Nonsymbiotic plant haemoglobin genes

I p

i

i

I 1:

I "

•1 B

I ,_

NONSYMBIOTIC PLANT HAEMOGLOBIN GENES

Carol Rae Andersson

February 1994

A thesis submitted for the degree of Doctor of Philosophy of

This thesis contains no material which has been accepted for the award of any

other degree or diploma in any University. To the best of my knowledge, this thesis

contains no material previously published, or the result of any work by another person,

except where due reference is made in the text.

The research described in this thesis is my own original work, with the following

exceptions.

Celia Miller embedded, sectioned and mounted tissue for dark field microscopy

(Chapter 2). S.B. Narasimhulu made some of the Parasponia haemoglobin promoter deletions (P-235 and below, Chapter 2). Janice Norman constructed and screened the

soybean genomic library and isolated the soybean nonsymbiotic haemoglobin gene

(Chapter 4). Erik Ostergaard Jensen conducted the Northern analysis of the soybean

nonsymbiotic haemoglobin gene (Chapter 4). Danny Llewellyn cloned the Arabidopsis

ACKNOWLEDGEMENTS

There are many people in the lab and throughout the Division who have helped

me at some stage during my PhD, either with practical help, advice or encouragement.

To them my grateful thanks. There are several people I would particularly like to thank.

Special thanks to my supervisors, Liz Dennis and Danny Llewellyn for their

support and advice throughout my PhD, from practical help to comforting words in

times of crisis. Also to Janice Norman for her encouragement and for teaching me

many techniques, and especially for practical help during the last few months. Thanks

to Jim Peacock for spurring me on and for critical reading of much of this thesis.

Thanks to Celia Millar for all her help with preparing tissue sections for dark field

microscopy, Stuart Craig for instruction and advice, Fraser Bergersen and lnara Licis

for generously providing soybean plants, Paul Keese for enlightening discussions,

Leigh Farrell and Tony Ashton for computing help, Robert Hill (University of Manitoba)

for making information regarding the barley haemoglobin available prior to publication

and to Cyril Appleby for his continued enthusiasm. Special thanks to Ron Wier for

keeping track of PhD progress and formal requirements.

Part of the work described in this thesis was conducted during a six month visit

to Professor Kjeld Marckers laboratory, University of Aarhus, Denmark. My thanks to

him, Jens Stougaard, Erik Ostergaard Jensen, Peter Lauridsen and Niels Bech

Laursen for vectors and bacterial strains and helpful advice about high efficiency

transformation of Lotus. Special thanks to Jens Stougaard for initiating the soybean

lbps1 study. Thanks to Erik Ostergaard Jensen for useful discussions and his

involvement in the soybean nonsymbiotic haemoglobin work.

Special thanks to Mary Clarke and Erika Merkel, whose efficiency kept the lab

running smoothly and made life easier for the rest of us.

Finally, thanks to my family for their encouragement and patience.

This research was conducted in the Division of Plant Industry, CSIRO.

acknowledge financial support from an Australian Postgraduate Research Award and

ABSTRACT

Haemoglobin is present in the nitrogen-fixing root nodules of both legumes and the non-legume Parasponia, where it facilitates the diffusion of oxygen to the respiring bacteria at a free oxygen concentration too low to inhibit the oxygen sensitive nitrogenase complex (reviewed by Appleby, 1984; Appleby et al, 1988). A closely related non-nodulating relative of Parasponia, Trema tomentosa, has a haemoglobin gene that is expressed in roots (Bogusz et al, 1988). Both the Parasponia and Trema

haemoglobin promoters direct expression of a reporter gene in the nodules of a transgenic legume (Bogusz et al, 1988). However, this thesis demonstrates that, unlike expression of the leghaemoglobins and endogenous Parasponia haemoglobin, these promoters confer high expression in uninfected tissues of Lotus nodules, and low expression in the bacteroid containing cells. This nodule expression pattern is similar to that of a nonsymbiotic haemoglobin gene from Casuarina, analysed concurrently with this study (Jacobsen-Lyon et al, in preparation), which is expressed in parenchyma of both roots and nodules. Thus, regulation of the Parasponia and Trema haemoglobin genes in a transgenic legume reflects aspects of both symbiotic and nonsymbiotic expression and suggests distinct signals control organ versus cell specificity.

The discovery of haemoglobin and haemoglobin genes in phylogenetically diverse non-legume genera, including Parasponia, Trema, Casuarina and Hordeum

(barley) (Appleby et al, 1983; Bogusz et al, 1988; Fleming et al, 1987; Christensen et al, 1991; Taylor et al, 1993) led to the proposal that haemoglobin may have a fundamental, but as yet unknown, function in all plants independent of its role in

symbiotic nodules (Appleby et al, 1988; 1990). This thesis explores the suggestion that legumes must have a haemoglobin gene expressed in nonsymbiotic tissue in addition to the nodule specific leghaemoglobin genes (Appleby et al, 1988; 1990). The apparent pseudogene /bps 1 has been suggested as the soybean nonsymbiotic haemoglobin gene (Nap and Bisseling, 1990b,c). However, no evidence of expression of lbps1, either in soybean or transgenic Lotus, was found in this study and it is likely

This thesis describes the isolation and characterisation of a novel soybean

haemoglobin gene that shows significant homology to other nonsymbiotic haemoglobin

genes and which is expressed in a variety of tissues. This haemoglobin gene

represents the elusive nonsymbiotic legume haemoglobin and supports the proposal

that the nodule specific leghaemoglobins arose from a preexisting nonsymbiotic

haemoglobin by gene duplication (Appleby et al, 1988; 1990). In addition, this thesis

describes the isolation of a haemoglobin gene fragment from Arabidopsis, which will

provide a powerful tool for the analysis of the general function of plant haemoglobin.

The taxonomic distribution of haemoglobins and the regulation of both symbiotic

and nonsymbiotic haemoglobin genes has implications both for the origin of plant

CONTENTS page

TITLE PAGE

CANDIDATES STATEMENT II

ACKNOWLEDGEMENTS 111

ABSTRACT IV

CONTENTS VI

LIST OF FIGURES XII

LIST OF TABLES XIII

CHAPTER 1

GENERAL INTRODUCTION 1

LEGHAEMOGLOBIN AND THE LEGUME-(BRADY)RHIZOBJUM SYMBIOSIS 1

The legume-(Brady)Rhizobium symbiosis 1

Leghaemoglobin facilitates oxygen diffusion within nitrogen fixing nodules 4 Leghaemoglobin as the archetype symbiotic protein

NODULINS ARE PLANT PROTEINS SPECIFICALLY INVOLVED

IN NODULATION

Early nodulins are involved in nodule formation

EN002 AND EN0012 ARE CELL WALL PROTEINS

Late nodulins are involved in nodule function

ENZYMES INVOLVED IN NITROGEN AND CARBON METABOLISM

PERIBACTEROID MEMBRANE NODULINS

Origin of nodulins

PARASPONJA HAEMOGLOBIN AND THE

PARASPONIA-(BRADY)RHIZOBIUM SYMBIOSIS

The Parasponia/(Brady)Rhizobium symbiosis

Haemoglobin is expressed in nodules of Parasponia and

6

9

9

10

11

1 1

12

13

13

HAEMOGLOBIN IN ACTINORHIZAL NODULES AND THE ACTINORHIZAL

SYMBIOSES 15

The actinorhizal symbioses 15

Haemoglobin in actinorhizal nodules 16

NONSYMBIOTIC PLANT HAEMOGLOBINS AND POSSIBLE FUNCTION 17 THE COMMON FUNCTION OF HAEMOGLOBIN IS REVERSIBLE

BINDING OF OXYGEN 18

Some haemoglobins are involved in oxygen transport 18

Some haemoglobins are linked to enzymatic domains 19

E.COLI AND YEAST HAEMOGLOBINS HAVE A REDUCTASE ACTIVITY 19

ASCARIS HAEMOGLOBIN CHANNELS OXYGEN INTO BIOSYNTHETIC PATHWAYS

Other haem proteins are also known to act as oxygen sensors

Rhizobium Fixl is part of a two-component regulator of

20 21

nitrogen fixation genes 21

ALL HAEMOGLOBINS EVOLVED FROM A COMMON ANCESTOR 22

Plant and vertebrate haemoglobins have a similar gene structure 23

Evolution of plant haemoglobin 25

HAEMOGLOBIN GENE REGULATION CAN BE DISSECTED USING TRANSGENIC PLANTS

Expression of leghaemoglobin genes involves quantitative

and qualitative promoter elements

SOYBEAN lbc3 SOYBEAN Iba

SESBANIA ROSTRATA g/b3

Gene expression requires trans-acting protein factors

A RHIZOBIAL PROTEIN INTERACTS SPECIFICALLY WITH A

27

28 28 30 32

33

LEGHAEMOGLOBIN PROMOTER 33

NODULE SPECIFIC HMG-LIKE FACTORS MAY BE SCAFFOLD PROTEINS 34

CASUARINA SYMBIOTIC HAEMOGLOBIN GENE REGULATION

PARASPONIA AND TREMA HAEMOGLOBIN GENES GENERAL AIMS OF THIS THESIS

CHAPTER 2

EXPRESSION OF THE PARASPONIA AND TREMA HAEMOGLOBIN PROMOTERS IN TRANSGENIC LOTUS REFLECTS ASPECTS OF BOTH

34 35

35

SYMBIOTIC AND NONSVMBIOTIC HAEMOGLOBIN EXPRESSION 37

INTRODUCTION 37

RESULTS 39

The Parasponia and Trema haemoglobin promoters confer expression

in nodules of Lotus 39

A distal positive element is required for high level, nodule

specific expression

The consensus motif GAAGAG is required for high levels of nodule

41

expression 46

5' deletion of the Parasponia and Trema haemoglobin promoters alters

the spatial pattern of GUS expression 46

The Parasponia and Trema haemoglobin promoters are also expressed

in the indeterminate nodules of white clover

DISCUSSION

Expression of the Parasponia and Trema haemoglobin promoters in

transgenic Lotus reflects aspects of both symbiotic and

nonsymbiotic haemoglobin expression

The promoter motif GAAGAG is involved in high level expression

The Parasponia and Trema haemoglobin promoters contain multiple,

47 48

48 50

interacting regulatory domains 51

Is the repressor control mechanism functionally significant? 52

The Parasponia and Trema haemoglobin genes are qualitatively similar 53

METHODS

Plasmids and Constructions

Site directed mutagenesis

Plant transformations

GUS histochemical assay

GUS fluorometric assay

CHAPTER 3

THE SOYBEAN LEGHAEMOGLOBIN GENE LBPS1 IS NOT THE PUTATIVE NONSYMBIOTIC HAEMOGLOBIN GENE

INTRODUCTION RESULTS

No /bps 1 transcript is detectable outside nodules

The Jbps1 promoter is unable to direct GUS expression in transgenic

Lotus roots or nodules DISCUSSION

METHODS

Plasmids

Northern analysis

Lbps1 promoter-GUS analysis in transgenic Lotus

CAT assays

Reporter gene analysis

CHAPTER 4

Amplification of a fragment of the soybean nonsymbiotic haemoglobin

gene by PCR 71

Isolation of soybean nonsymbiotic haemoglobin genomic clones 73

Soybean nonsymbiotic haemoglobin genome organisation 73

Expression pattern of the soybean nonsymbiotic haemoglobin gene 7 4

Isolation of a nonsymbiotic haemoglobin gene from Arabidopsis thaliana 75

Isolation of an Arabidopsis haemoglobin PCR fragment 75

Arabidopsis haemoglobin genome organisation 75

DISCUSSION 76

Novel haemoglobin genes from soybean and Arabidopsis 76

Evolution of plant haemoglobins 77

Possible functions of nonsymbiotic plant haemoglobin 79

MATERIALS AND METHODS 81

Isolation of genomic DNA 81

PCR reactions 82

Southern analysis 83

Isolation of soybean genomic clones 83

Northern analysis 83

CHAPTER 5

EVOLUTION OF PLANT HAEMOGLOBINS, NODULINS AND NODULATION 85

PLANT HAEMOGLOBIN GENE REGULATION 85

WHICH IS THE ANCESTRAL HAEMOGLOBIN GENE EXPRESSION

PATTERN IN SYMBIOTIC ROOT NODULES?

EVOLUTION OF PLANT HAEMOGLOBIN GENES

EVOLUTION OF NODULINS

REGULATION OF OTHER NODULINS AND THEIR HOMOLOGUES

POSSIBLE FUNCTION OF NONSYMBIOTIC HAEMOGLOBIN

87

88

91

93

EVOLUTION OF NODULATION 97 Symbiotic root nodules may have evolved as modifications of lateral roots 99

Nod factors may imitate plant signals 101

Nodulation as a controlled pathogenesis 101

Nitrogen-fixing symbioses involve both (Brady)Rhizobium and Frankia 103

CONCLUDING REMARKS 105

REFERENCES

LIST OF FIGURES following page

1.1. Alignment of the predicted amino acid sequences of various

leghaemoglobins. 4

1.2. Stages, sequence of events and involvement of nodulins in the

formation and function of a nitrogen-fixing root nodule. 9

1.3a. The basic 3-dimensional structure of the globins. 22

1.3b. The active centre of oxymyoglobin. 22

1.4. Alignment of key globin sequences of known structure. 22

1.5. Position of globin introns within the conserved a helices. 24

2.1. Alignment of the Parasponia andersonii and Trema tomentosa

haemoglobin promoter sequences. 39

2.2. Histochemical localisation of GUS activity. 40

2.3. Alignment of the 'nodulin box' motifs of haemoglobin gene promoters. 43

2.4. Site directed mutagenesis of promoter motifs. 43

2.5. GUS activity of mutant Parasponia and Trema haemoglobin promoters. 44

2.6. Summary of promoter elements involved in tissue specific regulation

of the Parasponia and Trema haemoglobin genes. 52

2.7. Schematic of Parasponia and Trema haemoglobin promoter-GUS

constructs. 56

3.1. Alignment of the predicted amino acid sequences of the

soybean leghaemoglobin genes. 59

3.2. Northern analysis of soybean /bps 1 expression. 61

3.3. GUS activity of the lbps1 promoter. 62

3.4. Alignment of the promoter regions of soybean lb genes. 64

3.5. Map of /bps 1 gene. 65

4.1. Alignment of the predicted amino acid sequences of various

plant haemoglobins. 72

4.3. Southern blot analysis of the soybean nonsymbiotic haemoglobin gene

and leghaemoglobin genes.

4.4. Two possible models of soybean nonsymbiotic haemoglobin

genome organisation.

4.5. Northern analysis of soybean nonsymbiotic haemoglobin gene

expression.

4.6. Sequence of the Arabidopsis haemoglobin 2f+4r2 PCR clone.

4.7. Southern blot analysis of the Arabidopsis haemoglobin gene.

4.8. Plant haemoglobin protein similarity tree.

4.9. Sequence alignment of the promoter regions of haemoglobin genes

from Casuarina, soybean, Parasponia and Trema.

5.1. A phylogenetic analysis of the polyphyletic class Hamamelidae.

LIST OF TABLES following page

2.1. Summary of GUS staining results for Parasponia and Trema

haemoglobin promoter constructs in transgenic Lotus.

4.1. Overall similarity between various plant haemoglobins.

5.1. Summary of nodule characteristics for legume, Parasponia and

actinorhizal plants.

73

74

74

75

76

77

78

90

41

71

page 4, line 14, change:

1984; to: 1984,

page 9, line 21, change:

15 40 to: 15, 40

page 15, line

12,

change:

taxanomically to: taxonomically

page 16, line 20-21, change: 'on

the tips of the hyphae of

Frankia' to: 'as terminal

swellings differentiating from short

branch

Frankia hyphae'.

page 16, line 25-26, change: 'which can

only fix nitrogen at very low ... ' to: 'which

cannot fix nitrogen even at very

low

...

'

page 20, line 3, change: et al to: et

al

page 30, line 26, change: requires

to: require

page 57, line 14, change: pIV21

to: pIV20

(Ramlov

et al, 1993)

page 58, line 18, change: 2-3um to:

2-3µm

following page 59, Figure 3.1

legend, line 5, change: proximal to: distal. line 6,

change: distal to: proximal.

page 65, line 14, change: absorbence

to: absorbance

page 102, line 9, change:

tumafaciens

to:

tumefaciens

t,:.:,,

'

..

:-;~

,

..

.~.

'i!

"" !,

i:

I

.

~a

"terminal

'

to:

t~. line o,

CHAPTER 1

GENERAL INTRODUCTION

LEGHAEMOGLOBIN AND THE LEGUME-(BRADY)RHIZOBIUM SYMBIOSIS

The legume-(Brady)Rhizobium symbiosis

The nitrogen-fixing root nodules of legumes develop as the result of a complex

interaction between the host plant and the invading bacteria. Within the controlled

environment of the nodule, the bacteria reduce atmospheric nitrogen to ammonia, thus

freeing the host plant from limitations in the soil nitrogen. In return, the bacteria are

supplied with carbohydrate and an environment compatible with nitrogen fixation.

The family Leguminosae (Fabaceae) is divided into three subfamilies:

Caesalpinioideae, Mimosoideae and Papilionoideae. Nodulation is general in the

Papilionoideae and Mimosoideae, but is less common in the Caesalpinioideae, with

only 23°/o of species examined found to nodulate (de Faria et al, 1989; Corby, 19_88).

The nodulating plants within the Papilionoideae have been the most extensively

studied, particularly members of the Loteae, Phaseloeae, Trifolieae and Viceae.

Nodule formation is induced by three genera of the Rhizobiaceae; Rhizobium,

Bradyrhizobium and Azorhizobium. These genera are quite distinct and are more

similar to nonsymbiotic relatives than to each other. For example, Rhizobium is closely

related to the plant pathogen Agrobacterium (Denarie et al, 1992). These diverse

bacteria are grouped in a single family by virtue of 'their common ability to establish a

nitrogen-fixing symbiosis with legumes' (Denarie et al, 1992).

Rhizobial genetics has been intensively studied (for review, Long, 1989).

Several groups of symbiotic genes have been defined, mostly by mutation. Nod

(nodulation) genes are required for nodule formation (Nod+ phenotype). Both nit and

fix genes are required for nitrogen fixation. Mutations in either produce ineffective

nodules (Nod+, Fix- phenotype). The nit genes probably encode components of the

nitrogen fixation machinery, while the fix genes are thought to be required for symbiotic

The interaction between legume species and Rhizobium strains is specific.

Bacterial strains nodulate a particular range of plants, having either a broad or narrow

host range. For example, R. meliloti nodulates alfalfa, B. japonicum nodulates

soybean, while Rhizobium sp. NG R234 is capable of nodulating a variety of tropical

legumes and Parasponia (Denarie et al, 1992).

Nodule formation is initiated by molecular signalling between a compatible

bacteria and plant. The legume produces flavonoids that induce expression of the

bacterial nodulation (nod) genes. Different legumes produce flavonoids with distinct

modifications that act as host determinants. Plant secreted flavonoids induce

expression qf the bacterial nodD gene, which in turn activates expression of other nod

genes, such as nodABC. nodABC are common nod genes, whereas other

host-specific nod genes are involved in determining host range (reviewed in Denarie et al,

1992; Spaink, 1992; Denarie and Cullimore, 1993). Together, the products of these

genes produce bacterial Nod factors, capable of initiating nodule organogenesis in the

host plant. Several bacterial Nod factors have been purified and shown to be

lipooligosaccharides (Lerouge et al, 1990; Spaink et al, 1991 ). Different bacterial species produce oligomers of ~-1,4-linked N-acetylglucosamine with different

substitutions, which have been shown to be important in determining host range.

The bacterial host-specific signals initiate homologous processes in different

legumes (Fisher and Long, 1992). The bacterial signal compound induces curling of root hairs in a susceptible zone behind the growing root tip. Only a small region close

to the zone of elongation is susceptible to infection by Rhizobia. Bacteria attach and invade the root hair by localised degradation of the cell wall. Formation of a specialised

structure called the infection thread allows the bacteria to penetrate the root.

The bacterial Nod factors also induce morphological changes in the plant root,

prior to bacterial infection. Cortical cell divisions are induced, giving rise to the nodule primordia prior to release of bacteria within the plant. Pericycle divisions are also

induced, and the two meristems fuse to produce a cluster of dividing cells (Brewin,

Infection threads enter individual nodule primordium cells. Endocytosis of the infection thread leads to formation of discrete structures, the symbiosomes, within the host cell cytoplasm. Within the symbiosomes, bacteria differentiate into bacteroids, their endosymbiotic form, and remain separated from the host cytoplasm by the host derived peribacteroid membrane (reviewed by Verma, 1992).

The mature nodule is derived from two cell lineages, the cortex and pericycle/ endodermis (Brewin, 1991 ). Differentiation of the nodule produces vascular connections and the various cell types needed for nodule function. The central nodule tissue contains both uninfected and infected, bacteroid containing cells. This tissue is surrounded by the nodule parenchyma, the nodule endodermis and the nodule cortex. The vascular bundles lie within the parenchyma.

The morphology and physiology of the mature root nodule creates an environment compatible with nitrogen fixation by the bacteroids. The tightly packed cells of the nodule inner cortex are thought to be important in the mechanical control of oxygen diffusion into the symbiotic zone (reviewed by Hunt and Layzell, 1993). Expression of the bacterial nitrogenase enzyme occurs late in the process. The mature nodule maintains a symbiotic zone where infected and uninfected cells assume different roles in nitrogen fixation, assimilation and transport. Fixed nitrogen in the form of ammonia is transported from the bacteroids to the plant cell cytoplasm and then to other parts of the plant (Nap and Bisseling, 1990b).

There are two main categories of nodule type (Nap and Bisseling, 1990b) although more complex classifications exist (Corby, 1988). Tropical legumes such as soybean, Lotus and Phaseo/us develop a spherical, determinate nodule, where the different stages in nodule development are separated temporally. Cell division is initiated in the outer cortex of the root and stops during nodule development and growth is a result of cell expansion rather than cell division. The vascular bundles meet,

persistent apical meristem that generates new cells that continue to be infected. In this

case, all developmental stages of nodule formation are present in a single nodule and

are separated spatially. A longitudinal section through an indeterminate nodule reveals

the entire series of developmental stages. The apical meristematic zone I is followed

consecutively by the zone of infection or prefixing zone II, the amyloplast-rich interzone

11/111, the nitrogen-fixing zone Ill, and the senescent zone IV (Vasse et al, 1990). Lupin

nodules are distinct in having an extensive attachment to the root, resulting in a

collar-like nodule that girdles the root (Corby, 1988).

Leghaemoglobin facilitates oxygen diffusion within nitrogen-fixing nodules

Plant haemoglobin was first identified in the nitrogen-fixing root nodules of

legumes (Kubo, 1939). The paradox of symbiotic nitrogen fixation is the requirement of

the bacteria for oxygen for respiration to meet the energy demands of nitrogen fixation

combined with a requirement for exclusion of free oxygen from the oxygen sensitive

nitrogenase enzyme complex (reviewed by Appleby, 1984; in press). Leghaemoglobin

facilitates the controlled diffusion of oxygen within symbiotic root nodules to respiring

bacteria while maintaining the local free oxygen as low as 10-20 nM. Within this low

oxygen environment, the concentration of leghaemoglobin-bound oxygen may be as

high as 600µM (Appleby, in press). The bound oxygen is available to the bacteroids

for respiration as it dissociates from the leghaemoglobin, but the free oxygen

concentration remains non-toxic.

All legumes so far examined appear to express high levels of nodule specific

leghaemoglobin (Konieczny, 1987; Marcker et al, 1984; Lehtovaara and Ellfolk, 1975;

Bogusz et al, 1987; Kiss et al, 1987; Gallusci et al, 1991; Richardson et al, 1975;

Lehtovaara et al, 1980). The amino acid sequences of many leghaemoglobins have

been determined, and all show extensive homology (see Figure 1.1 ). Leghaemoglobin

is the most abundant nodule protein, comprising up to 15-20°/o of total protein (Fuller et

leghaemoglobins.

Protein sequences were aligned using the GCG Pileup program. The

highly conserved residues involved in haem and ligand binding are in bold.

These are the distal and proximal histidines, phenyalanine CD1 and proline C2

(Riggs, 1991 ). lupin lb1 (Konieczny, 1987), lupin lb2 (Konieczny et al, 1987),

soybean Iba and lbc1 (Hyldig-Nielsen et al, 1982), soybean lbc2 and lbc3 (Wiborg et al, 1982), kidney bean lb (Lee and Verma, 1984), soybean lbps1 (Wiborg et al,

1983), Sesbania lbll (Metz et al, 1988), Sesbania lbVII (Welters et al, 1989), alfalfa lblll (Kiss et al, 1987), Medicago lb1 and lb2 (Gallusci et al, 1991 ),

broadbean lb (Richardson et al, 1975), pea lb (Lehtovaara et al, 1980).

,: .. r·r;:r,

,. •. -1·· ::i'~:;: .. :e:c .. t, ....

.

:;:"'a:'. r ... -:~· ,.;.,. ....

•• - .. 1,,, .. ;::-·

t:a.:a.: ""fl',.~,. ... _{... •;··}

~, ... , ""':.~· ;,.." ...

.... " .. ...

..

~=1·.x:

:;!·:tt .. ~c :•·>?1;:• ,.,. ... ! ...

"""'' 1°1::· \.,..,c ~ "' ..

~:: .. ~s .. ::::

...

::~:.:.: .. : ...

·:::::;: ..

..

.

!" ...

...

"•1 .. ,.~ .. : "1 ... ••

"!" ... :

~,:1,:::;

"~·· "]· ...

"··

:e;:-~::c

...

.. t,::

::\r~;:-~> ...

soylbc3 GAFTDKQEAL VSSSFEAFKT NIPQYSWFY TSILEKAPVA KDLFSFLAN . . GVDPTNPKL soylbc2 GAFTEKQEAL VSSSFEAFKA NIPQYSWFY TSILEKAPAA KDLFSFLSN . . GVDPSNPKL m. The soybeanlba VAFTEKQDAL VSSSFEAFKA NIPQYSWFY TSILEKAPAA KDLFSFLAN . . GVDPTNPKL

soybeanlbc GAFTEKQEAL VSSSFEAFKA NIPQYSWFY NSILEKAPAA KDLFSFLAN. . GVDPTNPKL . In cold. kidney bean GAFTEKQEAL VNSSWEAFKG NIPQYSWFY TSILEKAPAA KNLFSFLAN. .GVDPTNPKL

soylbpsl GAFTEKQEAL VNSSFEAFKA NLPHHSWFF NSILEKAPAA KNMFSFLGD . . AVDPKNPKL

11 e C2

sesblbvii .GFTEKQEAL VNASYEAFKQ NLPGNSVLFY SFILEKAPAA KGMFSFLKDF DEVPQNNPSL sesbanlbii .GFTDKQEAL VNASYEAFKK NLPGHSVLFY SFILEKEPAA KGLFSFLKDS DGVPQNNPSL

I, 1987), medicago2

.GFTEKQEAL VNSSWELFKQ N.PGNSVLFY TIILEKAPAA KGMFSFLKDT AGV.QDSPKL

alfalfalb .GFTDKQEAL VNSSWESFKQ N.PGNSVLFY TIILEKAPAA KGMFSFLKDS AGV.QDSPKL medicagol .SFTDKQEAL VNSSYEAFKQ NLSGYSVFFY TVILEKAPAA KGLFSFLKDS AGV.QDSPQL

rll!oor~

~r~ el al,

61 120

1 , 1989),

lupin2lb QAHAGKVFKL VYEAAIQLQV TGVWTDATL KNLGSVHVSK GVADAHFPW KEAILKTIKE lupinllb QAHAGKVFKL TYEAAIQLQV NGAVASDATL KSLGSVHVSK GVVDAHFPW KEAILKTIKE soylbc3 TGHAEKLFGL VRDSAGQLKA SGTW.IDA. .ALGSIHAQK AITDPQFVW KEALLKTIKE

I 1991), soylbc2 TGHAEKLFGL VRDSAGQLKA NGTW.ADA. .ALGSIHAQK AITDPQFVW KEALLKTIKE

soybeanlba TGHAEKLFAL VRDSAGQLKA SGTW.ADA. .ALGSVHAQK AVTDPQFVW KEALLKTIKA soybeanlbc TGHAEKLFAL VRDSAGQLKT NGTW.ADA. .ALVSIHAQK AVTDPQFVW KEALLKTIKE kidney bean TAHAESLFGL VRDSAAQLRA NGAW.ADA. .ALGSIHSQK GVNDSQFLW KEALLKTLKE soylbpsl AGHAEKLFGL VRDSAVQLQT KGLW.ADA. .TLGPIHTQK GVTDLQFAW KEALLKTIKE sesblbvii QAHAEKVFGL VRDSAAQLRA TGVWLADA. .SLGSVHVQK GVLDPHFVW KEALLKTLKE sesbanlbii QAHAEKVFGL VHDAAGQLRA TGVWLADA. .SLGSVHVQK GVTDPHFVW KEALLKTLKE medicago2 QSHAEKVFGM VRDSAVQLRA TGGWLGDA. .TLGAIHIQK GVVDPHFVW KEALLKTIKE alfalfalb QSHAEKVFGM VRDSAAQLRA TGGWLGDA. .TLGAIHIQK GVVDPHFAW KEALLKTIKE medicagol QAHAEKVFGL VRDSASQLRA TGGWLGDA. .ALGAIHIQK GVVDPHFVW KEALLKTIKE broadbean GAHAEKVFGM VRDSAVQLRA TGEWL.DG. .KDGSIHIQK GVLDPHFVW KEALLKTIKE pealb QAHAEQVFGL VRDSAAQLRT KGEWLGNA. .TLGAIHAGK GVTNPHFVW KEALLQTIKK

121 155

lupin2lb WGAKWSEEL NSAWTIAYDE LAIVIKKEMN DAA .. lupinllb WGDKWSEEL NTAWTIAYDE LAIIIKKEMK DAA .. soylbc3 AVGDKWSDEL SSAWEVAYDE LAAAIKKAF. .

....

soylbc2 AVGDKWSDEL SSAWEVAYDE LAAAIKKAF. .

....

soybeanlba AVGDKWSDEL SRAWEVAYDE LAAAIKKA .. .

....

soybeanlbc AVGGNWSDEL SSAWEVAYDE LAAAIKKA .. .

....

kidneybean AVGDKWTDEL STALELAYDE LAAAIKKAYA ... soylbpsl AVEDKWSEEL SNAWEVAYDE IAAAIKKAMA IGSLV sesblbvii AGGATWSDEV SNAWEVAYDE LSAAIKKAMS

...

...

medicago2 VSGDKWSEEL STAWEVAYDA LAAAIKKAMG ... alfalfalb VSGDKWSEEL NTAWEVAYDA LATAIKKAMV

...

....

cells (Verma and Long, 1983; Appleby, 1984), prior to the onset of nitrogen fixation

(Marcker et al, 1984; Verma et al, 1979; de Billy et al, 1991 ).

The soybean leghaemoglobins have been well characterised. Soybean nodules

contain four major leghaemoglobins, Lba, Lbc1, Lbc2 and Lbc3, as well as four minor

components, Lbb, Lbd1, Lbd2 and Lbd3, which result from N-terminal acetylation of the

major components (Fuschmann and Appleby, 1979). These leghaemoglobins are

encoded by a small family of four functional genes, one apparent pseudogene and two

or three truncated genes (Brisson and Verma, 1982; Hyldig-Nielsen et al, 1982; Wiborg

et al, 1982; 1983). The genes are arranged in two independent clusters, one

comprising 5_' Iba - /bc1 - lbps1 - lbc3 - 3' and the other 5' - lbps2- /bc2- 3' where lbps2

is a truncated gene (Bojsen et al, 1983; Brisson and Verma, 1982). The genes show

extensive homology extending into the 5' upstream region. The genes in both clusters

have the same transcriptional polarity (Bojsen et al, 1983). Induction of the four

soybean leghaemoglobin genes occurs sequentially in the opposite order to their

arrangement on the chromosome (Marcker et al, 1984). Transcription of lbc3, lbc2 and

lbc1 starts slightly before the appearance of visible nodules while Iba initiates after

another day or two. At around day 12, there is a dramatic increase in the level of

transcription of Iba, lbc3 and /be 1, concomitant with the transcription initiation of several

other nodule specific genes and prior to detectable nitrogenase activity. All four

leghaemoglobins continue to be expressed for as long as the nodule is functional. The

functional significance of this differential regulation of transcriptional activation is not

known. Expression of the apparent pseudogene /bps 1 has not been detected

(Marcker et al, 1984; Wiborg et al, 1983), but as the gene encodes an apparently

functional leghaemoglobin (Wiborg et al, 1983) it has been proposed that /bps 1 may

represent a functional haemoglobin gene (Nap and Bisseling, 1990b,c). This possibility

is examined in Chapter 3.

The leghaemoglobins of the tropical legume Sesbania rostrata have also been

extensively studied. The symbiosis between Sesbania and Azorhizobium caulinodans

stem nodules are unusual in that chloroplast containing cells are immediately adjacent

to bacteroid containing cells. Oxygen evolution associated with photosynthesis may

put an additional stress on the oxygen-sensitive nitrogenase enzyme complex, and it is

possible that stem nodules have adapted to the higher levels of oxygen by producing

modified or higher levels of leghaemoglobin (Dehio and de Bruijn, 1992). Sesbania

contains seven major leghaemoglobins in its stem and root nodules, of which at least

six are separate gene products (Bogusz et al, 1987). One form, LbVI appears to be

differentially regulated as it is five times more abundant in stem nodules than root

nodules (Bogusz et al, 1987) suggesting that it may play a specific role in stem

nodules. Two Sesbania leghaemoglobin genes have been isolated and studied, Srglb2

(encoding LbVI) and Srglb3 (LbVII) (Metz et al, 1988; Welters et al, 1989). The two

genes show a high level of homology, with only 6 nucleotide differences, leading to 4

predicted amino acid changes (Metz et al, 1988). The homology between the two

Sesbania genes, as well as the soybean leghaemoglobin genes, extends into the 5'

untranslated region.

Leghaemoglobin as the archetype symbiotic protein

The functional leghaemoglobin holoprotein consists of the monomeric globin

apoprotein and a haem prosthetic group. It has been clearly established that the

apoprotein is plant encoded, but the origin of the haem moiety is still unclear.

Biochemical studies localising 8-aminolevulinic acid synthase activity, the first

committed step in the haem biosynthetic pathway, to the bacteroid fraction of root

nodules (Nadler and Avissar, 1977; Cutting and Schulman, 1969) led to the idea that

leghaemoglobin was comprised of plant encoded globin and Rhizobium provided

haem. Leghaemoglobin has come to be regarded as the classic symbiotic protein, with

components provided by each symbiotic partner to produce a functional holoprotein.

Several studies have used haem deficient (Bradl)Rhizobial mutants in an effort

A 8. japonicum hemA mutant, deficient in 8-aminolevulinic acid synthetase,

induced fully effective soybean nodules that contained haem (Guerinot and Chelm,

1986). In contrast, a hemH mutant, deficient in protoporphyrinogen oxidase activity,

the penultimate step in haem biosynthesis, induced ineffective soybean nodules

(O'Brian et al, 1987). These conflicting results can be reconciled by evidence that the

hemA mutant bacteroids can synthesise haem from imported plant 8-aminolevulinic

acid (Sangwan and O'Brian, 1991 ). Nodules induced by the hemH mutant contained

the globin apoprotein but not the holoprotein, implying that bacterial haem synthesis is

required for leghaemoglobin but not apoleghaemoglobin synthesis (O'Brian et al, 1987).

This is the strongest evidence that the leghaemoglobin haem is provided by the

bacteria.

An R. meliloti hemA mutant formed ineffective nodules lacking leghaemoglobin

on inoculation of alfalfa (Leong et al, 1982). de Bruijn et al (1989) also found that

alfa.lfa nodules induced by a hemA mutant did not express leghaemoglobin. However,

Mohapatra and Puhler (1986) found that a hemA mutant of R. meliloti, although

ineffective, induced nodules apparently containing near wild-type levels of

haemoglobin. A hemA mutant of Azorhizobium caulinodans ORS571 also induced

ineffective root nodules lacking leghaemoglobin on inoculation of Sesbania (Pawlowski

et al, 1993).

These results were seen as further evidence that the haem group is bacterial in

origin. However, a recent reexamination of the R. meliloti hemA mutants used by

Leong et al (1982), Mohapatra and Puhler (1986) and de Bruijn et al (1989) found that

these nodules failed to express any of six nodule specific genes tested, including

leghaemoglobin (Dickstein et al, 1991 ). So it is unlikely that the ineffective symbiosis is

a "direct consequence of the failure of R. meliloti to supply the heme moiety of

hololeghemoglobin.11

Dickstein et al (1991) confirmed that symbiotic effectiveness of

the hemA mutants could be restored by an exogenous supply of 8-aminolevulinic acid

(Leong et al, 1982). They propose that the hemA mutant may be unable to properly

mutants induce nodules with atypical morphology and infection thread formation ( either

empty nodules with no infection threads or infection threads that failed to release

bacteria) (Dickstein et al, 1991; Pawlowski et al, 1993) and other evidence shows that

leghaemoglobin expression is dependent on bacterial release from the infection

threads (Dunn et al, 1988; Norris et al, 1988).

It appears -that bacterial haem synthesis is essential for an effective symbiosis

in the alfalfa-A. meliloti and Sesbania-A. caulinodans symbioses. However, in both

these cases the hemA mutation has multiple effects and its influence on

leghaemoglobin production appears general rather than specific. There are several

alternative explanations for the importance of bacterial haem in establishing an

effective symbiosis. Rhizobial bacteroids synthesise a terminal oxidase with high

oxygen affinity in order to function in the low free oxygen environment of the symbiotic

nodule (reviewed by Appleby, 1984). The B. japonicum symbiotic oxidase appears to

comprise several haem containing subunits including a haem binding oxidase subunit,

a dihaem cytochrome c and a monohaem cytochrome c (Preisig et al, 1993).

Mutations in the oxidase gene cluster, fixNOQP, result in defective nodules that are

white or greenish and contain few bacteroids, a phenotype similar to that of hemA

mutants. Also, the oxygen sensing regulatory system which triggers expression of

most of the genes required for nitrogen fixation includes an oxygen binding

haemoprotein, Fixl (discussed further on page 21 ). A haem-dependent

symbiosis-specific oxidase complex and haem-dependent activation of nitrogen fixation genes is

likely to account for the ineffective phenotype of haem deficient (BradY)Rhizobium

mutants.

Recent evidence demonstrating that plant synthesis of coproporphyrinogen

oxidase and 8-aminolevulinic acid increases concomitant with leghaemoglobin

expression, suggests the plant increases haem production to meet increased demand

in the nodules (Madsen et al, 1993; Sangwan and O'Brian, 1992) and thus that the

NODULINS ARE PLANT PROTEINS SPECIFICALLY INVOLVED IN NODULATION Nodule formation is the result of a coordinated reponse of both the bacteria and

the plant which requires the sequential induction of both bacterial and plant genes.

Many plant genes are required for the development and function of the nodule.

Host-encoded nodule-specific proteins, such as the well characterised leghaemoglobins, are

referred to as nodulins. By definition, nodulins are exclusively expressed during

development of the symbiosis, although there are many proteins that show enhanced,

rather than specific, nodule expression. Nodulins are distinguished for convenience on

the basis of time of expression. Early nodulin genes are expressed during

development 9f the nodule structure. Their gene products are thought to be involved in

bacterial invasion and in nodule formation, although the precise function of most early

nodulins is not known. Late nodulin gene expression, such as that of leghaemoglobin,

generally follows release of bacteria but precedes the onset of nitrogen fixation. Late

nodulins probably function in establishing and maintaining a nodule environment

compatible with nitrogen fixation (reviewed by Nap and Bisseling, 1990b,c; Verma and

Miao, 1992). The relationship of nodulin expression and nodule development and

function is summarised in Figure 1.2. Nodulins have been most extensively studied in

the legume-(Brady)Rhizobium symbiosis.

Early nodulins are involved in nodule formation

Several early nodulins (ENODs) have been isolated and characterised,

including ENOD2, 3, 5, 13, 15 40 and 55 (Franssen et al, 1987, 1990; Scheres et al,

1990a, b; Vijn et al, 1993; de Blank et al, 1993). Activation of early nodulin expression seems to be independent of the attachment of bacteria as purified Nod signal compounds can induce some of these genes, as can auxin-transport inhibitors or

'

PRElNFECTION INFECTION ANO NODULE FORMATION

STAGE I STAGE lI

-·· .;_

-

-~-~

·---~

V

,;sJ.;,

.A TT .A CHM ENT

/ / IACTERI.AC. )olUl Tll'LJCA. TION

RELEASE OF BACTERIA MOOUL( IHITI.A TIOH

IHF'ECTION THR(.AO GROWTH

ll'IF(C TION THRE.AO FOR>,4A TIOH

CORTICJ.C. cnL OIVlSIOHS

NODULE FUNCTION ANO MAINTENANCE

STAGE m

NODULE SENESCENC~

STAGE I7

MEMBIUN[ OISIH rEGIU TIOH

CC:Ll OISIN fEC.RA TIOH

HOOULE BRE.AXOOWH

Figure 1.2. Stages, sequence of events and involvement of nodulins in the formation

and function of a nitrogen-fixing legume root nodule. From Gloudemans and Bisseling

(1989). Many early and late nodulins have been characterised (see text for details).

ENOD12 appears to be involved in the preinfection stage (Scheres et al, 1990b; Pichon

et al, 1992). A protease inhibitor expressed specifically in the infected cells of

senescent nodules has been identified (Manen et al, 1991).

developmental stages in pea nodules. ENOD12 transcript is 'present in every cell of

the invasion zone, whereas ENOD5, ENOD3 and ENOD14 transcripts were restricted

to the infected cells in successive but partially overlapping zones of the central tissue'

(Scheres et al, 1990a).

ENOD2 AND ENOD12 ARE CELL WALL PROTEINS

ENOD2 is expressed exclusively in the parenchyma (inner cortex) of soybean,

pea and alfalfa nodules (van de Wiel et al, 1990a). mRNA expression is detectable as

soon as meristem cells differentiate into inner cortical cells. In soybean, expression is

transient, wh~reas in pea, ENOD2 transcript accumulates during development. This

may reflect the different developmental pattern of determinate nodules, where mitotic

activity ceases shortly after the onset of nitrogen fixation, in contrast to the persistent

meristem of indeterminate nodules (Nap and Bisseling, 1990b). Expression of alfalfa

ENOD2 is also induced in empty (bacteria-free) alfalfa nodules formed by R. meliloti

mutants defective in exopolysaccharide synthesis, in pseudo-nodules induced by auxin

transport inhibitors (Van de Wiel et al, 1990b), and in the spontaneous nodules formed

on some alfalfa roots in the absence of Rhizobial infection (Truchet et al, 1989).

Expression of the ENOD2 gene of Sesbania is induced by a variety of cytokinins (Dehio

and de Bruijn, 1992).

The presence of a signal transit peptide and the characteristic praline-rich

pentapeptide repeat of ENOD2 (Franssen et al, 1987; 1990) resembles that of

extensin, a major structural component of the cell wall, and a soybean praline rich

protein, 1A10, expressed in the cell wall of germinating seedlings. It is therefore likely

that ENOD2 is a structural cell wall protein. The expression of ENOD2 in the nodule parenchyma, as well as its expression in soybean nodules devoid of bacteria and infection threads (Franssen et al, 1987) suggests a role in the formation of the nodule

de Bruijn, 1992; Verma and Miao, 1992) possibly by limiting cell wall expansion,

producing the small tightly packed cells of the inner cortex (Brewin, 1991 ).

ENOD12 also contains a putative signal peptide and a series of two

pentapeptide repeats (Govers et al, 1991 ). ENOD12 is expressed in the root cortical

cells containing the infection thread, in cells preparing for infection thread passage and

in dividing inner cortical cells that form the nodule primordium (Scheres et al, 1990b;

Pichon et al, 1992). ENOD12 is expressed transiently in root hairs of the infection

zone, while expression is enhanced and maintained in root hairs that are subsequently

infected (Pichon et al, 1992). In nodules, transcript is present in the invasion zone and

at decreasing_ levels in the early symbiotic zone. Because of its homology with ENOD2

and 1A10, and as all root and nodule cells containing ENOD12 transcript are sites of

new cell wall synthesis, ENOD12 is assumed to also be a cell wall protein (Scheres et

al, 1990b). As ENOD12 is expressed at a different stage and in different cell types to

ENOD2 it probably fulfils a different role in nodule formation.

Late nodulins are involved in nodule function

Leghaemoglobin is the most abundant and best characterised late nodulin.

Many other late nodulins function as the key enzymes of nitrogen assimilation and

carbon metabolism. Factors involved in the activation of late nodulins are clearly

different from the early nodulins. Expression of many late nodulins occurs prior to nitrogen fixation. While expression does not occur without the formation of infection

threads and release of bacteria, it does occur in nodules induced by some

Fix-mutants. Induction is triggered either by bacterial infection or in some way by the

unique nodule environment created during the symbiotic state. The expression and

regulation of late nodulins has been reviewed recently by Sanchez et al, 1991; Verma and Miao, 1992; Nap and Bisseling 1990b, c.

There are nodule-specific or nodule-abundant forms of several enzymes of the

nitrogen and carbon assimilation pathways. These include glutamine synthetase,

glutamate synthase, sucrose synthase, phosphoenolpyruvate carboxylase, aspartate

aminotransferase, uricase, malate dehydrogenase and xanthine dehydrogenase.

Glutamine synthetase (GS) catalyses the first reaction in the assimilation of

ammonia produced by the bacteroids. Cytosolic GS is encoded by a small family of

genes. Several legumes, such as soybean, alfalfa and lupin, have nodule specific

isoforms (Roche et al, 1993; Dunn et al, 1988). However, in pea, Phaseolus and

Medicago truncatula, GS isoforms show enhanced nodule expression but are also

expressed i~ other parts of the plant (Forde et al, 1989; Stanford et al, 1993). It

appears that the nodule GS form is located exclusively in the cytosol of the infected

cells, while another, highly homologous GS is expressed in uninfected nodule tissue

and in other parts of the plant (Forde et al, 1989). Differentially expressed GS genes

produce functionally similar enzymes (Nap and Bisseling, 1990b).

A nodule specific form of sucrose synthase has been isolated from soybean

nodules (Thummler and Verma, 1987). Sucrose synthase is the key enzyme involved

in breakdown of sucrose to the monosaccharides glucose and fructose and plays three

key roles in nodules: to sustain respiration in the plant and bacteroids; to provide

substrate for cell wall synthesis and; to provide carbon skeletons for the assimilation of

fixed nitrogen (Thummler and Verma, 1987).

PERIBACTEROID MEMBRANE NODULINS

The host-derived peribacteroid membrane (PBM) surrounding the bacteroids is the main control point for nutrient and signal exchanges between the Rhizobium and the host. The PBM nodulins are likely to be involved in transport across the membrane, as is the dicarboxylic acid transporter identified in the soybean PBM

N-26 is a transmembrane protein with six membrane-spanning domains. The

protein is cotranslationally glycosylated and inserted into membranes and is

phosphorylated near the carboxy terminus (Miao et al, 1992). The phosphorylation may be directly related to the function of N-26. Based on the homology of N-26 to

several eukaryotic and prokaryotic intrinsic channel-type proteins, it has been

suggested that N-2b may form a channel translocating specific molecules between the

bacteroids and the host cytoplasm (Miao et al, 1992; Miao and Verma, 1993).

Origin of nodulins

It ha~ been hypothesised that nodulin genes evolved by gene duplication of

preexfsting plant genes to fit the developmental and physiological requirements of

symbiotic nitrogen fixation (Nap and Bisseling, 1990b,c; Verma and Miao, 1992).

Preexisting genes may have been modified and brought under nodule developmental

control. These nodulin genes remain recognisably homologous to the progenitor gene

(Nap and Bisseling, 1990b). It is therefore expected that all nodulin genes will have a

nonsymbiotic counterpart expressed in non-nodule tissue. This is the case for many of

the well-characterised nodulins that have been assigned a biochemical role, such as

glutamine synthetase, glutamate synthase and sucrose synthase, as well as ENOD2

and N-26 (Dehio and de Bruijn, 1992; Miao and Verma, 1993). The nonsymbiotic form

of the archetype nodulin, leghaemoglobin, has been difficult to identify. This thesis describes the isolation of a soybean haemoglobin gene homologous to the

nonsymbiotic haemoglobin genes previously identified in nonlegumes (Chapter 4).

PARASPON/A HAEMOGLOBIN AND THE PARASPON/A-(BRADY)RHIZOB/UM

SYMBIOSIS

The Parasponia/(Brady)Rhizobium symbiosis

legumes, infection of Parasponia roots by (Brady')Rhizobium occurs in the zone of elongating cells directly behind the root tip which is transiently, highly susceptible to

infection (Scott and Bender, 1990). However, there is no root hair curling or formation

of infection threads (Lancelle and Torrey, 1984; Scott and Bender, 1990).

Caetano-Anolles and Gresshoff (1991) are of the opinion that bacteria invade the host by taking

advantage of the emergence of a lateral root. However, others suggest that cells

exposed by emerging lateral roots are not susceptible to invasion (Lancelle and Tor~ey,

1984; Scott and Bender, 1990). Scott and Bender (1990) have observed degradation

of the mucilage layers in the primary cell walls of epidermal cells in association with

bacterial contact. Erosion of the root surface results in localised root swelling

producing a break in the epidermis (Lancelle and Torrey, 1984; Scott and Bender,

1990). The bacteria induce cortical cell divisions, forming the prenodule, which

emerges through the epidermal break, forming an exposed site for intercellular

colonisation by bacteria. These initial cortical divisions cease and are followed by

pericycle divisions, giving rise to the nodule lobe primordia, which is invaded by

Rhizobium (Lancelle and Torrey, 1985). The mature nodule tissue is derived from these pericycle, rather than cortical, cell divisions. Thus, the Parasponia nodule has the same ontogeny, and so similar morphology, to that of a lateral root (Caetano-Anolles and Gresshoff, 1991 ). The mature nodule has a central vascular bundle and

persistent apical meristem (Trinick, 1979; Lancelle and Torrey, 1985; Scott and Bender,

1990). The central zone of the nodule contains both infected and uninfected cells. Within the infected cells, the Rhizobia remain within host-derived thin-walled 1

infection1

threads, more appropriately called fixation threads, rather than differentiating into

symbiosomes (Trinick, 1979; Lancelle and Torrey, 1985; Scott and Bender, 1990).

Haemoglobin is expressed in nodules of Parasponia and also in roots of Parasponia and Trema

conserved amino acid residues (Appleby et al, 1983; Kortt et al, 1988). The

Parasponia haemoglobin has similar binding kinetics to the leghaemoglobins (Appleby

et al, 1988) and functions, like leghaemoglobin, to provide oxygen to the bacterial

symbiont at a low free oxygen concentration. lmmunogold labelling has localised

Parasponia haemoglobin expression to the infected cells within the nodule (Trinick et

al, 1989). P. andersonii has a single haemoglobin gene which is differentially

expressed to give high expression in nodules (Landsmann et al, 1986), and also

approximately 1000 fold lower expression in roots (Bogusz et al, 1988).

The genus Parasponia (7 species) is the only member of the subfamily

Celtidoideae (12 genera) to form nodules. The related genus Trema (55 species) is so

morphologically close to Parasponia that the two genera were initially confused

taxanomically (Akkermans et al, 1978a,b). Trema tomentosa is non-nodulating in the

wild and also lacked nodule formation after inoculation with 98 representative test

strains of (Brady)Rhizobium under controlled conditions (Bogusz et al, 1988). Trema

tomentosa also has a single haemoglobin gene with 93°/o homology to that of Parasponia, which is also expressed in the roots (Bogusz et al, 1988). This represents

the first isolation of a functional haemoglobin gene from a non-nodulating plant. The

close taxonomic relationship of Parasponia and Trema suggests the possibility that

Trema has recently lost the ability to nodulate, but still retains a functional haemoglobin

gene homologous to that of Parasponia. However, a Parasponia haemoglobin DNA

probe also detected haemoglobin-like sequences in the genome of Ce/tis australis, another non-nodulating member of the Ulmaceae (Bogusz et al, 1988).

HAEMOGLOBIN IN ACTINORHIZAL NODULES AND THE ACTINORHIZAL SYMBIOSES

The actinorhizal symbioses

Well known members of the group include Casuarina glauca (river she-oak), Myrica

gale (sweet gale) and A/nus rubra (alder). The actinorhizal plants rival legumes in

overall amounts of biological nitrogen fixation, but the symbiosis is less well understood (reviewed by Newcomb and Wood, 1987).

The nitrogen-fixing endophyte, Frankia, is a gram positive, filamentous

prokaryote which often exhibits hyphal growth. It is not yet clear whether

host-specificity exists to the same extent as in the (Brady)Rhizobia, although there are at

least two major inoculation groups (Newcomb and Wood, 1987).

As in the legumes, the first step in nodule initiation is usually invasion of a

deformed root hair, but can also occur by intercellular penetration of the root epidermis

(Newcomb and Wood, 1987). As in Parasponia nodules, transitory cell division is stimulated in the root cortex, forming the prenodule, followed by pericycle divisions,

giving rise to the nodule primordium. Actinorhizal nodules also resemble modified

lateral roots. The nodules are generally multi-lobed with each lobe being a modi.fied

root with a central vascular cylinder {Tjepkema et al, 1986). The nodules are

indeterminate, having a persistent apical meristem. As the nodule differentiates, the

intercellular bacteria invade the newly divided cells of the nodule cortex (Mullin, 1993).

Frankia hyphae are separated from the host cytoplasm by a host-derived structure resembling an infection thread (Newcomb and Wood, 1987).

The nitrogenase enzyme complex is expressed in symbiotic vesicles formed on

the tips of the hyphae of Frankia. These vesicles are also formed in vitro under

aerobic, but not hypoxic, conditions. Cultures of Frankiae are capable of fixing nitrogen

at atmospheric concentrations of oxygen, despite the oxygen sensitivity of the nitrogenase. It appears that the cell wall layers of the vesicles provide a physical

barrier against oxygen diffusion. This is in contrast to free-living Rhizobia, which can only fix nitrogen at very low concentrations of oxygen.

The observation that Frankia can fix nitrogen at atmospheric concentrations of oxygen may suggest that actinorhizal symbioses do not require haemoglobin to act as

an oxygen buffer. Haemoglobin is required in legume nodules, not only because

Rhizobia have no means of buffering against oxygen, but because the structural barrier of the legume nodule results in a low free oxygen concentration, requiring facilitated

transport of oxygen to the endophyte within the nodule. Many actinorhizal nodules do

not have such a diffusion barrier and have atmospheric concentrations of oxygen within

the cortex. There is no need for facilitated oxygen diffusion and haemoglobin would

presumably have little effect. However, haemoglobin is found in varying concentrations

in actinorhizal nodules, and is known to occur at relatively high concentrations in the

nodules of Casuarina, A/nus and Myrica (Tjepkema, 1983; Tjepkema et al, 1986). This correlates with the low free oxygen concentration known to exist in the nodules of

Myrica and Casuarina (Tjepkema, 1983b; Newcomb and Wood, 1987).

Casuarina glauca, an Australian tree, has a small family of symbiotic haemoglobin genes expressed exclusively in the root nodules (Fleming et al, 1987; Jacobsen-Lyon et al, in preparation). C. glauca also has a distinct haemoglobin gene expressed in roots and at a lower level in leaves/stems and nodules (Christensen et al,

1991; Jacobsen-Lyon et al, in preparation). The predicted amino acid similarity between the symbiotic and nonsymbiotic haemoglobins is 64°/o.

NONSYMBIOTIC PLANT HAEMOGLOBINS AND POSSIBLE FUNCTION

Recently, the serendipitous discovery of a haemoglobin cDNA from barley

(Hordeum vulgare) was reported (Taylor et al, 1993). The deduced amino acid sequence shows significant homology to other nonlegume haemoglobins as well as to haemoglobins generally (Taylor et al, in press). The barley haemoglobin gene is expressed in the roots as well as coleoptiles and the aleurone layer. Presence of a functional haemoglobin gene in a monocot, as well as expression of the Parasponia

function other than in nitrogen-fixing symbioses, and that haemoglobin is likely to be

present throughout the angiosperms (Appleby et al, 1988; 1990).

The function of nonsymbiotic plant haemoglobin is not known. Two possible

roles have been proposed: as a facilitator of oxygen diffusion in rapidly dividing cells, or

as an oxygen sensor involved in switching plant metabolism to anaerobic pathways

during hypoxic stress (Appleby et al, 1988; Bogusz et al, 1990). These possibilities will

be discussed further in Chapters 4 and 5. The possible functions of nonsymbiotic plant

haemoglobins may best be considered with regard to the known functions of

haemoglobins in other systems.

THE COMMON FUNCTION OF HAEMOGLOBIN IS REVERSIBLE BINDING OF

OXYGEN

Haemoglobin, present in vertebrates, many invertebrate phyla, bacteria, fungi

and some higher plants, can be broadly defined as a haem protein capable of

reversibly binding oxygen. The precise function of haemoglobin varies in different

systems, ranging from oxygen storage and transport, sensing of oxygen tension and

acting as a terminal oxidase (reviewed by Riggs, 1991; Wittenberg and Wittenberg, 1990).

Some haemoglobins are involved in oxygen transport

Almost all vertebrates have a tetrameric haemoglobin, comprised of two a and

two ~ subunits, which is expressed in erythrocytes and functions in intercellular oxygen

transport through the cardiovascular system. Vertebrates also have a monomeric myoglobin that facilitates diffusion of oxygen to the mitochondria of muscle and heart

tissue, making oxygen available to the respiratory machinery at low free oxygen tensions (reviewed by Wittenberg and Wittenberg, 1990). There is evidence that

The filamentous bacterium Vitreoscilla produces a cytoplasmic homodimeric

haemoglobin that shows 24°/o amino acid sequence homology to that of lupin

leghaemoglobin (Wakabayashi et al, 1986). Haemoglobin levels increase 50-fold when

the bacteria are grown under hypoxic conditions. Expression of Vitreoscil/a

haemoglobin is regulated by oxygen at the level of transcription (Dikshit et al, 1989).

The cooperative binding of Vitreoscilla dimeric haemoglobin has led to the suggestion

that the deoxy/oxy state of the protein may be the sensor molecule of oxygen

concentration leading to stimulation of its own production (Dikshit et al, 1989). An

associated protein, NADH-methaemoglobin reductase, serves to keep haemoglobin in

the active, ferrous form (Dikshit et al, 1989). The Vitreoscilla globin gene expressed in

E. coli allows the cells to grow faster and to greater cell densities (Khosla and Bailey,

1988). Vitreoscilla haemoglobin may function as an oxygen trap, facilitating diffusion to

the respiratory membranes (Wakabayashi et al, 1986).

Some haemoglobins are linked to enzymatic domains

Several haemoglobins are known to be two-domain proteins with an N-terminal

haem binding group linked to domains with various enzymatic activities:

dihydropteridine reductase in Escherichia coli (Vasudevan et al, 1991 ), and a

flavoprotein reductase in Alcaligenes (Weihs et al, 1989; Zhu and Riggs, 1992),

Saccharomyces (Zhu and Riggs, 1992) and Candida (lwaasa et al, 1992). Ascaris

haemoglobin also has a reductase activity that has not yet been localised (Sherman et

al, 1992b). The fusion, or association, of a globin domain with an unrelated protein may serve as a molecular switch controlling the enzymatic activity of the second domain through conformational changes of the globin domain associated with its oxygenation status (Zhu and Riggs, 1992).