the deoxygenated form.

Hemoglobin (Hb) is a general term for hemeproteins regardless of animal or plant origin.

Accession refers to a sample in a gene bank; its number is unique.

Cultivar refers to a cultivated variety.

Ml generation refers to the first mutated generation, i.e. plants grown from the treated seeds.

Nitrogen refers to the gaseous element N2; should be correctly termed 'dinitrogen'.

Total N refers to the total amount of nitrogen fixed.

(References: Appleby 1984, Date 1978, Summerfield 1988.)

.ix ACKNOWLEDGEMENT

I would like to thank Dr. Galway from Cambridge

University, U.K., and Dr. Michaels from the University of

Guelph, Canada, for the plant material.

Furthermore, I wish to thank my thesis supervisor Dr.

F.B. Holl for his guidance and support throughout this study. Thanks are extended to my committee members, Dr.

A.J. Griffiths, Dr. V.C. Runeckles, Dr. M. Upadhyaya, and my external examiner Dr. D.P.S. Verma for their time and effort for reviewing this research.

I also wish to thank Dr. G. Eaton for his assistance with the statistical analysis of this experiment.

Special thanks to my husband, Claude Lapointe, for his support and encouragement without which I might never have finished this project.

Finally, thanks to all those friends who made me laugh and thus made the situation a bit more bearable.

x I. GENERAL INTRODUCTION

The successful establishment of nitrogen fixing no•

dules relies on a complex sequence of physiological pro•

cesses, many of which involve interaction between the as• sociated Rhizobium bacterium and its host plant. A cha•

racteristic of this symbiotic process is the presence of a red pigment which was first shown to be a hemeprotein by

Kubo in 1939. He proposed that the physiological signifi• cance of this protein could relate to the transport and

storage of oxygen similar to the monomeric myoglobin.

These findings were later confirmed by Keilin 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 is synthesized by the bacteroids, and a globin component which is encoded by the host plant and

synthesized on plant ribosomes (Bergersen 1977, Ellfolk

1972, Verma and Bal 1976).

Globins are widely distributed in nature and occur in many different 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 in root nodules of

nitrogen fixing non-legumes (Appleby et al. 198 3a, Goodman

et al. 1988, Hunt et al. 1978, Magnum 1976, Terwilliger

1980).

Recently, hemoglobins have also been found in root

nodules formed by" the actinorrhizal Frankia endophyte with

genera as diverse as Alnus, Casuarina, Ceanothus,

Comptonia, Eleagnus and Myrica (Roberts et al. 1985,

Tjepkema 1983). In addition, Landsmann et al. (1986) re•

ported that Parasponia Lb complementary DNA (cDNA) hybri•

dizes to sequences in Trema, a species which is related to

Parasponia but non-nodulating. Hattori and Johnson (1985)

also detected cross-hybridizing DNA sequences in Betula and

Ceratonia, neither of which fix nitrogen. Consequently,

leghemoglobins or closely related proteins seem to be widespread in the plant kingdom and may be present and

functioning in some as yet undiscovered capacity in all plants (Roberts et al. 1985).

All nitrogen fixing symbioses involving the bacterium

Rhizobium require the presence of leghemoglobin since mu•

tants deficient in hemoglobin synthesis are ineffective in nitrogen fixation (Fuller and Verma 1984, Lang-Unnasch et al. 1985).

The establishment of a symbiotic nitrogen fixing re-

2 lationship as reviewed by Sprent (1979) and Bergersen

(1982), begins with the release of plant compounds from the germinating root into the soil. These compounds attract

the associated rhizobia bacteria and induce them to multi• ply and thus lead to a build-up of a bacterial population within the rhizosphere.

The first obvious symptom of plant infection is usually observed as a curling of the root hair due to the release of a bacterial product (Yao and Vincent 1969).

Sprent (1979) has suggested that curling might delimit the area where essential substances accumulate or that entry holes made in the crook of a curl may be more easily plug• ged to prevent entry of pathogens.

Once the bacteria are properly aligned and bound to the root hair, they enter by localized digestion of the cell wall (Callaham and Torrey 1981) . The infection pro• ceeds with the formation of infection threads which consist of rows of dividing bacteria enclosed by a host membrane derived from the plasmalemma. The plant root reacts to bacterial synthesized auxins with cell enlargement and di• vision which reflects the initiation of nodule formation.

The thread continues to grow between and through the cortical cells until it reaches the inner cortex (Sprent

1979, Bergersen 1982, Sprent and Minchin 1985). There, the bacteria are released into the plant cell cytoplasm but

3 they remain enclosed, singly or in groups, within the host

membrane, also called the peribacteroid membrane (Bergersen

and Goodchild 1973a).

The next step in a successful symbiosis involves the

enlargement and differentiation of the bacteria into bac•

teroids. At this latter stage, they are capable of fixing

atmospheric nitrogen into ammonia, which is then released

into the host cytoplasm and incorporated into plant pro•

teins via the glutamine/glutamate pathway (Schubert 1986,

Sprent 1979).

The actual process of nitrogen fixation is catalyzed

by the enzyme nitrogenase which consists of two complex

subunits. The process of reducing nitrogen requires con•

siderable amounts of energy in the form of ATP, which is

produced by nodule respiration coupled to oxidative phos• phorylation with oxygen as the terminal electron acceptor

(Appleby et al. 1975). However, the occurrence of the ne• cessary amounts of free oxygen in the cell would irrever•

sibly inactivate the oxygen-sensitive nitrogenase enzyme complex. Hence, nitrogen fixing plants have solved this problem through the production of restrictive structures which limit oxygen diffusion into the nodule (Minchin et

al. 1985, Sheehy et al. 1985, Hunt et al. 1988) and by the

synthesis of leghemoglobins which function by facilitating oxygen transport and thus distribution (Wittenberg et al.

4 1974) . Consequently, a rapid flux of oxygen is maintained throughout the nodule, sufficient for ATP production, but maintaining a concentration of free oxygen sufficiently low that the nitrogenase complex is not affected (Appleby et al. 1975, Bergersen and Turner 1975a,b, Wittenberg et al.

1974).

Phaseolus vulgar is L. (kidney bean) has traditionally been regarded as a poor nitrogen fixer in comparison to other species such as Vicia faba L. (broad bean) and

Glycine max L. (Merr.) (soybean), especially during the important late pod-filling stage, although this view may reflect the absence of specific selection for this trait

(Graham 1981, Rennie and Kemp 1983). Breeding for improved nitrogen fixation has been focused on three targets: the bacteria, the host, or coincident selection of both.

Rhizobium strains differ in traits such as the time required for nodule development, cold and nitrogen ferti• lizer tolerance, nitrogenase activity, and the presence of an uptake hydrogenase system which recovers some of the energy lost due to hydrogen evolution (Attewell and Bliss

1985, Barnes et al. 1984, Hardarson et al. 1984, Ligero et al. 1987, Rennie and Kemp 1981) . Consequently, one or a combination of these traits could be used to select im• proved strains. However, the major problem of this strate• gy is the difficulty in establishing a particular Rhizobium

5 strain in the soil where indigenous strains are already established (Johnson et al. 1965, Roughley et al. 1983,

Weaver and Frederick 1974). In addition, even if this problem can be overcome successfully, the introduced strain may not necessarily make up the majority of the nodules

(Caldwell and Vest 1970, Vest et al. 1973).

The same problems apply to coincident selection of host and microbe. Results have indeed shown that some cultivars fix considerably more nitrogen with one or few strains (Heichel 1982, Rennie and Kemp 1983, Mytton and

Livesey 1983). However, in addition to difficulties in establishing specific strains, Mytton et al. (1984) ob• served that non-additive interactions can account for over

70% of the total variation and thus make the phenotypic expression of nitrogen fixation subject to substantial and unpredictable variation when different Rhizobium genotypes nodulate a plant.

Given these impediments, a promising target for im• proving nitrogen fixation constitutes the host itself.

Because of the large amounts of energy required for nitro• gen fixation, it has been proposed that the supply of car• bohydrates constitutes an important limiting factor to no• dule function (Brunner and Zapata 1984, Smith et al. 1980).

Hence, selection 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• tion (Attewell and Bliss 1985, Barnes et al. 1984, Graham and Temple 1984, McFerson et al. 1981).

However, some authors have pointed out that nitrogen fixation might also be oxygen-limited (Denison et al. 1988,

Suganuma et al. 1987, Tajima et al. 1986). Tajima et al.

(1986) and Denison et al. (1988) proposed that oxygen sup• ply may be a more limiting factor for nitrogen fixation than the demand of energy. Furthermore, a correlation has been observed between the ability of nodules to fix nitro• gen and their Lb content, suggesting that this hemeprotein plays a fundamental role in nitrogen fixation (Appleby

1984, Wittenberg et. al 1974). The importance of leghemoglobin may also be reflected in the observation that up to

40% of the total soluble protein of the nodule is Lb (Nash and Schulman 1976).

In addition, Lb genes are inherited codominantly and are stably expressed (Holl et al. 1983) in contrast to many quantitatively inherited nitrogen fixing traits which often show low heritability and large environmental interactions

(Mytton et al. 1984). Thus, leghemoglobin because of its importance in the nodule would seem to be an obvious target for selection in breeding for enhanced nitrogen fixation of

P_. vulgar is.

7 Many structural genes occur in multiple copies in the genome of higher eukaryotes. If such a group of genes descended from a common ancestral gene, then they consti• tute a gene family (Lewin 1985). The members of such a family may be clustered on the same or dispersed on dif• ferent chromosomes, or a combination of both. If the co• pies are (almost) identical, then they usually provide a source of increased (amplified) gene products. However, if the genes have diverged during evolution, then gene fami• lies can provide slightly different proteins for particular circumstances (Lewin 1985, Suzuki et al. 1986).

Hemoglobin, myoglobin and leghemoglobin constitute such a globin "super-family", derived from a common ances• tral gene (Lewin 1985). With the exception of myoglobin, which is represented only by a single gene in the human genome, hemoglobins and leghemoglobins usually occur in multiple copies (Lewin 1985, Suzuki et al. 1986).

An excellent example of a globin gene family is the leghemoglobin genes in soybean. Lee and Verma (1984) identified four loci containing four functional, two pseudo and two truncated leghemoglobin genes. Proteins coded by the four functional genes differ slightly in their amino acid sequence, the homology being over 90%. The functional genes encode four distinct products which can be separated by isoelectric focusing into four major (Lba, Lbc^, Lbc£,

8 Lbc3) and four minor components, the latter being posttranslational modifications of the major components

(Fuchsman and Appleby 1979, Whittaker et al. 1979).

In contrast, P_. vulgaris was shown to contain four globin genes which might be arranged in one locus but are not closely linked (Lee and Verma 1984). However, if leghemoglobin is extracted from common bean nodules and sepa• rated by cellulose acetate electrophoresis (CAE) or iso• electric focusing (IEF), then only one major Lb component

(Lba) can be detected (Lehtovaara and Ellfolk 1975a). This observation raises the question of whether only a single gene is expressed and the other three are silent, or whether all four genes encode a single component?

There is evidence to support expression of a single gene in the structural details available; the complete amino acid sequence of P_. vulgaris Lba has 85% identity with G_. max Lbc^ and 79% identity with G_. max Lba indi• cating their close evolutionary relationship (Lehtovaara and Ellfolk 1975b, Lee and Verma 1984). In addition, the intragenic structure of the P_. vulgar is globin gene shows the same intron/exon arrangement as that of the G_. max leghemoglobin genes, including extensive sequence homology in both coding, and 5' and 3' non-coding regions (Lee and

Verma 1984) .

Experimental evidence suggests that the Lbc^gene of

9 soybean is activated before the Lba gene and that Lbc^ is activated after the Lbc3 gene (Fuchsman and Appleby 1979,

Verma et al. 1983). The amount of Lbc in young nodules is five to eight times as great as the amount of Lba. This ratio changes when the nodules mature to about 1.5:1

(Fuchsman and Appleby 1979). The genes are apparently transcribed in reverse order of their arrangement on the chromosome and their rate of synthesis is regulated sepa• rately indicating that leghemoglobin heterogeneity is functional (Marcker et al. 1984, Jensen et al. 1988, Verma et al. 1979) .

Soybean Lba was found to have a higher oxygen-binding capacity than Lbc and is, therefore, more effective in oxygen transport. The relative increase in Lba content might be necessary for the regulation of oxygen concentra• tion when the nodule structure becomes more complex with age (Fuchsman and Appleby 197 9) .

Fuchsman and Palmer (1985) have studied Lbs from a genetically diverse selection of cultivated and wild soy• beans. They concluded that the conservation of both leg• hemoglobin heterogeneity and also all four major globin structures provided strong circumstantial evidence that this heterogeneity is 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 is an evolutionary adaptation for more

effective nitrogen fixation".

Leghemoglobin variability is widespread among nitrogen

fixing plant genera e.g. Glycine, Lathyrus, Lens, Lupinus,

Medicago, Ornithopus, Pisum, Sesbania, and Tr ifolium

(Cutting and Schulman 1971, Dilworth 1969, Holl et al.

1983, Kortt et al. 1987, Kuhse and Punier 1987, Richardson

et al. 1975). Thus, P. vulgaris and its relative P_.

coccineus, which contain only a single globin component each, seem to be rare among the nitrogen fixing species.

If, indeed, Lb heterogeneity is an evolutionary adap•

tation for more effective nitrogen fixation and this capa•

bility could be transferred to P_. vulgaris, one might an•

ticipate the development of a more efficient nitrogen fi•

xing bean species. Such a modified plant type might be

expected to have a higher fertilizer-use efficiency and/or

to be more cost efficient in nitrogen fertilizer use.

Therefore, this study was conducted to assess the

possibility of introducing leghemoglobin heterogeneity into

Phaseolus vulgar is and to evaluate such changes as a means

to improve the nitrogen fixation efficiency of this

species. The specific objectives of this investigation were:

11 To introduce or create variability for leghemoglobins in Phaseolus vulgar is.

To determine whether leghemoglobin variability introduced into Phaseolus vulgar is enhances the nitrogen fixation capability of this crop species.

12 II. GENERAL LITERATURE REVIEW

2.1. HEME

The prosthetic group of bean leghemoglobin consists

of protoheme IX (Lehtovaara and Ellfolk 1975a). The iron

atom is in a trivalent state and coupled to the nitrogen

atoms of the four pyrrole rings which lie in a plane around

it. The other two bonds of the iron are perpendicular to

and on either side of the heme. The fifth ligand is bound

to an imidazole side chain of histidine of the globin chain

and the sixth position is available for binding oxygen '•' •T

(Ellfolk 1972, White et al. 1973).

In the nodule, the iron is maintained in a physiolo•

gically active ferrous form due to the presence of reduc•

tases which probably originate in the periplasmic space

between the bacteroid plasmamembrane and the plant cell wall (Appleby 1984, Rigaud 1983).

The biosynthetic pathway for protoheme IX formation

involves the aminolevulinic acid (ALA) synthetase pathway

and was initially postulated to take place in the bacte• roids (Tait 1968), a proposal which was confirmed by

O'Brian et al. (1987). The authors obtained a transposon-

induced Bradyrhizobium mutant, deficient in protoporphyri-

nogen oxidase activity which is needed for the penultimate

13 step in heme biosynthesis. This mutant induced nodules on soybeans which lacked leghemoglobin. However, experiments by Guerinot and Chelm (1986) with a heme mutation in the first step of the ALA synthesis pathway showed that part or all of the heme synthesis can also be carried out by the host plant and hence the authors proposed that there may be a coordinated effort of plant and bacteria to produce the necessary heme.

Even though the bacteroids have now been identified 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 is inserted into protoheme IX by a bacteroid ferrochelatase (Porra 1975). The heme moiety lies within a hydrophobic cavity created by the globin chain. Protoheme

IX is structurally the same in most plant or animals and thus, differences in affinity for oxygen and other charac• teristics of the hemoglobins are attributable to variation in the amino acid composition of the globin chain (White et al. 1973). Such differences would be consistent with the suggestion that Lb heterogeneity may have functional sig• nificance (Fuchsman and Palmer 1985) .

14 2.2. GLOBIN

In addition to the heme group, leghemoglobins are comprised of a globin apoprotein. Phaseolus vulgar is Lba has a molecular weight of 16,900 daltons and consists of one polypeptide chain with 145 amino acids (Lehtovaara and

Ellfolk 1975a).

With the exception of pea, alfalfa, lupin, and broad bean, no other leghemoglobins have been found to include methionine (Ellfolk 1972, Dilworth and Appleby 1979).

Cysteine has not been reported in any leghemoglobin, ana• logous to mammalian myoglobins but not hemoglobins (Hunt et al. 1978). Usually, myoglobins and hemoglobins have a gly• cine or valine residue as N-terminal amino acids; kidney bean Lb contains glycine (Lehtovaara and Ellfolk 1975b).

Like Glycine max, P_. vulgar is contains two histidines in position 61 and 92. The histidine in position 61 is most likely the fifth ligand to heme (Appleby 1974).

Ellfolk (1972) pointed out that prolines are situated close to points where the globin chain is folded and are thus important for the tertiary structure of the protein.

Hence, the author proposed that leghemoglobins are folded similarly to vertebrate globin chains. Four out of five prolines are in the same position in P_. vulgaris and G. max.

15 Ollis et al. (1983) observed similar structures for soybean Lba and other monomeric hemoglobins except that the

D-helix is missing in leghemoglobins. Consequently, leg- hemoproteins consist only of seven helical 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, Ellfolk 1972, Lehtovaara and Ellfolk

1975a,b). Appleby (1974) speculated that the E-helix might have some ability to move in a vertical direction and thus contribute to its high oxygen affinity in contrast to other hemeproteins. In addition, the hydrophobicity of the heme cavity influences the reversible binding of oxygen to a large extent (Ellfolk 1972).

Lee and Verma (1984) determined that the intragenic structure of bean Lba showed three intervening sequences in the same positions as those in soybean, although they are, however, shorter. These authors also reported the presence of four hybridizing bands indicating the presence of four leghemoglobin genes in the Phaseolus genome. The bean Lb locus, like soybean, contains a common sequence specific to the 5' and 31 end. Since only one copy of each sequence was found in Phaseolus, the authors proposed that the four bean globin genes are arranged in one locus.

16 2.3. BIOSYNTHESIS, ASSEMBLY AND DEGRADATION

Leghemoglobins are only synthesized when plant and

Rhizobium develop in symbiosis. Recently, small amounts of

Lb have been detected in uninfected interstitial cells of

soybean root nodules (VandenBosch and Newcomb 1988). How•

ever, the authors could not determine whether it 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 is formed after the infection. Verma et al. (1979) demonstrated that the initiation of the globin genes does not depend on the appearance of nitrogenase since leghemo• globin was detected six days after infection by in vitro translation of nodule polysomes and thus, prior to the ap• pearance of nitrogenase activity. Furthermore, a transposon

Tn5-induced cytochrome-deficient mutant which could not ca• talyse the penultimate step of the heme biosynthesis path• way still contained apoleghemoglobin (O'Brian et al. 1987).

Thus, the authors concluded that heme is not necessary for apoglobin synthesis. However, O'Brian et al. (1987) did not rule out a regulatory role for a heme precursor.

Marcker et al. (1984) investigated the activation of the soybean leghemoglobin genes and concluded that leghe• moglobins contain two discrete activation sites with which two possible different regulatory molecules interact in a

17 cooperative manner. These results were confirmed by Jensen

et al. (1988) who mapped two distinct regions in the 5'

upstream region of the soybean Lbc^ gene which strongly

bind a nodule specific factor. The initial activation

would then be due to the interaction at one site and a se•

cond interaction at the second site would explain the ob•

served large increase in transcription of the Lbc^ gene

after about 12 days. However, the origin of the initial

activation remains to be elucidated.. It has been suggested

that the activation of leghemoglobin genes is 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 al. 1984).

Since the location of leghemoglobin is still disputed

(see chapter 2.4.), it is not certain where the assembly of heme and globin takes place. Verma et al. (1979) concluded

that since the apoprotein does not have a transit sequence,

it is most likely that the globin remains in the host cy•

toplasm after translation from plant ribosomes and that heme is excreted into the cytoplasm by the bacteroids. In

this scenario, assembly likely takes place in the host no• dule cytoplasm.

Once heme is present, the polypeptide and the prosthetic group can combine spontaneously (Ellfolk and

Sievers 1965). They are rapidly and strongly bound and

18 only low levels of free heme are found in healthy nodules

(Porra 1975).

Legumes with meristematic nodule tissue like faba

beans and lupins, actively synthesize leghemoglobin at one

end of the nodule while it is being degraded at the other end. Nodules lacking meristems like common beans and soy•

beans synthesize leghemoglobin only over a brief period of nodule formation and the concentration remains about the

same until nodule senescence (Dilworth and Appleby 1979,

Dilworth 1980).

Based on pulse-chase experiments with J-^c-labelled proteins, Coventry and Dilworth (1976) estimated that lupin

Lb had an apparent half-life of about 18 days. In con• trast, Bisseling et al. (1980) determined that pea Lb had an average turnover-rate of about two days. The authors used two methods, kinetics of 35s incorporation into pro• tein and chloramphenicol inhibition of bacteroid protein synthesis, which gave similar estimates. A third method,

pulse-chase experiments with 35grj 2-f was found unsuitable since this procedure tended to overestimate half-life of proteins due to re-utilization of released amino acids de• rived from protein catabolism. Thus, leghemoglobin and bacterial proteins seem to have the same turnover rates

(Bisseling et al. 1980) .

In general, degradation of leghemoglobin starts with

19 flowering and its decrease is concomitant with but not correlated to the decrease in the nitrogenase activity

(Klucas 1974, Virtanen and Miettinen 1963). Pladys et al.

(1988) reported that senescence (and nitrate treatment) induced a decrease in the intracellular pH of bean nodules.

This decline in nodule pH was found to stimulate Lb hydro• lysis and oxidation which contributed to depression of nitrogen fixation in ageing nodules of this species.

2.4. INTRACELLULAR LOCATION

Bacteroids are located singly or in groups within membrane-bound vesicles (Bergersen and Briggs 1958,

Goodchild 1977) which arise from the plasmalemma following endocytotic processes during nodule development (Dixon

1967).

In 1949, Smith determined that, in soybean, leghemo• globin was located in the bacteroid-containing cells of the nodule tissue. However, the exact intracellular location of the hemeprotein remains a controversial issue.

One group of researchers has reported that the hemo• globin is located exclusively in the plant cytoplasm of different legumes using various methods. For example,

Verma and Bal (1976) and Verma et al. (1978) isolated in• tact membrane envelopes which contained no material immu- noreactive to leghemoglobin antibodies. The authors con-

20 eluded that Lb was localized in the host cell cytoplasm.

Similarly, when Robertson et al. (1978) isolated complete

membrane-enclosed bacteroids they could find only traces of

leghemoglobin within the membranes; they also concluded

that the protein must be located in the host cytoplasm.

In contrast, it has also been proposed that leghemo•

globin is exclusively located in the peribacteroid space.

Dilworth and Kidby (1968) concluded from electron micros•

copic autoradiography studies using 59pe-labelled

Serradella nodules that leghemoglobin was located in the peribacteroid membrane and thus in direct contact with the

bacteroids. Bergersen and Goodchild (1973a) used oxidized

diaminobenzidine staining to visualize soybean leghemoglo•

bin for electron microscopy. They confirmed that the heme

protein was present in 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 results.

Recently, Robertson et al. (1984) demonstrated that

leghemoglobin can be detected in the plant cytoplasm and

the host nucleus but not in the peribacteroid spaces, using

immuno-gold staining. Since leghemoglobin is not synthe•

sized as a precursor molecule for transport across a mem• brane (Verma et al. 1979) and since the nodule cytosol

21 contains enzymes both for protection of the heme from oxi•

dation and for leghemoglobin reduction (Puppo et al. 1982,

Saari and Klucas 1984), it seems certain that the hemepro-

tein is indeed located in 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, ni•

trogenase activity would be greatly reduced, whereas the

presence of the globin exclusively in the peribacteroid

space had no effect on enzyme activity levels. However,

since the authors observed a discrepancy between the level

of oxygenation required and that observed experimentally by

Appleby (1969c), they suggested that leghemoglobin is

likely present in both cytoplasm and peribacteroid space.

2.5. LEGHEMOGLOBIN FUNCTION

Keilin and Wang (1945) investigated leghemoglobin

spectroscopically and observed that the red pigments in nodules have 'an affinity 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 purified

soybean leghemoglobin were plotted as rectangular hyper•

bolae; such curves would be expected for monomeric hemo• globins without heme-heme interaction (Behlke et al. 1971,

22 Imamura et al. 1972). In 1973, Bergersen and coworkers added leghemoglobin to a bacteroid suspension and found that stimulation of nitrogenase activity was much greater than stimulation of oxygen uptake, suggesting that there may be a more energy-efficient ATP producing pathway.

However, it was Wittenberg et al. (1974) who demons• trated in in vitro 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 ratio, the reduction of acetylene to ethylene

(a measure of the nitrogenase activity), and the incorpo• ration of labelled into ammonium. These authors con• cluded from equilibria and kinetic measurements that oxygen was responsible for the increase in bacteroid phosphoryla• tion and thus nitrogenase activity.

In addition, Wittenberg et al. (1974) also reported that other oxygen carriers such as vertebrate and invertebrate myoglobins and hemoglobins, hemerythrin and hemocyanin could substitute for leghemoglobin in the enhancement of acetylene reduction although proteins with high molecular weight, low affinity or slow oxygen dissociation-rates were less efficient.

Leghemoglobin shows an unusually high oxygen affinity

23 due to a combination of a fast oxygen association-rate constant and a relatively slow dissociation-rate constant

(Imamura et al. 1972, Wittenberg et al. 1972). Thus, this protein facilitates the transport of oxygen to the bacteroids at a rate which is sufficient to allow bacteroid phosphorylation, as reflected in enhanced levels of ATP,

ATP/ADP ratio and nitrogenase activity (Appleby et al.

1975, Wittenberg et al. 1972), but low enough to avoid damage to nitrogenase (Bergersen and Turner 1975a,b).

However, the interpretation of in vitro experiments like those by Wittenberg et al. (1974) and Appleby et al.

(1975) is limited because dissolved oxygen concentrations and fractional oxygenation could not be measured and be• cause leghemoglobin could have been facilitating the dis• tribution of oxygen within the shaken liquid which might have no relevance to its in vivo function (Bergersen 1978,

1982). Thus, Bergersen and Turner (1975a,b) developed a no-gas phase assay in which deoxygenation of Lb was spec- trophotometrically monitored. The authors showed that in contrast to previous results, Lb increased oxygen uptake with increasing oxygen concentration (Bergersen and Turner

1975a). Furthermore, the authors reported that maximum nitrogenase rates occurred in a range of oxygen concentra• tions which supported no activity in the absence of leghe• moglobin. Nitrogenase activity was enhanced because of

24 higher ATP/ADP ratios due to a more efficient respiratory pathway. The dependence of nitrogen fixation on the presence of molecular oxygen (Bergersen and Turner 1975a,b) and studies with the oxidative phosphorylation inhibitor carbonyl cyanid m-chlorophenylhydrazone (Appleby et al.

1975) confirmed that ATP generation is linked to electron transport and not to substrate level phosphorylation.

In the absence of leghemoglobin, bacteroid respiration is restricted due to limited diffusion of free oxygen. The high concentration of leghemoglobin in the nodule buffers the bacteroids against fluctuations in oxygen concentration which may occur through external perturbation or as a con• sequence of fluctuations in the supply of energy yielding substrates (Bergersen 1978). Stokes (1975) also recognized the considerable storage and buffering capacity, and thus the stabilization of delivered oxygen tension, to be an important point of the leghemoglobin function.

The abundance of oxygenated leghemoglobin in the cy• toplasm constitutes a large store of oxygen which would ensure a uniform supply for bacteroid respiration. In ad• dition, the concentration of free oxygen in the cytoplasm will be kept low because of the extraordinary affinity of leghemogobin for oxygen, and thus damage to the nitrogenase protein may be avoided (Wittenberg 1980).

Oxygen enters the nodules via lenticels on their sur-

25 face (Pankhurst and Sprent 1975). From its point of entry,

the oxygen diffuses through the nodule via intracellular

spaces (Bergersen and Goodchild 1973b) with a possible

diffusion barrier in the cortex (Tjepkema and Yocum 1973).

Hunt et al. (1988) developed a model which predicted that a diffusion barrier in form of water plugs was present in the

intracellular spaces of a layer of cells between the inner and outer cortex. The presence of oxygen diffusion bar•

riers together with high respiratory oxygen consumption

keeps the oxygen concentration in infected cells low (Hunt et al. 1988, Minchin et al. 1985, Tjepkema and Yocum 1973,

1974). Sheehy et al. (1985) concluded from their mathema• tical model that, as a consequence of the primary need for nitrogenase protection, nodules function under a degree of oxygen limitation.

Membrane envelopes are thought to be impermeable to leghemoglobin but not to free dissolved oxygen (Bergersen

1982). The results of Bergersen (1980) suggested that a small amount of leghemoglobin present in the envelope space would ensure an even distribution of oxygen to all bacte• roids. The author concluded that in the absence of leghe• moglobin in the envelope space, oxygen would be deficient through much of the space and optimum bacteroid respiration could not be maintained.

In contrast, other researchers have advocated that

26 there is no leghemoglobin present in the envelope space

(see chapter 2.4.). In the latter case, the question ari•

ses of how oxygen is delivered to the bacteroid terminal

oxidases.

The experiment by Bergersen and Turner (1975b) con•

firmed the presence of two different nodule terminal oxi•

dase systems; a high affinity system, sensitive to inhibi•

tion by N-phenylimidazole and a low affinity, inhibitor-

insensitive system. When oxygen was supplied at a stabi•

lized low concentration, the high affinity pathway produced

up to five times greater bacteroid ATP concentrations than

the low affinity oxidase pathway. The authors proposed

that cytochrome P450 was involved in the high affinity

system. However, Inozemtseva et al. (1979) proposed that

cytochrome P450 does not directly receive oxygen from leg• hemoglobin.

Findings reported by Melik-Sarkisan et al. (1982) showed that in order to fix nitrogen, bacteroids require

the presence of the peribacteroid membrane. In contrast, removal of the peribacteroid membrane did not affect the

rate of oxygen consumption by bacteroids and thus must not be involved in bacteroid respiration. They concluded that

the reduction of oxygen is accomplished by electron trans• porters present in the peribacteroid space and not by bacteroid oxidases.

27 It is difficult to comprehend how oxygen is deli•

vered in a regulated manner to the bacteroids. Wittenberg

(1980) suggested that if the Brownian movement of the

bacteroids is fast, then their 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 al. (1989) reported that bacteroids are indeed in close contact with the peribacte-

roid membrane.

Many researchers have attempted to elucidate the structural features which confer upon leghemoglobin its , unusual combination of rapid combination rate with oxygen and a moderately slow dissociation rate and thus its very high oxygen affinity (Appleby 1962, Imamura et al. 1972).

One characteristic of leghemoglobin is its ability to bind bulky ligands such as nicotinic acid, reversibly (Appleby et al. 1973). Peive et al. (1972) suggested that such binding is due to a greater degree of accessibility of the heme pocket. X-ray analysis by Vainshtein (1978) confirmed that the size of the heme pocket in leghemoglobin is larger on the distal side of histidine than in other globins.

Similarly, Ollis et al. (1983) observed an exceptionally large cavity on the distal side of the heme in soybean leghemoglobin. When nicotinic acid is bound, histidine is 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 affinity of leghemoglobin

(Appleby et al. 1980).

Ollis et al. (1983) found that the heme group in soy• bean Lba is closer to the G and H helices and further from the B and C helices than in myoglobin. This shift also causes an increase in the size of the heme pocket on the distal side. The authors also proposed that there may be two routes by which ligands have access to the heme. The first pathway lies between the G helix and the junction of the B and C helices (Gin 101=G3 and Lys 36=B16); the second lies between the distal histidine 61 (E7) and leucine 65

(Ell).

However, Armstrong et al. (1980) argued that heme distortion is not the primary factor leading to rapid re• action with oxygen. They suggested a strong ligand field could lower the energy required for spin pairing in the transition from the high spin deoxy to the low spin oxyleghemoglobin and/or stabilize the complex by decreasing the dissociation rate constant and thus facilitate the re• action of leghemoglobin with oxygen. Appleby et al. ,(1980) also considered the perturbation of the heme electronic structure in conformational changes of the protein as a likely cause of the high oxygen affinity.

29 Laser flash photolysis analysis by Stetzkowski et al.

(1985) showed that the faster association rate in leghemo•

globin compared to myoglobin is due to faster bond forma•

tion. These authors observed that the migration of oxygen

from the solvent to the heme pocket was much faster in

leghemoglobin. Rapid transfer was facilitated by the pre•

sence of solvent molecules in the heme pocket which gained

access via an open channel to the pocket.

However, the question still remains as to the expla•

nation for the large differences observed in the off-rate

constants for leghemoglobin in comparison to hemoglobin or

myoglobin. Appleby et al. (1983a) suggested that the high

affinity of leghemoglobin could be due to protonation of

the imidazole of the distal histidine (his 61) which would

lead to formation of a hydrogen bond with bound oxygen and

thus affect the electronic configuration of the heme. This

conformational change would cause a decrease in the rate of oxygen dissociation but would have no effect on the oxygen

combination rate.

The importance of oxygen, and thus leghemoglobin, for optimum nitrogen fixation activity has been emphasized by

the findings of Suganuma et al. (1987). The results of their study implied that even in the presence of oxygenated leghemoglobin, oxygen-limiting conditions prevail in the nodule. Similarly, Tajima et al. (1986) and Denison et al.

30 (1988) recognized that oxygen supply may be a more impor• tant factor than the demand of energy for nitrogen fixation.

2.6. QUANTITATIVE RELATIONSHIP BETWEEN LEGHEMOGLOBIN

AND NITROGEN FIXATION

The discussion in the previous chapter (2.5.) des• cribed the fundamental role of leghemoglobin in nitrogen fixation. However, does this role imply a quantitative relationship between the amount of leghemoglobin in the nodule and the amount of nitrogen fixed by bacteroids?

Virtanen et al. (1947a,b) and other researchers

(Graham and Parker 1961, LaRue and Child 1979) reported such a correlation between leghemoglobin content and acetylene reduction rate. Similarly, Becana et al. (1986) observed a close relationship between the hemeprotein content and nitrogen fixation activity in alfalfa nodules during the vegetative growth period which followed an exponential curve. These authors suggested that the- functional state of leghemoglobin could have an effect on the decline of nitrogenase activity in ageing nodules.

Patil and Rasal (1985) reported a good correlation between leghemoglobin content and nitrogen fixed at all stages of crop development in chickpea. In contrast,

Koundal et al. (1987) could not confirm this relationship

31 for the chickpea cultivar in their study. They suggested

that the nitrogenase activity was limited by the sugar

content, and hence the energy supply, of the nodule.

In vitro experiments by Wittenberg et al. (1974)

showed an increase in both oxygen uptake and acetylene re•

duction by bacteroid suspensions with increasing concen•

tration 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 in Lb concentration to

limitations of in vitro experiments (as discussed in chap•

ter 2.5.). Increasing Lb concentration above 50 .UM had no

effect on nitrogenase activity at a given oxygen concen•

tration (Bergersen and Turner 1975a).

Troitskaya et al. (1979) measured the content of the hemoglobin in different 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, fruit formation) of each

species. The same authors reported that maximum nitrogen

fixing capacity of the nodules was not correlated with the maximum leghemoglobin content of the different crops.

32 Species which accumulated the largest amount of hemeprotein did not achieve the highest level of nitrogen fixation and vice versa. Furthermore, the time of maximum nitrogen fix• ation rate and maximum leghemoglobin content showed a lack of coincidence with the stage of plant development and was different for different species. For example, in fababeans maximum leghemoglobin content preceded, whereas in soybeans it lagged behind, the period of maximum nitrogen fixation activity. Kidney beans were the poorest nitrogen fixer with only 1 to 1.5% of the activity of soybean nodules although the former species contained the second highest amount of leghemoglobin (Troitskaya et al. 1979).

Nash and Schulman (1976) showed that leghemoglobin content in 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 diffusion pathway. Nash and Schulman (1976), how• ever, pointed out that such compensation is not fully effective and that other factors necessary for maintenance of nitrogenase activity become more limiting.

In summary, variability for leghemoglobin content exists between and within different species. However, the correlation between Lb content and nitrogen fixation is not simple or linear. One might speculate that at least part of

3 3 the effect which confounds this relationship could be at• tributed to differences in oxygen affinity of Lb components from different species, e.g. a species containing highly effective Lb components might not need as much Lb as an• other species with less effective components.

2.7. LEGHEMOGLOBIN HETEROGENEITY

Leghemoglobin variability is widespread among nitrogen fixing species with very few exceptions (Appleby 1974,

1984). For example, alfalfa (Medicago sativa L.) and

Lathyrus contain four and Lupinus polyphyllus produces three leghemoglobin components (Holl et al. 1983).

Tr ifolium subterraneum was separated by ion exchange chro• matography into four distinct components (Holl et al. 1983,

Thulborn et al. 1979).

The most frequently studied loci in soybean show similar heterogeneity for the leghemoglobin gene family.

The four soybean leghemoglobin genes encode four distinct gene products which can be separated by isoelectric 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 arise from the major components by cleavage of the N-terminal valyl residue and subsequent acetylation of the ex-

34 posed alanyl residue and are thus post-translational modi• fications of the major isomers (Whittaker et al. 1979).

Experimental evidence suggests that the Lbc^ gene is activated before the Lbc^ gene and that Lbc2 is activated at about the same time as the other two Lbc genes (Marcker et al. 1984). Thus, during the very early stages of nodule development only Lbc type mRNAs are synthesized. After a few more days, the Lba gene is activated. Consequently, soybean leghemoglobin genes are activated in reverse order of their chromosomal arrangement (Marcker et al. 1984).

The amount of Lbc in young nodules is more than seven times as great as the amount of Lba. This ratio changes when the nodules mature, to less than two for three-week old nodules due to the increase in biosynthesis of Lba

(Fuchsman and Appleby 1979, Fuchsman et al. 1976, Verma et al. 1979, 1983). The rate of synthesis of each Lb compo• nent is regulated separately from the others, indicating that leghemoglobin heterogeneity is functional (Marcker et al. 1984, Fuchsman and Appleby 1979).

Lba was found to have a higher oxygen binding capacity than Lbc and is, therefore, more effective in enhancing nitrogen fixation and oxygen consumption. The relative increase in Lba content might be required for the regula• tion 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 genetically diverse selection of cultivated and wild

soybeans by isoelectric focusing. They concluded that the

conservation of both leghemoglobin heterogeneity and also

all four major leghemoglobin structures in an otherwise

diverse population provides strong circumstantial evidence

that leghemoglobin variability is functional and that this

heterogeneity is an evolutionary adaptation for more ef•

fective nitrogen fixation.

Uheda and SyOno (1982a) detected two major (Lbl and

LblV) and three minor (Lbll, Lblll, and LbV) leghemoglobin

components in pea using disc gel electrophoresis. In young

nodules, Lbl is synthesised at a higher rate than LblV

whereas in older nodules, LblV is predominantly produced.

Thus, the ratio of the two major components (I/IV) de•

creased with increasing age. Similar to soybeans, the

component which is synthesized at a higher rate in older

nodules (LblV) also has a higher oxygen binding affinity.

Hence, since low oxygen tension seems to prevail in older

nodules, the higher oxygen binding capacity of LblV would

result in a better capacity for oxygen transport. The

authors suggested that changes in the relative amounts of

the leghemoglobin components during nodule development

contribute to more effective nitrogen fixation which is

36 mediated by changes in the capacities for oxygen transport within the nodule (Uheda and Syono 1982b).

Holl et al. (1983) described a line of Tr ifolium subterranean which contained only three leghemoglobin com• ponents; Lbl, a major component of all other T. subterranean cultivars, was missing. The amount of Lbll was approximately twice that usually found in nodules, suggesting a compensating response for the lack of Lbl or a reflection of mutation in the Lbl component which caused it to coelute with Lbll in chromatographic and electrophoretic separations. Although differences in nitrogen fixation were observed, the widely different phenological patterns of the three- and four-banded lines precluded any definite comparisons.

Lupins contain two major (Lbl and Lbll) and one minor

(Lblll) component, the latter being a posttranslational modification of Lbl (Szybiak-Strozycka et al. 1987). In early nodule development, Lbl was predominant whereas in later stages Lbll prevailed (Sikorski et al. 1986). The oxygen affinity for each component is not known; however, the authors proposed that leghemoglobin variability has a physiological role.

Sesbania rostrata is a nitrogen fixing legume with both stem and root nodules. Stem nodules contain photo- synthesizing chloroplasts with potential for oxygen evolu-

37 tion adjacent to bacteroid-containing tissue (Dreyfus and

Dommergues 1981). Anion exchange chromatography demons•

trated that there are seven major leghemoglobin components

in both root and stem nodules although in different pro•

portions. At least six of these components are separate

gene products (Bogusz et al. 1987).

Twelve days after inoculation, Sesbania stem nodules contain about equal amounts of Lb2, Lb3, and Lb5 while Lb4 predominates and Lbl is barely detectable. This relation• ship changes in mature nodules such that Lb2 and Lb5 are predominant, Lb3 and Lb4 are moderately abundant and Lbl is

a minor component (Lajudie and Huguet 1986). Bogusz et al.

(1987) found that the amount of Lb2 was highest in both stem and root nodules about 20 to 30 days after inocula•

tion. Decreasing relative amounts were detected for Lb2,

Lb7, Lb5, Lb6, Lb4, Lbl and Lb3 in stem nodules and for

Lb2, Lb7, Lb3, Lb5, Lb4, Lbl and Lb6 in root nodules.

The predominant Sesbania Lb2 component had the highest oxygen affinity yet recorded for any leghemoglobin as de• termined by Wittenberg et al. (1985). The concentration of free oxygen in the nodule was extremely low. The associa• tion of the high oxygen affinity with the predominant com• ponent is consistent with observations for soybean and pea leghemoglobins.

Kortt et al. (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 similar indicating that very small changes must be responsible for the higher oxygen affinity.

Furthermore, Fleming et al. (1987) also reported he• moglobin heterogeneity for Casuarina glauca, a non-legume which forms root nodules in association with the actino- mycete Frankia. Isoelectric focusing separated the hemo• globin into three major and multiple minor components, re• flecting a multigene family. Molecular weights were simi• lar to other plant globins and since these hemproteins can bind oxygen reversibly and have a high oxygen affinity, it is likely that hemoglobin in Casuarina is functionally active in nitrogen fixation.

An exception to leghemoglobin heterogeneity is

Parasponia, a member of the Ulmaceae which forms nodules with Rhizobium. However, it contains an unusual dimeric hemoglobin, the only one known in plants (Appleby et al.

1983b, Landsmann et al. 1986). Kinetic measurements re• vealed sufficiently fast oxygen association and dissocia• tion rate constants and extremely high oxygen affinity to allow Parasponia hemoglobin to function similarly to known leghemoglobins.

In conclusion, the literature has shown considerable variation for leghemoglobin in almost all species examined.

39 Hence, it is surprising and noteworthy that _P_. vulgar is,

which is so closely related to G_. max, contains only one

component.

2.8. EVOLUTION OF THE GLOBIN GENES

The origin of globin genes in nitrogen fixing plants

is disputed. Three possible sources have been suggested

from which leghemoglobins might have evolved: a transfer

from Rhizobium, a viral vector, or the plant itself.

Appleby (1969a) reported that Bradyrhizobium contain a

pigment with the characteristics of a hemoglobin. Thus,

Broughton and Dilworth (1973) proposed that a primitive -

hemoglobin was transferred from Rhizobium to the host plant

similar to the natural Agrobacterium Ti plasmid system

which then evolved into an efficient oxygen carrier.

However, leghemoglobin genes contain introns (Lee and

Verma 1984, Hyldig-Nielson et al. 1982) whereas prokaryotic

genes do not (Lewin 1985) and thus, a bacterial origin for

the globin gene is unlikely. Furthermore, Appleby (1969b)

pointed out that the bacteroid hemeprotein he discovered

was unrelated to leghemoglobin.

The second theory by Jeffreys (cited in Lewin 1981)

suggests that globin genes were transferred by an insect-

borne plant pathogenic virus to an ancestral legume.

Nucleotide substitution experiments by Brown et al. (1984)

40 and X-ray crystallographic studies by Vainshtein et al.

(1975) estimate the time of divergence between plant and animal globin genes to about 900 and 1400 million years.

Goodman et al. (1975, 1988) placed the duplication of the globin gene after the emergence of the vertebrates about

680 million years ago. Since it seems unlikely that the globin genes of a donor would have diverged prior to this , time, this would exclude the possibility of transfer from an insect donor.

Further evidence against such a horizontal transfer comes from the fact that animal globin genes contain only two introns unlike leghemoglobins which possess three (Go

1981). Verma et al. (1983) observed that exon fusion must have occurred before the duplication of the globin genes, about 500 million years ago. Thus, the authors reasoned that a vectoral transfer occurring long before the emer• gence of legumes (about 140 million years ago) seems un• likely.

In addition, Lee et al. (1983) argue that if such a horizontal gene transfer has taken place, then leghemoglo• bins should show a close correlation to one particular do• nor. However, analysis of 245 globin genes by Goodman et al.(1988) using the parsimony principle indicate that there is no such close correlation.

Lee et al. (1983) explain that a more rapid divergence

41 of globin genes in contrast to animal hemoglobin could ac• count for the equally close correlation of leghemoglobin to all animal globin genes. However, Goodman et al. (1975) reported that in the beginning, mutations which improved hemoglobin function also took place at an accelerated rate in animals and only decelerated when all functional oppor• tunities had been exploited.

The third theory invokes a plant origin for leghemo• globins either by d_e novo development or due to a primitive globin common to all hemoglobin carrying organisms.

Appleby (1974) advocated a de novo evolution of leg• hemoglobin from a duplicated plant hemoprotein like cyto• chrome due to the need to improve an oxygen-limited inef• fective symbiosis. However, Broughton and Dilworth (1973) argue that, since leghemoglobins are significantly related to myoglobins and primitive hemoglobins and since the he• terogeneity in leghemoglobins follows the pattern of legume evolution, it does not seem likely that they evolved de novo. Consequently, it is possible that all hemoproteins arose from a common original globin gene before the sepa• ration of the animal, fungi and plant kingdom.

Brown et al. (1984) estimated that the separation of the three eukaryotic groups dates back about 1.4 to 1.5 billion years. Ramshaw et al. (1972) came to similar con• clusions from their studies with cytochrome c. However,

42 Verma et al. (1983) determined that legumes appeared only about 140 million years ago. Hence, Appleby (1974) argues that "an apparently useless gene would not have survived for over 1 billion years before its first expression".

Estimation of time scales are often only educated guesses (Goodman et al. 1975). Axelrod (1958) and Heywood

(1971) reported finding fossilized legumes in tertiary- formations (around 50 million years ago). Since legumes consist of soft material, they might not h.ave been well preserved and thus their age could have been substantially underestimated (Dilworth and Appleby 1979) similar to other angiosperms. Ramshaw et al. (1972) reported that * angiosperms originated at least several geological periods before the Cretaceous, which is the earliest period in geological record in which their fossils have been found.

Another strong argument for a plant origin comes from the occurrence of globin genes outside the legume family.

Southern blot experiments by Hattori and Johnson (1985) and

Roberts et al. (1985) showed that non-nodulating legumes and even non-legumes have sequences which cross-hybridize to soybean leghemoglobin probes. Globin genes occur in such distantly related families as Rosiflorae

(Casuarinaceae, Betulaceae, Myricaceae), Malviflorae

(Ulmaceae, Elaegnaceae), Rutiflorae (Coriariaceae), and

Fabiflorae (Leguminosae) according to Dahlgren (1980) and

43 Landsmann et al. (1986). Hattori and Johnson (1985) also suggested that globin genes might be more widespread in the plant kingdom than expected and were at some time, or still are present, in all plants.

Further support for this theory comes from western blot analyses by Fleming et al. (1987) who detected strong reciprocal cross-reactions between hemoglobins from

Casuarina and Parasponia, two nitrogen fixing non-legumes which belong to widely separated plant orders. Parasponia is a plant which fixes nitrogen in association with

Rhizobium. Its leghemoglobin cDNA hybridizes to genes in the genome of Casuarina, a genus which forms a nitrogen fixing association with Frank ia. Because of the high degree of hemoglobin homology between these two species and their different symbiotic associations, Landsmann et al. -

(1986) suggested that a separate evolution of plant leg• hemoglobin and nitrogen fixing symbioses might have taken place.

All these findings support the theory that hemeproteins have an ancient origin and that globin sequences be• came silenced in plants which have not aquired a nitrogen fixing capability or do not form actinorhizal associations

(Appleby et al. 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 al. 1988). However, there are very few amino acids in the hemeprotein which are preserved in all globins

(Ellfolk 1972, Guy 1985, Lee and Verma 1984, Ollis et al.

198 3).

Hurrell et al. (1979) investigated 55 hemoglobins.

Only two amino acid residues out of 145 were found to be invariant in all species. In contrast, size, helix content and disposition and thus, the overall geometry of the he• moglobin molecule, were remarkably similar for molecules from such diverse origins as mammals and plants.

Guy (1985) compared crystal structure and sequences of vertebrate hemoglobins, myoglobins, insect larva globins and plant leghemoglobins to determine their evolutionary relationship. Analysis of the globin sequences showed that very few residues in homologous positions were identical and thus were not very useful for evaluating structural relationships.

Usually, globin chains consist of eight helical rods with the exception of leghemoglobins where the short

D-helix is missing (Hurrell et al. 1979). During evolu• tion, changes are more likely to occur at the interface of the molecule with its environment than in the interior which incorporates the heme group since changes in the heme environment are more likely to be disruptive and thus cause loss of functional activity. When changes occurred, they

45 were usually conformationally conservative, i.e. amino acids were substituted by residues with similar properties

(Hurrell et al. 1979). In addition, Lehtovaara et al.

(1980) demonstrated that relatively few mutations have in• 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 in the heme cavity and in the terminal regions of different hemo• globins does not imply that these globins have the same evolutionary origin. The author points out that according to Margoliash et al. (1968) two requirements have to be fullfilled to show ancestral relationship; it has to be proven that sequences are genetically related to a greater degree than can be expected by chance, and it must be shown that similarities are greater in 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 difficult to distinguish between con• vergence of unrelated proteins during evolution (analogy) and descent from a common ancestor (homology) (Ramshaw et

46 al. 1972). However, in 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 in addition to the typical eukaryotic 'CAAT1 and 1TATAA1 boxes, the 5' region of leghemoglobin genes contain an imperfect, tandemly re• peated sequence which seems necessary for all globin func• tion (Verma et al. 1983). In addition, Brown et al. (1984) showed that this region also contains a sequence comple• mentary to the 3' end of 18S rRNA. Yamaguchi et al. (1982) proposed that such a region might enhance the rate of ini• tiation of complex formation in protein synthesis. Hence,

Verma et al. (1983) concluded on the basis of the conser• vation of globin regulatory sequences that all globin genes have a common origin.

The coding regions of leghemoglobins are interrupted by three introns. Similarly, Parasponia (Landsmann et al.

1986) and Trema (Bogusz et al. 1988), the latter is a non- nodulating species, contain hemoglobins with three introns in identical positions to those in leghemoglobins. In con• trast, all other hemoglobins contain only two intervening sequences. The two common introns of g-globin and leghe• moglobin genes are in similar positions. The third intron of leghemoglobin divides the central exon which encodes the proximal and distal heme contacts (Go 1981).

47 Blake (1978) argued that if exons correspond to a

folded protein fragment, then mutational changes would be much more likely to yield stable globular conformations.

Thus, Go (1981) proposed that the central intron was ex• cised from an ancient ancestral hemoglobin gene. Nishioka et al. (1980) reported the presence of an a, -globin-1 ike pseudogene in mice which lacks the two introns that are normally present in hemoglobins and thus a mechanism for splicing out introns must exist in eukaryotes. Based on these findings, Appleby et al. (1988) suggested that all globin genes have evolved from a primitive globin-carrying proto-organism.

Furthermore, Appleby et al. (1988) speculated that the expression of hemoglobin in Trema and in un-nodulated

Parasponia and Casuarina roots ensured its persistence du• ring evolution. The authors suggested that the small amount of hemoglobin in the un-nodulated roots may be at• tributed to "the sensing of anaerobiosis and the initiation of anaerobic response mechanisms, rather than facilitation of oxygen diffusion".

In summary, the close evolutionary relationships of leghemoglobins with other hemoglobins as well as the simi• larities in intron position and regulatory sequences and the presence of globin sequences in non-nitrogen fixing species provide strong evidence that all hemoglobins have

48 indeed evolved from a common ancestral gene.

49 III. INDUCED MUTAGENESIS FOR LB VARIABILITY

3.1. INTRODUCTION

Breeding implies selection of variants which either arose spontaneously or were induced by mutagenic agents.

In general, a plant breeder would first screen cultivated or wild accessions to determine if a desired character is present within the species or genus. If variation in the character exists, then hybridization and backcrossing will establish the trait in a crop. If, however, a variant cannot be found or if all hybridization attempts fail, then inducing variability by means of mutagenic agents is often the only solution (Simmonds 1981) .

A mutation is defined as a "heritable change in the genotype of an organism" (Lewin 1985). Accordingly, a mutagen may be any agent which increases the rate of muta• tion to a higher than spontaneous level (Lewin 1985).

Spontaneous mutations and subsequent recombination are the basis for existing variability in plant evolution.

Most mutations are recessive in nature and natural muta• tions generally occur at low frequencies (10*6 per genera• tion). Such changes are likely a response to ultra-violet and background radiation, errors in DNA replication, ageing of the DNA and transposable genetic elements (Simmonds

50 1981).

For breeding purposes, the overall rate of spontaneous mutation is often too low to be of practical use; further• more, mutations of the desired type may be particularly rare. Increasing mutation rates with the help of mutagenic agents like radiation or chemical mutagens increases the probability of creating and enhances the potential for finding the desired mutant.

The foundations of induced mutagenesis were laid by De

Vries (1909, 1910) at the beginning of this century while working with Oenothera lamarckiana. Schliemann (1912) was the first to investigate the effects of chemical mutagens on Aspergillus niger. The experiments of Muller (1927) and

Stadler (1928) showed that X-rays directly increased the frequency of mutations. Since then, the induction of mu• tations has often been suggested as a tool to enlarge variation in a crop (Gottschalk and Wolff 1983).

However, since most mutagenic agents are non-selec• tive, any treatment will produce a random array of muta• tions from which one has to sort out those of interest.

Adequate selection procedures among the mutagenized popu• lation are an essential component of a successful mutation breeding program. The choice of a method usually depends on the facilities available. In P_. vulgaris, radiation with gamma-rays and chemical treatment with alkylating

51 agents such as ethyl methane sulfonate (EMS) have been more commonly used for mutation induction (Crocomo et al. 1973,

Delgado de la Flor 1978, Hussein and Disouki 1976, Motto et al. 1975).

52 3.2. LITERATURE REVIEW

All radiation releases energy as it passes through matter. The energy is absorbed in the atoms of the target and is released in form of fast charged particles. These particles can interact with the electrons of the atom by expelling electrons out of orbit. The atom thus becomes ionized which can affect the whole molecule in such a way to initiate a change in configuration. Such changes may result in the loss of the functional activity of the molecule (Timofe"ef f-Ressovsky 1940) .

Timofe"eff-Ressovsky et al. (1935) developed the theory that only one "hit" would be necessary for a gene mutation to be induced. Consequently, the number of mutations in• creases linearly at low to medium doses. Early theories were developed in the absence of knowledge about the DNA.

Since then, their 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 hit to occur, the first break may have been repaired. Hence, the fre• quency of chromosome breakage does not show a simple linear increase; the response curve reflects an increase in breakage approximately reflecting the square of the dose.

It seems that the lethal effects of radiation are often due to double strand breaks. Single hit events lead to dele-

53 tions, gene mutation, and sister chromatid unions (IAEA

1977, Muller 1954a).

In general, gamma-rays have a shorter wavelength and

therefore possess more energy per photon than X-rays. In

addition, X-rays are always less homogeneous and hence,

gamma-rays (obtained from Cobalt-60 or Caesium-137) are

more frequently used for the induction of mutations (IAEA

1977).

Beside the direct effects on genetic material, the

experiments by Stone et al. (1947) and Wyss et al. (1947,

1948, 1950) showed that at least part of the mutations

produced are the result of an indirect chemical effect of

radiation. The authors found that irradiating the media

with UV-light, on which Staphylococcus aureus was subse•

quently grown, caused mutations. This radiochemical effect

involves the formation of hydrogen peroxide and free reac•

tive chemical intermediates from water in the presence of molecular oxygen.

Beside the polarization of water molecules, water is also important as a diffusion medium for free radicals and

oxygen (Weiss 1944, Muller 1954b). Consequently, the pre• sence or absence of water and/or oxygen has a profound in•

fluence on the effectiveness of radiation. Konzak et al.

(1970) pointed out that radiochemical damage can be pre• vented by using presoaked seeds for gamma radiation treat-

54 ment. However, soaking seeds in water also induces germi• nation and growing tissue has been shown to be more affec• ted by radiation than inactive tissue (Savin et al. 1968).

There are numerous factors beside oxygen and water which modify the effects of radiation. Temperature is an important secondary factor which influences the extent of radical movement and thus interaction. For example, pre- and post-irradiation heat treatments have been shown to reduce radiation-induced damage in seeds while increasing the mutation frequency (Ehrenberg 1955, IAEA 1977).

In addition, it seems that the effect of radiation also depends on the trait studied. For example, Crocomo-et al. (1973) demonstrated that a dosage of 6 - 7 Krad was optimal for mutagenesis for increasing the total amount of bean protein, whereas at least 7 Krad was necessary for specifically increasing the methionine level. Thus, com• parisons between different experiments of even the same species are extremely difficult due to the number of variables affecting an experiment.

In 1943, Oehlkers demonstrated the mutagenic effects 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 principal categories of chemical

55 mutagens are alkylating agents, base analogues, intercala•

ting agents and deaminating agents. Alkylating 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 is sufficiently high, alkyla•

ting agents such as EMS can transfer their reactive alkyl

group(s) either to the phosphate backbone, resulting in an

unstable phosphate triester which might lead to chromosome

breakage, or to the nitrogeneous bases which in turn might

cause point mutations due to pairing errors (IAEA 1977,

Suzuki et al. 1986).

The effective 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 biological

system (IAEA 1977). The uptake of alkylating agents de•

pends on simple diffusion laws and requires that the con• centration of the chemical in solution is greater than that

in the plant cell.

In addition, the effects of treatment are also influ• enced by temperature. EMS hydrolyses and has a half-life

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 ability (Froese-Gertzen et al. 1964).

56 Consequently, extensive post-treatment washing is recom• mended to remove the potential effects of the EMS hydro• lysis products. Konzak et al. (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 tightly bound to lipids within the tissue.

Pre-treatment of seeds by soaking them in water seems to facilitate the diffusion of mutagenic substances through membranes and to achieve better infusion of the chemicals into the tissue (Konzak et al. 1970). However, Mikaelsen et al. (1967) reported that the uptake of EMS was indepen• dent of presoaking time.

Other factors influencing the effects of alkylating agents include post-mutagenic treatments such as reported by Mikaelsen et al. (1967) who found that chlorophyll mutations and chromosome aberrations increased when seeds were dried and stored after the mutagenic treatment. These effects depended on storage time, temperature and also on the initial level of damage.

Gaul (196 2) proposed that EMS is the most powerful chemical for the induction of chlorophyll mutations in 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 lethal

57 than X-rays under similar experimental conditions.

However, the authors also found that the effect of EMS showed a large dependence on prevailing environmental con• ditions and thus often precluded satisfactory experimental reproducability.

An experiment on the effects of EMS (0.1% and 0.2% w/v) and gamma rays (0-100 Gray) on five P_. vulgar is varieties was performed by Hussein and Disouki (1976).

They reported varietal differences in mutagen-sensivity, with the variety '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 in the Ml generation. However, EMS induced a higher degree of sterility than the gamma-ray treatment, which the authors attributed to the physiological damage produced by EMS and its hydrolysis products. They suggested that P_. vulgar is, like other legumes, should be treated with 0.1% (w/v) or less EMS in order to obtain a reasonable number of Ml survivors. However, the authors recommended the use of EMS and gamma rays as "the most efficient physical, respectively chemical agents available nowadays" for the induction of mutations (Hussein and

Disouki 1976).

Chemical as well as radiation treatments are usually applied to seeds, which is essentially a treatment of the

58 embryo meristems. Since the meristematic regions of an

embryo usually consist of several initial meristematic

cells, any mutation induced will only affect the part of

the plant which arises from this mutated cell. Hence,

mutagenic treatments often give rise to chimeras

(Gottschalk and Wolff 1983, IAEA 1977).

The experiment by Motto et al. (1975) showed chimeric

Ml P_. vulgaris plants with often only a small mutated sector. Based on the segregation ratios obtained, the

average number of initial cells present in the apical meristem was estimated to be eight. However, in heavily damaged plants, the average number of initial cells de- * creased to three. In one particular mutant, a single meristematic initial cell generated the whole plant.

Delgado de la Flor (1978) reported that EMS tended to in• duce a larger size of chimera than gamma-rays.

59 3.3. MATERIALS AND METHODS

Approximately 4000 seeds of P_. vulgar is L. cultivar

•Contender' were used. Kidney beans are diploid and are

normally self-pollinated. 'Contender* is an early bush bean

variety with buff to brown seeds. The average thousand

seed weight is about 387g. Seed protein content ranges

from 20-22 % on a dry matter basis (Adams et al. 1985).

'Contender' was released from the USDA Southeast Vegetable

Breeding Laboratory in 1949. The cultivar was derived from crosses involving Commodore x Streamliner x US#5 Refugee.

'Contender' is resistant to common bean mosaic and powdery mildew (J.L. Morris, personal communication).

The experiment was carried out in the greenhouses of the Department of Plant Science, University of British

Columbia in 1986 and 1987. Well developed seeds of about the same seed size were selected for the experiment. For either mutation treatment, the Ml survival rate was tar• geted at 20%-30%.

For the gamma-ray treatment, seeds were soaked over• night in distilled water and treated in a Gammacell 220 irradiation facility using Cobalt-60 in the Faculty of

Agricultural Sciences, University of British 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 lasting

18 hours, seeds were washed thoroughly under running water for one hour.

All seeds from both treatments were immediately seeded in 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 fertilizer. A total of 708 Ml and 571 Ml plants were obtained from the gamma-ray treatment and the EMS treat• ment, respectively.

At least one seed from every Ml plant (and thus the M2 generation) was seeded in a styrofoam cup containing

Turface® (Montmorillonite clay). The M2 plants were inocu• lated with a commercial Rhizobium leguminosarum biovar phaseoli inoculant and fertilized twice weekly with a ni• trogen-free nutrient solution (Holl 1975). After three to four weeks, nodules from each M2 plant were excised, crushed in 0.1 M sodium phosphate buffer pH 7.4 and centrifuged at 12,400 x g in an Eppendorf microcentrifuge for 2 minutes. The supernatant containing leghemoglobin was used for cellulose acetate electrophoresis (CAE). The CAE was run for 15 minutes at 420 volts (Holl et al. 1983). After separation, the Sepraphore® strips were stained for the presence of protein with Ponceau S and counterstained for the presence of heme using the method of Owen et al. (1958)

61 as described in Holl et al. (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 survival rates, chlorophyll mutations, changes in leaf size and shape, alterations in growth habit from de• terminate to indeterminate and varying degrees of sterility

(Hussein and Disouki 1976, Motto et al. 1975, Rubaihayo

1975).

On average, plants of the Ml generation obtained from the gamma ray treatment produced 5.28 seeds per plant, the variation ranging from 0-23 seeds, whereas EMS-induced Ml mutants produced 4.98 seeds per plant on average, the va• riation ranging from 0-10 seeds.

About 780 M2 seeds derived from the gamma-ray treat• ment and 620 M2 seeds derived from the EMS treatment were sown for leghemoglobin testing. Approximately 55 M2 seeds did not germinate, while 20 M2-gamma and 36 M2-EMS plants either did not develop any nodules or developed ineffective nodules without leghemoglobin. Other nitrogen fixing mu• tants were also detected. For example, variations were observed in nodule number and size compared to seed-derived control plants. Thus, both mutagens proved to be effective in inducing mutations in P_. vulgaris.

Nevertheless, although 1400 plants were screened for variability in leghemoglobin, no heterogeneity for this

63 trait could be detected. This observation is not incon• sistent with previous reports of genetic conservation for this protein (Holl et al. 1983, Fuchsman and Palmer 1985).

There are a number of possible explanations for the failure to induce any mutational changes in Lb pattern.

Firstly, cellulose acetate electrophoresis separates proteins according to their electric charge and is not the most discriminating method. Isoelectric focusing (IEF) can distinguish proteins differing by only 0.01 pH unit in their isoelectric point (Fuchsman 1983). However, unlike

CAE, IEF is a more costly, time-consuming procedure which cannot be routinely used as a rapid screening technique for large nodule numbers. Thus, if mutations had been induced which effected only a slight change in the charge of the Lb molecule, they would not likely 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. Theoretically, any gene can mutate, but such a mutational event is a matter of chance and the expected rate of mutation at any specific locus is difficult to es• timate (IAEA 1977). Micke (1984) estimated that the muta• tion rate in a particular single gene occurs with a fre• quency of 10*4 to 10per generation. Given that this frequency range is a reasonable estimate, a population of

6 4 10,000 - 100,000 plants would be required to detect muta• tion in a single Lb gene, a much larger sample than was available for this experiment.

However, this estimate is based on the occurrence of a single active gene. Lee and Verma (1984) proposed that there might be four active genes present in the Phaseolus genome, which would imply that a four-fold decrease in the population size should be adequate. Consequently, the probability of finding the desired mutant in 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 all mutagenic treatment is damaging and higher doses are also likely to produce too many sterile plants and/or too high a level of mortality. In the literature, treatment of P_. vulgar is with gamma-rays has been reported using 30 - 400

Gray, and treatment with EMS using from 0.02 - 0.2 M

(Crocomo et al. 1973, Hussein and Disouki 1976, Motto et al. 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 6-12 hours

(Delgado de la Flor 1970, 1978, Moh 1969).

As noted in the introduction to this section, in ad• dition to the absolute dose of mutagen, mutation rates are influenced by many other factors, including the cultivar

65 (Hussein and Disouki 1976) , temperature (IAEA 1977) , water and oxygen content (Weiss 1944, Muller 1954b), pre- and post-treatment conditions (Conger et al. 1969, Delgado dela

Flor 1970, Konzak et al. 1970) as well as the number of initial meristematic cells in the growing point of the seed

(Gottschalk and Wolff 1983, Motto et al. 1975).

In this experiment, usually only one seed per plant was tested. However, an average of five seeds per Ml plant was harvested. According to Motto et al. (1975) , it is likely that the mutagenic treatment of P_. vulgaris induced chimeras since the number of initial cells present in the apical meristem of this species was estimated to be eight.

If the production of chimeras for Lb variability is consi• dered a possibility, then every single nodule would have to be screened. However, since over 4000 seeds were harves• ted, the logistics of such an analysis were beyond this research.

The results of this mutagenesis work again raise the question of whether all four leghemoglobin genes in P_. vulgaris encode a single identical component or if only one gene is active and the other three are silent (Lee and

Verma 1984). About 1400 seeds were grown and screened for leghemoglobin variability. If there are four active genes, one might speculate that it should have been possible to detect a mutant. However, the fact that 56 mutants did not

66 form any nodules or were deficient in leghemoglobin pro• duction gives some indication that only one gene might be active. Thus, one might further speculate that in case only a single gene is active, it is quite possible that inducing a mutation in the Lb gene is an extremely rare event and/or if it occurs, it has most likely a lethal effect since any mutation will affect the structure as well as the function of the leghemoglobin molecule. Probably only very few mutations are acceptable which will not dis• rupt its function, and even fewer of those will improve its oxygen-carrying capability.

In conclusion, many plant breeders and geneticists are similarly sceptical with regard to the effectiveness of mutagenesis for plant improvement (Stadler 1930, Micke

1984, Simmonds 1981). Micke (1984) pointed out that many positive results obtained so far with mutation breeding are due to easily identifiable, 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 is L. is 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 origin and from there beans spread to Europe and Africa during the sixteenth century.

Archeological findings date the domestication of beans between about 7680 and 10,000 BC (Kaplan et al. 1973).

The genus Phaseolus belongs to the Leguminosae

(Fabaceae) family, subfamily Papilionoideae and tribe

Phaseoleae. P_. vulgar is is a. diploid species with 2n=22 chromosomes. In view of their common morphological cha• racters, _P_. coccineus L. (scarlet runner bean) and _P. acutifolius Gray (tepary bean) are close relatives. P_. lunatus L. (lima bean) is more distantly related as re• flected in the failure of interspecific crosses with _P. vulgar is. Beans are autogamous with the exception of P_. coccineus which shows predominant cross-fertility (Bliss

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, it should be noted that, accor• ding to their results, P_. vulgar is also showed two compo• nents. This has not been confirmed by our results using cellulose acetate electrophoresis (CAE) or by the use of the more sophisticated technique of isoelectric focusing

(IEF) (Holl and Lulsdorf, unpublished data). These latter methods demonstrated that nodules of bush beans contain only one major leghemoglobin component and a minor compo• nent which is likely a deamination product of the major one, as suggested by Lehtovaara and Ellfolk (1975a).

Since leghemoglobins in the genus Phaseolus have never been investigated systematically, this project was conduc• ted to evaluate representatives of the genus and to deter• mine if globin heterogeneity is present. If variation for leghemoglobin exists between species, then this plant ma• terial could be used to transfer the variant leghemoglobins into P_. vulgaris in order to determine the impact of Lb heterogeneity on the fixation efficiency of the altered bean.

Furthermore, knowledge of the arrangement of leghemo• globin genes in 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• lysis by Sullivan and Freytag (1986) determined that P_. vulgar is is closely related to P_. coccineus (runner bean) , less closely to P_. acutifolius (tepary bean) and only dis• tantly to P_. lunatus (lima bean) . P_. f iliformis can be grouped between tepary and lima beans in this geneology.

A genus is an excellent source of genetic variability for transfer of characters not otherwise available within a crop species. Mendel (1865) accomplished the first suc• cessful interspecific hybridization between P_. vulgaris and

P_. coccineus. Since then, many crosses have been performed between these two species because of the relative ease with which hybrids can be obtained.

For example, Lamprecht (1941) reported extensive hy• bridization studies between P_. vulgar is and P. coccineus

(multiflorus) carried out during the nineteen thirties.

Since fertility of the hybrids was reduced, the author proposed that hybridization barriers due to cytoplasmic factors caused the high gamete sterility in the Fl genera• tion. From further studies, Lamprecht (1957) concluded that the larger pollen of runner beans carried over a small but sufficient quantity of cytoplasm to permit restoration of fertility whereas the reciprocal cross using P_. vulgaris

70 pollen remained infertile due to the smaller cytoplasmic

content of p. vulgar is pollen grains.

In contrast, interspecific hybridization with other

species in the Phaseolus genus has been more difficult, and

embryo rescue methods are generally required. Honma (1956) crossed P_. vulgar is ('Great Northern') with _P_. acutifolius

in order to transfer common blight resistance. Three to twenty-four days after pollination, tissue culture of the aborting embryos was required. However, the resulting hybrids were self-fertile and produced viable seeds.

Al-Yasiri and Coyne (1964) reported that applying naphthaleneacetamide and potassium gibberellate to the base of pollinated flowers improved seed set in interspecific crosses. Pod growth was stimulated and embryo abortion delayed, but embryo rescue was still necessary for plant survival. Similarly, Smartt (1970) obtained hybrids of common and tepary beans and their reciprocals, although the level of success was low. A few individuals were obtained without the help of tissue culture. The author concluded that the lower viability of hybrids in an acutifolius plasmon was due to cytoplasmic effects.

Mok et al. (1978) also produced immature embryos from reciprocal crosses of P_. vulgaris ('Great Northern' and

'Gallatin 50') and P.. acutifolius. Cotyledon development of the hybrids was uneven and the rate of growth as well as

71 the final size of the hybrid embryos were found to be dependent on the genotype of both parents.

Further studies by Rabakoarihanta et al. (1979) re• vealed that fertilization in interspecific crosses might be completed but incompatibility arose during endosperm for• mation; development was especially delayed if tepary bean was the female parent. Cytological observations indicated bivalent and univalent chromosomes during Metaphase I. In addition, Fl pollen fertility was found to be low and sel- fing of the hybrids was not possible (Rabakoarihanta et al.

1980).

Pratt et al. (1985) reported that using heterozygous female P_. vulgar is parents almost tripled the frequency at which interspecific hybrids could be produced. However, all Fl plants were sterile 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 relative success of the hybridization procedure.

These somewhat contradictory findings by different authors were clarified by the experiment of Parker and

Michaels (1986) in which an examination of nine parental P_. vulgar is lines indicated that common beans carry an allele for interspecific incompatibility which interacts with a nuclear factor in the P. acutifolius genome. Thus, some

72 cultivars like 'lea pijao' and 'Sacramento Light Red

Kidney' do not contain this allele and are able to produce

fertile hybrids.

In contrast to P_. acu tifolius, hybridization between

P_. vulgar is 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 failed

within seven days of pollination. A further attempt using

an intraspecif ic heterozygous P_. vulgar is hybrid as a fe•

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 interspecific hybrids because

he was unable to reproduce these results in similar expe•

riments. The author suggested that the progeny were pro•

ducts of either self-fertilization or apomixis.

In order to examine these contradictory reports,

Leonard et al. (1987) investigated the effect of maternal

heterozygosity on the subsequent development and growth of

interspecific hybrids. They concluded that the maternal P_.

vulgaris genotype affected the number as well as the size

of the embryos produced. However, in contrast to the fin•

dings by Honma and Heeckt (1959) , intraspecific maternal

hybrids resulted in significantly smaller embryos than pure

73 maternal lines. In addition, since no reciprocal effects were observed, a cytoplasmic influence was ruled out.

Savova and Zagorska (1987) confirmed in their experi• ment that the success of interspecific hybridization de• pends on the heterozygosity of the female parent.

Mok et al. (1978) rescued P_. vulgaris x P_. lunatus hybrid embryos in the pre-heart-shape stage. Reciprocal crosses did not succeed. These authors also observed that hybrid embryo development depended on the parental geno• types. Similarly to P_. acutifolius crosses, fertilization occurs but endosperm division is either delayed or does not take place. It appears that these hybrids cannot complete differentiation (Rabakoarihanta et al. 1979). A few viable plantlets were obtained from these experiments and trans• ferred to soil. However, the plantlets did not continue to grow beyond the four trifoliate leaf stage (Mok et al.

1986) suggesting an incompatibility barrier.

Cytoembryological studies by Molkhova and Tsoneva

(1986) indicated that the incompatibility of kidney and lima bean crosses is reflected in abnormal development of the embryo and suspensor haustorium and in hypertrophy of the tapetum of the integument. Thus an effective incompa• tibility barrier exists between the two species.

Finally, the literature on hybridization between P_. vulgar is and P_. f iliformis is scarce. Tau et al. (1986)

74 produced viable Fl plantlets from this cross using embryo rescue techniques. Reciprocal crosses were not successful.

The Fl hybrids were completely sterile due to non-viable pollen. Backcrossing also failed. Treatment of the Fl hy• brids with colchicine 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 in this study consisted of a selection of Phaseolus germplasm obtained from Dr. N.

Galway of the Cambridge University, U.K., two P. acutifolius accessions from the Centro Internacional de

Agricultura Tropical (CIAT) , and the P_. vulgaris cultivar

'lea pijao' from the University of Guelph, Canada. Furth• ermore, one of the scarlet runner bean (P_. coccineus) cultivars, the cultivars 'Contender', 'Gallatin 50' (P_. vulgaris) , and 'Lima Hendersons' (P_. lunatus) were obtained from Buckerfield's Ltd., Vancouver, B.C.. Table 1 lists all accessions and their geographical source.

Beans are diploid and are normally self-pollinated with the exception of P_. coccineus (Adams et al. 1985,

Brucher 1977). The average thousand seed weight ranged from about 8 g for P_. filiformis to about 1150 g for P_. lunatus (Market Sample). Seed coat color varied from white, through beige, brown, purple to black and variegated types. All plants with the exception of 'Contender' and

'Gallatin 50' were pole beans.

Some of the wild accessions with hard seed coats nee• ded scarification and/or soaking in 0.5 mM gibberellic acid

76 Table I. Phaseolus germplasm accessions

SPECIES VARIETY CULTIVAR or GEOGRAPHICAL PLANT INTRODUCTION SOURCE

P_. vulgar is V 2851 Europe 840 Eur ope 841 Europe 868 Eur ope 869 Europe 871 Eur ope 872 Europe

P_. vulgar is 2249 Central Amer ica 2832 Central Ame r i c a 2860 Central America 2895 Central America 2960 Central America 3701 Central America 3782 Central America 3798 Central America 3807 Central America

P. vulgar is wild N 1695 wild N 1735 - wild N 11002 - wild N 11042 - wild N 11089 - wild N 11091 -—

P. vulgar is 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 GEOGRAPHICAL PLANT INTRODUCTION SOURCE

P. vulgar is 'Contender' USA 'Gallatin 50' USA 1 lea pijao'

P. coccineus 'Hammond's Dwarf* NC44 'Crusader' C196 'Exhibition Scarlet Prizewinner1 C184 'Scarlet Runner'

P. acutifolius CIAT 79069 Mali CIAT 40107 Mexico latifolius N 1775 N 1602

P_. lunatus Market Sample 'Lima Hendersons'

P_. filiformis N 1600

P_. microcarpus N 1708

78 to induce germination.

Following the results and recommendations by Parker and Michaels (1986), the P_. vulgaris cultivar 'lea pijao1 was used as a female parent for all P_. vulgar is x P_. acutifolius interspecific crosses. All other crosses were attempted with *Ica pijao*, 'Gallatin 50', or 'Contender' as the female parent. The nitrogen fixation experiment was conducted only with hybrids derived from crosses involving

1 lea pijao'.

4.3.2. GROWING CONDITIONS

The experiments were conducted in the greenhouses and growth chambers of the Department of Plant Science,

University of British Columbia from 1986 to 1988.

Plants were grown initially in growth cabinets with day and night temperatures of 22°C and 18°C, respectively.

All P_. acutifolius accessions, except P. acutifolius Mali, are short-day plants and thus required a maximum day-length of 10 hours for flowering; all other accessions were grown under a 16 hour day/ 8 hour night regime.

Depending on the season and the size of the plants, the accessions and the hybrids were transferred to the greenhouse and supplemented with fluorescent light during the fall and winter months according to their day-length requirements. Spider mites and white flies were controlled

79 when necessary.

For the hybridization experiments and hybrid multi• plication, each plant was grown in a .15 cm diameter flexi• ble plastic pot with a soil : peat moss (4:1 w/w) mix con• taining Osmocote®, a 14-14-14 slow release fertilizer.

After about two months of growth, the plants were ferti• lized twice weekly with Peters®water soluble fertilizer

(20-20-20). Throughout the growth period, all plants were watered with tap water as required.

According to the recommendations by Mok et al. (1986) for hybridizations with _P_. lunatus, the female P_. vulgar is parent was grown in hydroponic culture in a medium supple• mented with 10 >uM benzyladenine. Nutrient solutions were prepared according to Murashige and Skoog (1962) at one- half strength; one liter per plant. Aeration was provided from a compressed air line, at about one liter per minute.

All female parent plants were maintained in 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 in 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 this inocu• lum, this species was inoculated with a R. leguminosarum

80 bv. phaseoli strain TAL 22, specific for lima beans, ob• tained from the University of Hawaii, NifTAL Project. All plants were fertilized twice weekly with a nitrogen-free nutrient solution.

For the nitrogen fixation experiment, well-rooted cuttings from each hybrid were grown in 8 cm diameter plastic pots filled with Turface®, inoculated and ferti• lized similar to the CAE and IEF procedure.

4.3.3. LEGHEMOGLOBIN TESTING

Three to five weeks after inoculation, nodules from each accession were excised, crushed in 0.1 M sodium phosphate buffer, pH 7.4 and centrifuged at 12,400 x g in an Eppendorf microcentrifuge for 2 minutes. The superna• tant containing leghemoglobin was either used directly for

CAE and IEF or further purified according to methods des• cribed by Holl et al. (1983) via ammonium sulfate precipi• tation, treatment with potassium ferricyanide and dialysis.

Samples were concentrated using Centricon® microconcentrators.

The CAE was run for 15 minutes at 420 volts (Holl et al. 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 in

Holl et al. (1983) .

81 IEF was performed in 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

Calibration Kit®, pH 2.5-6.5) was used for calibration.

The total running time including prefocusing was 500 Volt- hours at 15°C and lasted about 25 minutes. One gel was stained with Coomassie Blue for protein according to the recommendations in the manual for the Phast Gel® IEF system

(Phast Development Technique File No. 200); the other gel was stained for heme using the method by Owen et al. (1958) as described by Holl et al. (1983).

4.3.4. INTERSPECIFIC HYBRIDIZATION

Flowers of P_. vulgar is were emasculated one day before opening (Bliss 1980) and pollinated immediately using the hooking method described by Buishand (1956). To prevent dessication, pollinated buds were enclosed in cellophane tape as recommended by Bliss (1980) . Only one flower per raceme was pollinated; all other flowers were removed to decrease competitive effects. The number of interspecific pods growing at the same time on one plant was limited to five. Pods showed visually discernible growth about three to four days after pollination.

82 4.3.5. EMBRYO RESCUE AND HYBRID GROWTH

Developing pods for embryo rescue were removed from the P_. acutifolius hybrids 23 days after pollination

(Parker and Michaels 1986), and 12 to 15 days after anthe- sis from crosses involving P_. lunatus (Mok et al. 1978,

Leonard et al. 1987). P_. filiformis hybrids were rescued

15 to 20 days after pollination.

Pods were rinsed with 95% ethanol for one minute, and surface sterilized for 15 minutes by immersion in an aqueous solution of commercial bleach (20% v/v) containing two drops of Tween-20®. All subsequent procedures were carried out in a laminar flow hood. The sterilized pods were rinsed twice with sterile distilled water. Ovules were removed from pods and embryos dissected under a binocular dissecting microscope.

The excised P_. acutifolius embryos were placed in culture vials containing 15 ml of B5 medium (Gamborg et al.

1968) supplemented with 3% (w/v) sucrose and 0.8% (w/v) agar. Following the procedure of Parker and Michaels

(1986), the test tubes were placed in the dark at 25°C un• til hypocotyl elongation took place. Then, embryos were transferred to a growth chamber with a 16 hour photoperiod.

After 12 to 15 days, vigorously growing plantlets were transferred to 8 x 8 cm square clear plastic containers filled with 80 ml of the same B5 medium.

83 About one month later, well-developed plantlets with roots were transferred to 8 x 8 cm square flexible plastic pots containing a soil : sand : peat moss (3:1:1, w/w/w) mixture with Osmocote® fertilizer, a 14-14-14 slow release fertilizer. The plants were placed on culture trays in closed aquariums filled with about 3 cm of water (plants were not in contact with water) to maintain high relative humidity. Later, the aquariums were partially opened until the plants were adapted to ambient growing conditions; the adjustment required about two to three weeks. P_. filiformis hybrids were treated similarly to P_. acutifolius embryos except for the tissue culture medium.

Excised P_. filiformis and P. lunatus interspecific embryos were placed in culture vials containing 15 ml of a three quarter strength Murashige and Skoog (1962) embryo rescue medium prepared according to Mok et al. (1978) , with

0.125 /iM benzyladenine, 0.7 mM glutamine, 0.8% (w/v) agar, and 3% (w/v) sucrose.

P_. lunatus interspecific embryos were placed in growth chambers with continuous light at 28°C for five weeks

(Leonard et al. 1987) and then maintained with a 16 hours photoperiod and a temperature of 26°C.

84 4.3.6. NITROGEN FIXATION EXPERIMENT

The nitrogen fixation experiment was conducted with

cuttings obtained from P_. vulgar is x P. acutifolius and P.

vulgaris x P_. filiformis interspecific hybrids (all had

'lea pijao* as female parent) and from each of the parents.

Cuttings were taken from healthy plants, including at least

one growing point, and dipped in Stim-Root® No.l, a rooting

hormone powder containing 0.1% indole butyric acid, and

planted in 8x8 cm square pots containing a soil : sand

mixture (4:1, v/v). The plantlets were placed in aquariums

as described in chapter 4.3.5. and grown under fluorescent

lights (16 hour photoperiod) until they were well rooted^

(about two weeks).

Three-week old rooted cuttings of about the same size

were selected for the experiments. The soil mixture was

carefully washed off the roots, and cuttings planted in 8cm

diameter plastic pots filled with Turface®, inoculated with

Rhizobium leguminosarum biovar phaseoli and watered twice

weekly with nitrogen-free nutrient solution.

The nitrogen fixation tests were analysed as rando• mized complete block designs (RCBD) with seven replicates

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 in 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 carried out using a closed system and analysis by flame ionization gas chromatography as described by Holl et al.

(1983) . This method is based on the ability of the nitro• genase to reduce acetylene to ethylene (Dilworth 1966).

The ARA is a kinetic measurement of activity which deter• mines the nitrogen fixing rate of a plant at the time of measurement (Boddey 1987, Upchurch 1987).

Directly after 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• lysis to determine total nitrogen (Lepo and Ferrenbach

1987). Total nitrogen gives an estimate of the cumulative nitrogen fixation activity over the period of plant growth.

The determination of total nitrogen was conducted in the

Department of Soil Science, University of British Columbia.

Statistical analysis of the nitrogen fixation experi• ment was carried out according to Cochran and Cox (1960).

All other statistical procedures were as described in Steel and Torrie (1980).

86 4.3.7. PROTEIN SEQUENCING

Leghemoglobins from P_. lunatus 'Lima Hendersons' and from an interspecific P_. filiformis hybrid were extracted and purified as described in Chapter 4.3.3. After purifi• cation, Lbs were applied to a 1.5 x 26 cm column of DE-52

(Whatman®) for cellulose ion-exchange chromatography (Holl et al. 1983). Leghemoglobins from the P_. filiformis hybrid and from P_. lunatus were eluted with a linear gradient of 5

- 150 mM sodium acetate (pH 5.4) in a 250 ml volume. Sam• ples were collected and the absorbance monitored at 405 nm to determine the elution of Lb components. The collected fractions were concentrated in Centricon® microconcentrators using an IEC® clinical centrifuge. Salts were removed by desalting with the same microconcentrators.

Apoproteins for the Lb components were prepared by the acid-acetone procedure (Ellfolk 1961). Precipitated pro• teins were re-dissolved in dilute sodium bicarbonate solu• tion (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_. filiformis hybrid. Sequencing of the

N-terminal end of the proteins was carried out by the

Protein Sequencing Laboratory of the University of Victoria using an automated Edman degradation.

87 4.4. RESULTS AND DISCUSSION

4.4.1. LB VARIABILITY IN THE GENUS PHASEOLUS

All Phaseolus accessions (see Table I, page 77) were screened for Lb variability using CAE and IEF, three to five weeks after inoculation. The results of both methods were identical in regard to numbers of heme-stained Lb bands. In addition, the only difference between purified and unpurified Lb extracts was that the bands were sharper with the former samples. Isoelectric points were estimated from IEF gels using protein standards.

All P. vulgaris accessions (including P_. vulgaris wild and P. vulgar is aborigineus) as well as P_. coccineus ac• cessions and P.. microcarpus showed only one Lb band in the same position as P.. vulgar is controls. Occasionally, a second band was visible which has been identified as a de- amidation product of the major component (Lehtovaara and

Ellfolk 1975a). Lehtovaara and Ellfolk (1975a) reported isoelectric points of 4.7 for Lba and 4.55 for Lbb from P. vulgaris. The isoelectric points estimated from the pre• sent isoelectric focusing study confirmed those results.

All P_. acutifolius accessions also contained one major component; however, this band migrated more slowly in the electrophoretic systems in comparison to P_. vulgaris

88 Fig. 1. CAE Lb profile showing P_. vulgar is (C) , P_. acuti fol iu s N1602 (A ^) , and _P. acutifolius ssp. latifolius (A j_) •

89 (Fig. 1). The basic principle of CAE, as reviewed by Chin

(1970), is 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 its net charge. Consequently, the dif• ferent migration pattern result from changes in one or more amino acids. Isoelectric focusing results gave an estimate of 4.8 for the P_. acutifolius Lb isoelectric point.

In contrast, both P. filiformis and P_. lunatus acces• sions contain more than one Lb component. P_. filiformis

(Fig. 2) shows two bands when separated by CAE. Both com• ponents migrate more slowly than that of P. vulgar is. One

(the faster of the two) migrated to a position similar to that of P_. acutifolius Lb (Fig. 3) . The isoelectic points of the two filiformis components were estimated to be about 4.8 and 4.9.

The most interesting species examined in the survey was P_. lunatus (Fig. 4), since this species produced two major and probably three minor components. The major com• ponents migrated faster than P_. vulgar is and their iso• electric points have been estimated as 4.5 and 4.4. One minor component seems to be in a similar position as the P_. acutifolius band, the other is in a position similar to the

P.. vulgar is band; the third one has an isoelectric point of approximately 4.4.

90 Fig. 2. CAE Lb profile showing P. vulgaris wild (Pw),

P. f iliformis (P p) , P. microcarpus (PM ) and P. vulgaris 'Gallatin' (G).

91 Fig. 3. CAE Lb profile showing P_. vulgar is x P. f iliformis hybrid (1), P. acutifolius (2), P. vulgar is x P.. acutifolius hybrid (3) and p_. vulgaris (4).

92 93 The only report of intra-specific leghemoglobin vari• ability has been described by Holl et al. (1983) who dis• covered a Tr ifolium subterranean line with a major compo• nent missing. In contrast, Fuchsman and Palmer (1985) who studied leghemoglobins from a genetically diverse selection of cultivated (G_. max) and wild soybeans (G_. soja) could not detect any differences even though they used IEF, a more sensitive method.

This present study is the only one to screen a genus in which differences between species for leghemoglobin genes have been discovered. Sullivan and Freytag (1986) investigated seed protein patterns in the genus Phaseolus and similarly found very little variation in protein pro• files within most species, while considerable variation was evident among species. However, the same authors reported considerable variation within the P. acutifolius species which did not have a single easily recognizable protein profile. In contrast, the present study did not detect any heterogeneity for Lb between P_. acutifolius subspecies.

This investigation studied only a small number of species within the Phaseolus genus which comprises 31

(Mare"chal et al. 1978) to 150 (Briicher 1977) species. It would be interesting to determine if further variation exists in this genus since, in general, it would appear

94 that the more distantly related the species, the more dif• ferent are their Lb profiles. The only exception to this 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 closely related to P. lunatus than to P_. vulgar is (Sullivan and Freytag 1986) .

Leghemoglobin components from many species have been shown to differ in their oxygen affinity and thus in their enhancement of bacteroid oxygen consumption and support of bacteroid nitrogen fixation (Fuchsman and Appleby 1979,

Uheda and Syono 1982a,b, Wittenberg et al. 1985). Often, only very small changes in the amino acid composition were found to be responsible for this higher oxygen affinity

(Kortt et al. 1987). Unfortunately, we could not determine oxygen association and dissociation rates of the different leghemoglobins in Phaseolus but if differences in oxygen affinity could be confirmed in this genus, then those fin• dings would further support the theory that Lb heteroge• neity is functional and that it is an adaptation for more effective nitrogen fixation as suggested by Fuchsman et al.

(1976) and Uheda and Syono (1982a,b).

95 4.4.2. INTERSPECIFIC HYBRIDIZATION

Based on the results obtained from CAE and IEF, P_. acutifolius, V. f iliformis, and _P. lunatus were chosen as parents for transfer of Lb variability into P_. vulgar is. A total of about 500 crosses was performed, from which about

450 developed into pods. However, many of those pods con• tained seeds with aborted embryos, especially when P_. lunatus was one of the parents. This observation is in agreement with that by Alvarez et al. (1981) and Mok et al.

(1978). Hybrid embryos from P_. lunatus crosses only deve• loped to an oval-rod-shape stage similar to reports by Mok et al. (1978) . Furthermore, growth of the interspecific plantlets in tissue culture was slow. Due to a growth chamber malfunction which destroyed the interspecific hy• brids obtained from this cross, this part of the research project was discontinued.

In contrast, embryos from interspecific hybridizations involving P. acutifolius ssp. (ssp. acutifolius, ssp. latifolius, ssp. acutifolius Mexico and ssp. acutifolius

Mali) developed to the cotyledon stage. As reported by Mok et al. (1978), these embryos often showed characteristic asymmetric development of the two cotelydons and typical retarded endosperm development (Rabakoarihanta et al.

1979). Hybrids grew well in tissue culture and transfer to soil and adaptation to ambient growing conditions resulted

96 in very few losses. However, even though all interspecific

plants from this cross flowered, the hybrids were generally

sterile, with little pollen developed in the anthers. The

exceptions to this sterility were two P_. vulgaris x P_.

acutifolius Mexico plants which were self-fertile and de•

veloped seeds. Similarly, Haghighi (1987) reported ob•

taining some selfed seeds from this 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 is x P_. f iliformis hybrid pods

contained seeds with embryos sufficiently large for exci•

sion. Both P_. vulgar is cultivars 'Contender' and 'lea

pijao' were successfully used as female parents, and hy•

brids from both crosses grew well in tissue culture and

soil. Leaf shape, size and texture of the hybrids were

intermediate between the two parents. Plants flowered

profusely but again, all hybrids were completely sterile.

Tau et al. (1986) also reported obtaining sterile hybrids

from this cross, due to formation of nonviable pollen^

These authors used colchicine to produce tetraploids in

order to restore partial fertility and to induce seed de• velopment. Interspecific 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 in Table II.

In the analysis of variance shown in Table III highly

significant differences (P< 0.01) were detected for 'Plant

Species' in the ARA experiment, indicating that species

differed in their nitrogen fixation rates. Further parti•

tioning of the sums of squares for 'Plant Species' showed

highly significant differences among the five groups and

within group V (involving P_. f iliformis) . Calculation of

single degree contrasts among groups (Table IV) revealed

that groups I to IV (involving P_. acutifolius ssp.) dif- ,

fered significantly (P< 0.01) from group V (involving _P.

filiformis), indicating that group V had a higher nitrogen

fixation rate than any of the other groups (Table V).

Furthermore, contrasts calculated within groups of the ARA

experiment (Table VI) showed that in group I, both parents

(P_. vulgaris (1) and P_. acutifolius N1602 (6) ) differed

significantly from their associated hybrid (11). Thus, the

hybrid mean (Table V) is significantly higher than either parent. Whether this difference is due to heterosis or a

consequence of parent II (P_. acutifolius N1602) remains a

matter of speculation, since the difference between parent

I and II was not significant (Table VI).

98 Table II. Grouping of 'Plant Species' for analysis.

Group1 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. latif. (9) P.v. X p. a.latif. (14)

V P.v. (5) P. filif. (10) P.v. X p. filif. (15)

x For analysis, 'Plant Species' were coded from 1 to 15 (numbers in parentheses) and grouped into five groups, each consisting of parent I, parent II and the respective hybrid.

2 Abbreviations: P. vulgaris = P.v.; P. acutifolius = P.a.; latifolius = latif.; P. filiformis = P. filif.

99 Table III. Analysis of variance for ARA of P. vulgaris, P_. acutifolius, P. filiformis and interspecific hybrids. Additional analysis for the partitioned sums of squares for 'Plant Species' is also shown.

Source of Variation DF MS

Replicates 6 558,778.4 0.864 Plant Species 14 1,662,522.5 2.570** Error 84 646,859.2

Among Groups 2,492,596.8 3.853**

Within Group I 2 1,710,954.0 2, 645 Within Group II 2 1,015,737.8 1, 570 Within Group III 2 97,770.3 0.151 Within Group IV 2 193,964.3 0.230 Within Group V 2 3,634,037.8 5.618**

*,** Significant at the 0.05 and 0.01 level of probability.

100 Table IV. Orthogonal contrasts among groups for the ARA exper iment.

Compar ison of Groups rlc2 MS(Q) F i

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 vs.Ill 21(12) 249,026. 8 0 .385

.** Significant at the 0.01 level of probability.

101 Table V. Mean acetylene reduction (nm/plant/hour) for each 'Plant Species' and group measured in 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 between group 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**

*,** Significant at the 0.05 and 0.01 level of probability.

103 The most interesting results occurred within group V.

Both parents (P_. vulgar is (5) and P_. f iliformis (10) ) dif• fered significantly from their associated hybrid (15)

(Table VI). Their respective means (Table V) showed that their hybrid had a higher nitrogen fixing rate than either parent. In addition, parent I (P_. vulgar is) differed sig• nificantly (P< 0.01) from parent II (P. filiformis) (Table

VI). The mean rate of nitrogen fixation (Table V) of the

P. filiformis parent is about four times higher than that of the P.. vulgar is parent indicating that the hybrid de• rived its higher nitrogen fixing ability from the P_. filiformis parent.

The analysis of variance (Table VII) also detected highly significant differences among 'Plant Species' in the

Total N experiment, indicating that some species accumu• lated more nitrogen during their growth period than others.

Partitioning the DF and sums of squares for 'Plant Species'

(Table VII) revealed that there were significant differen• ces among groups and within group V for the total amount of nitrogen accumulated over the growing period. To determine the source of the differences, orthogonal contrasts were carried out among and within groups. Table VIII shows that groups I to IV (involving _P. acutifolius ssp.) differed significantly (P< 0.01) from the P. filiformis (V) group.

104 Table VTI. Analysis of variance for Total N in P_. vulgar is, P.. acutifolius, P_. filiformis and interspecific hybrids. Additional analysis of the partitioned sums of squares for 'Plant Species' is also shown.

Source of Variation DF MS

Replicates 6 451.4 0.855 Plant Species 14 2,507.4 4.749** Error 84 527.9

Among Groups 6,827.2 12.933**

Within Group I 2 891.3 1.688 Within Group II 2 188.5 0.357 Within Group III 2 345.0 0.654 Within Group IV 2 204.8 0.388 Within Group V 2 2,273.0 4.306*

*,** Significant at the 0.05 and 0.01 level of probability.

105 Table VIII. Orthogonal contrasts among groups for the Total N experiment.

Compar ison of groups rl 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 vs.Ill 21(12) 1,868. 5 3.54

*,** Significant at the 0.05 and 0.01 level of probability.

106 Table IX. Means (mg total 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 five groups

(Table IX) show that the P_. filiformis group accumulated more nitrogen during the growth period than either of the

P_. acutifolius groups. Furthermore, group I (involving P_. acutifolius N1602) also differed significantly (P< 0.05) from groups II, III and IV (Table VIII). Finally, con• trasts calculated within groups (Table X) showed signi• ficant differences only in group V between the parents (P. vulgar is (5) and P_. f iliformis (10)) and their associated hybrid (15). Their respective means (Table IX) confirmed that the hybrid accumulated more nitrogen than either parent. Whether this result is attributable to the P_. filiformis parent as in the ARA experiment is not clear since the means of the parents do not differ statistically.

However, it is tempting to speculate that the higher rate of ARA in P_. filiformis may contribute to greater N accumulation in the hybrid.

In summary, 'Plant Species' differed in their nitrogen fixation rate as well as the total amount of nitrogen fixed over the growing period. Results from both methods showed that group V (involving P_. filiformis) outperformed all other groups. Notably, the hybrid P_. vulgaris x P_. filiformis had a higher acetylene reduction rate and thus a higher nitrogen accumulation rate than all other 'species'.

However, only in the ARA experiment could this be attribu-

108 Table X. Orthogonal contrasts within groups for the Total N experiment involving P_. vulgaris, P. acutifolius. P_. filiformis and interspecific hybrids.

Compar ison within between 2 group species 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

* Significant at the 0.05 level of probability.

109 ted to the JP. filiformis parent. In addition, the P_.

acutifolius N1602 hybrid also had a higher nitrogen fixing

rate at the time of measurement, although the total amount

of nitrogen fixed did not differ significantly.

A correlation analysis conducted between the ARA and

Total N method, gave a correlation coefficient of 0.40,

indicating that the acetylene reduction assay is a poor

predictor of the amount of nitrogen fixed. Nitrogen fixa•

tion rates vary during the growth period as well as during

the day (Rennie and Kemp 1981, Goh et al. 1978). Rennie

and Kemp (1981) found no significant correlation between

ARA and Total N in their experiments either. In contrast,

Hungria and Neves (1987) reported a highly significant

correlation between these two parameters when ARA was mea•

sured from flowering to mid pod-fill stage of different

bean cultivars. From further studies with _P_. vulgaris,

Rennie and Kemp (1984) concluded that ARA severely under•

estimates nitrogen fixation in this species. Similarly,

Pacovsky et al. (1984) concluded that the amount of assi• milated nitrogen is a better measure of symbiotic effi•

ciency than the reduction of acetylene. Recent studies of

nitrogen metabolism in kidney bean suggest that the rela•

tionship between acetylene reduction activity measurements

and nitrogen accumulation are likely to be strongly influ• 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 in

this experiment and thus might have contributed to the poor

correlation between ARA and Total N, was the evolution of

hydrogen in the acetylene reduction assay. Pacovsky et al.

(1984) pointed out that the results of the ARA should be corrected for this energy loss. Piha and Munns (1987) re• ported hydrogen evolution rates of 17-27 ymol/pot/hour for

P_. vulgar is, 15-16 jjmol for P_. acutifol ius, 21-25 ymol for

the P_. vulgaris x P. acutifolius hybrid, and 7 jimol for P_.

filiformis. However, the authors also estimated the ni•

trogen fixation rate for P_. filiformis as 18 jjmol/pot/h, which was much lower than either P_. vulgar is, P_.

acutifolius 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 replicates varied considerably. However, the mean of P_. filiformis activity highly significantly (P< 0.01) exceeded P_. vulgar is using either method.

The present study used cuttings instead of plants de• rived from seeds. When plants rely on nitrogen fixation for their sole N supply, they have to overcome an initial lag period, in which they function under nitrogen stress.

However, plants grown from seeds have some nitrogen re-

Ill sources available in the endosperm. In contrast, cuttings have to rely entirely on the nitrogen which can be remobi- lized from leaves, stem or roots until nitrogen fixation sets in. This puts cuttings under a more severe nitrogen stress and hence, restricts their growth. P_. filiformis and its hybrids seemed to overcome this limitation much faster than either P_. vulgaris or V_. acutifolius.

Another confounding effect which could not be taken into account in this experiment, resulted from differences between the species in respects other than nitrogen fixa• tion. In order to eliminate this source of error, one

would have to use isogenic lines which differ only in Lb ',t components. Thus, one cannot conclude decisively from these experiments that the higher acetylene reduction rates and the higher amounts of nitrogen fixed by P_. filiformis and its hybrids are due to more efficient function of Lb components in those materials. However, these results suggest that it might be worthwhile to explore this hypothesis further.

4.4.4. Protein Sequencing

Cellulose ion exchange chromatography resolved Lbs from a P_. filiformis hybrid into three peaks (Fig. 5 ).

Peak II was chosen for the N-terminal analysis. This peak corresponds to the second Lb band in CAE (Fig.2 )» it mi-

112 X IE

no oo uo i

FRACTION NUMBER

Fig. 5. Elution profile for P. vulgar is x P. filiformis hybrid Lb components on DE52-cellulose columns. Experimental conditions are described in section 4.3.7. , page 87.

113 grated to a position similar to the band from Phaseolus acutifolius.

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 results of the protein sequencing showed that the first 37 amino acids of the N-terminal matched the known amino acid sequence of P_. vulgaris (Lehtovaara and Ellfolk

1975b). Bean Lbs consist of 145 amino acids and show a close homology to both soybean Lba and Lbc3. The first Lb exon consists of 32 amino acids in both species. Four of those in Lbc3 and 5 of those in Lba differ from the bean sequence (Lee and Verma 1984). The major changes were found in the third exon. Consequently, it is possible that the changes which caused the different Lb profiles with CAE from P. acutifolius, P. filiformis and P_. lunatus also af• fected mainly the heme cavity rather than the amino termi• nal end. Migration patterns in CAE can differ due to a single amino acid change similar to human hemoglobin (Chin

1970). Since only small differences can result in changes in the oxygen affinity and these changes would most likely take place in the heme cavity, it is not surprising that

114 20

FRACTION NUMBER

Fig. 6. Elution profile for P_. lunatus 'Lima Hendersons' Lb components on DE52-cenulose columns. Experimental conditions are described in section 4.3.7., page 87.

115 the N-terminal sequences of all Lb components investigated

in this study are similar.

116 V. GENERAL DISCUSSION AND CONCLUSIONS

Beans have generally been considered to be poor ni• trogen fixers in comparison to species such as soybean.

Among the explanations for this apparent phenomenon is the fact that kidney beans contain only one nodule Lb component which might be less effective in supporting the nitrogen fixing bacteroids. In contrast, more efficient nitrogen fixing crops like soybeans or peas contain several Lb com• ponents which differ in their oxygen affinity and in their efficiency in supporting nitrogen fixation (Fuchsman et al.

1976, Uheda and Syono 1982a,b). On the basis of their work with soybeans (Fuchsman et al. 1976) and peas (Uheda and

Syono 1982a,b), these authors have concluded that Lb he• terogeneity is functional and that it constitutes an adap• tation for more effective nitrogen fixation. Consequently, the present study was conducted to determine whether such

Lb heterogeneity can be achieved in P_. vulgaris and if this variability indeed enhanced the nitrogen fixing capability of this crop.

Leghemoglobin heterogeneity in P_. vulgaris was ad• dressed using a dual strategy. On the one hand, beans seeds were treated with mutagenic agents (gamma rays and

EMS) in order to induce mutations in the existing Lb gene(s). From their study with kidney beans, Lee and Verma

117 (1984) have concluded that there are four Lb genes present

in the Phaseolus genome. However, the authors could not determine if only one gene was active, or if all four genes produced an identical component. About 1,400 M2 plants were tested in the present study for Lb variability using

CAE. No mutation which affected the migration pattern of

the Lb protein was detected. If there are indeed four ac• tive genes present in the bean genome, it should have been possible to detect mutation in the sample tested. The ab• sence of detectable mutation suggests that only one gene in

the bean genome may be active and the other three are si•

lent or, that the gene is not highly mutable. It is likely

that the number of plants screened was too small to detect a low frequency variant, and if the method (CAE) was not

sensitive enough, then mutation induction is too laborious to consider as an alternative method for achieving hetero• geneity for plant breeding of this species.

On the other hand, plant breeders often screen a genus

for a desired trait and use intra- and interspecific hy• bridization to transfer such characters into an acceptable crop background. The Phaseolus genus was screened for the presence of existing Lb variability and considerable vari• ation for this trait was revealed. P_. acutifolius nodules also contained a single Lb component which migrated to a different position in comparison to the P_. vulgaris con-

118 trol, suggesting differences in the amino acid sequence.

Furthermore, nodule extracts of P_. filiformis, a desert species, contained two Lb components, both migrating dif• ferently from P_. vulgar is. _P. lunatus apparently produced two major and probably three minor Lb components, also showing a different electrophoretic profile from P_. vulgaris. Thus, the present study constitutes the first report of Lb heterogeneity occurring within the genus.

A further study was initiated to determine if Lb va• riability enhanced nitrogen fixation in interspecific crosses. Interspecific hybrids were obtained from P.. vulgar is x P.. acutif ol ius and from P_. vulgar is x P_. filiformis. Parents and their respective hybrids were ve- getatively propagated for evaluation of nitrogen fixation capability. Two nitrogen fixation tests were used to assess the performance of the plants; ARA which gives an estimate of the nitrogen fixing rate at the time of mea• surement and Total N which reflects the total amount of nitrogen fixed over the growth period in an N-free environ• ment. P_. vulgar is x P. f iliformis hybrids had a higher nitrogen fixation rate as well as accumulated significantly more nitrogen over the growing period than either parent.

However, only in the ARA experiment could this be clearly attributed to the P_. filiformis parent. In addition, the

P_. vulgaris x P_. acutifolius N1602 hybrid performed better

119 than its parents in the ARA experiment but data for the

Total N experiment showed no significant difference between parents and the hybrid.

Interpretation of the data from the nitrogen fixation tests should be made with caution. Comparisons in this experiment involved different species; differences in per• formance may be confounded by the different genetic back• ground of the species involved in a particular hybrid. De• finitive tests of the contribution of Lb heterogeneity to nitrogen fixation efficiency require the use of isogenic lines, differing only in the genes defining the Lb compo• nents. Nevertheless, the within-group comparison of the P. vulgar is x P_. filiformis hybrids and the respective pa• rents, shows transgressive segregation for both ARA and

Total N in the hybrid. It is tempting to speculate that at least part of the improved fixation level is a consequence of the heterogeneous Lb pattern in the hybrid.

N-terminal sequencing of one of the P. filiformis components (peak II) and two of the P_. lunatus 'Lima

Hendersons' Lbs (peak I and II) showed no differences be• tween the first 37 amino acids of the three proteins and those of P_. vulgaris. However, since only one amino acid need vary in order to affect the migration pattern in CAE

(Chin 1970) , these data are not unexpected and are consis• tent with the other observations of conserved sequences in

120 Lb (Appleby 1984). Furthermore, it is likely that changes

leading to an improvement in Lb efficiency will be located

in the sequences associated with the heme cavity rather

than at the N-terminal end.

This study has identified different Lb components in

the genus Phaseolus. However, no tests were made to eval•

uate the components for differences in oxygen affinity or

to determine whether the molecules are regulated indepen•

dently. Independent regulation would be in accord with most other species studied so far and thus, this point

clearly needs further investigation.

Furthermore, funding limitations precluded sequence

analysis of the three Lb components beyond the 35-40

N-terminal amino acids. Hence, sequencing of all Lb mole•

cules detected in this study should be carried out to fur•

ther our understanding of sequence changes that may cause different oxygen affinities.

The difficulties in obtaining fertile interspecific hybrids from P_. vulgaris x P. lunatus clearly limits the

potential transfer of genetic traits between those species.

Even though plantlets were obtained from this cross, the plants did not develop beyond the formation of four trifo•

liate leaves. Leghemoglobin genes have been transferred

from soybean to Lotus corniculatus using an Agrobacterium

system; the chimeric gene was found to be expressed in root

121 nodules formed on the transformed Lotus (Jensen et al.

1986). Hence, this latter method could be used to transfer

Lb genes from lima to kidney beans if the P_. lunatus Lbs are identified to be superior in their oxygen affinity.

This procedure would also have the advantage of producing isogenic lines without further backcrossing.

Testing Lbs from a single nodule is also an easy method to determine if hybrids obtained from interspecific crossing are, indeed, hybrid. Thus, simple electrophoretic analysis could have resolved the doubts expressed by Smartt

(1979) about plants obtained by interspecific hybridization of P_. vulgar is x P. lunatus in experiments by Honma and

Heckt (1959) .

An interesting question is, how Lb variability arose in the genus Phaseolus from an evolutionary or geneological perspective. Did P_. vulgar is and P_. coccineus lose their

Lb variability after the initial speciation events, or did other Phaseolus species only develop Lb heterogeneity fol• lowing speciation? In the latter case, Lb heterogeneity must have been a relative recent evolutionary event. In this context, it is also interesting to note that P_. microcarpus which has been described as more closely rela• ted to P_. lunatus than P. vulgar is (Sullivan and Freytag

1986) contains only one Lb component which migrates to the same position in CAE as P. vulgar is. Based on the simila-

122 rities of their Lb profiles, one might speculate that P_.

microcarpus is more closely related to P_. vulgaris and P.

coccineus rather than _P. lunatus. However, since crossing

data are not available, this apparent contradiction cannot

be resolved at present.

Only relatively few species in the genus Phaseolus have been screened for Lb heterogeneity in this study. A

further analysis of the species in the genus might reveal more variation and thus further our understanding of the

evolution of leghemoglobins genes. In addition, this in• vestigation might also resolve the controversy about the phylogenetic relationship of some species which cannot be clearly attributed to either the genus Phaseolus or the genus Vigna on the basis of their morphological traits

(Briicher 1977) .

In conclusion, although Lb heterogeneity is not evi• dent in common cultivars of P_. vulgar is, it does occur in

the genus Phaseolus and thus, these findings are consistent with the general theory that Lb heterogeneity is functional

and that it constitutes an adaptation for more effective nitrogen fixation. If the Lb components found in the genus are shown to have different oxygen affinities which are reflected in more effective nitrogen fixation, then any P_. vulgaris breeding program should utilize interspecific hy• bridization or gene transfer and selection for this trait.

123 VI. SUMMARY AND CONCLUSIONS

Phaseolus vulgar is seeds were mutagenized using gamma rays and ethyl methane sulfonate. Among 1400 M2 offsprings derived from the treated plants, no mutation in the legemo- globin (Lb) gene was detected. The inability to induce a mutation in this locus suggests that leghemoglobin consti• tutes a strongly conserved protein.

Fifty accessions were screened for the presence of existing Lb variability in the genus Phaseolus. Heteroe- neity was evident in some of the species investigated.

Therefore, the single Lb component found in P. vulgaris constitutes an exception among the nitrogen fixing species.

Interspecific hybrids were produced in order to trans• fer the existing Lb variability into P. vulgar is and to de• termine if this heterogeneity indeed enhances nitrogen fixation. Significant differences in the nitrogen fixing capabilities of the different hybrids were determined.

Therefore, it was concluded that the introduction of leg• hemoglobin variability into P. vulgaris should be a target for selection in plant breeding projects.

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