FIXATION AND TRANSLOCATION CF NITRCGEN IN LICHENS WITH SPECIAL REFERENCE TO PELTIGERA SPECIES A thesis presented for the Degree of Doctor of Philosophy of the University of London by C. J. B. Hitch, M. Sc. Department of Botany Imperial College of Science and Technology London November 1976 ACKNO";;LEDGEMENTS I would like to express my gratitude to Professor Rutter., for allowing me to use .the facilities of the Botany Department, Imperial College, freely in the furtherance of this Degree. I would. also like to thank the members of the Department generally, for advice and to Dr. K. L. Alvin in particular, for the use of his phote-microscopic equipment, without which some of this work would not have been possible. Further, I would like to thank the staff of the Workshop for their assistance in the development and construction of various pieces of apparatus and also to Hr. Rodney Dawes for his patience in making and mending glassware and to the Photographic Department, for help in the production of photographs and slides. In conclusion I would like to express my sincere thanks to my supervisor, Dr. J. W. IIillbank, for his great generosity and thoughtful help and discussion during this period of research. TABLE CP CONTENTS Section 1 1.1.1 ABSTRACT 1 1.2.1 GENERAL INTRODUCTION 3 1.2.2 MATERIALS AND COLLECTION SITES 7 Section 2 SCHE ASPECTS OP LICHEN STRUCTURE THE PHOTOSYNTHETIC ALGAL CELL 2.1.1 INTRODUCTION 11 2.1.2 METHODS 13 2.1.3 ERRORS INVOLVED IN THE CALCULATICN a? ALGAL POPULATIONS 18 2.1.4 A COMPARISON OF CELL VOLUMES 28 THE HEqhROCYST 2.2.1 INTRODUCTION 30 2.2.2 METHODS 33 2.2.3 THE HETEROCYST FREQUENCY IN LICHENS WITH ONE PHYCOBICNT 35 2.2.4 THE HETEROCYST FREQUENCY IN LICHENS WITH TWO PHYCOBIONTS 44 Section 3 NITRCGEN FIXATION — USE (F ACETYLENE 3.1.1 INTRCDUCTICN 52 3.1.2 PMii0Ds IN NITRCGEN FIXATION 58 3.1.3 DISCUSSION ON AS.FLCTS CF NITRCGEN FIXATION 64 ,- ASPECTS OF NITROGENASE ACTIVITY 3.2.1 INTRCDUCTICN 77 3.2.2 METHODS 79 3.2.3 VARIATIONS IN NITROGEN FIXING ACTIVITY OVER THE THALLUS AREA 80 -- Section 4 15 N NITRCGEN FIXATION — USE CF 2 4.1.1 INTRODUCTICU 98 4.1.2 METHODS 106 4.1.3 DISCUSSICU OF THE 15N —UPTAKE EXPERIMENTS 116 - Section 5 SOME FACTORS AFFECTING NITROGEN FIXATION, PHOTOSYNTHESIS AND RESPIRATION IN WHOLE 'PHALLI 5.1.1 INTRODUCTION 143 5.1.2 METHODS. 150 5.1.3 SOME INTER—RELATIONSHIPS AND EFFECTS OP CO2, pH, LIGHT AND INHIBITORS ON NITROGEN FIXATION, PHOTOSYNTHESIS AND RESPIRATION 154 5.1.3.1 Oxygen and its affect on nitrogen fixation 154 5.1.3.2 Light and darkness on nitrogen fixation 159 5.1.3.3 The effect of pH on nitrogen fixation 165 5.1.3.4 Effects of DCMU and CN— on nitrogen fixation, photosynthesis and respiration 169 5.1.3.5 Variability of photosynthesis and respiration 173 CONCLUDING REMARKS 197 --- SUMMARY OF RESULTS 201 •APPENDICES 210 REFERENCES 219 1 Section 1 1.1.1 ABSTRACT 60 lichen species were examined in this investigation, Peltigera canina and P. polydactyla in detail, the remainder to a much lesser extent, for the purposes of comparison or survey. Nostoc cell numbers in the two principal species were of the 7 order of 10 cells ( 1 sq cm thallus ) with a recorded maximum of 2.5 x 107 in P. canina. Heterocyst numbers were also examined from many lichens with a blue—green algal phycobiont and photomicrographs were prepared. In lichens where the blue—green algae formed a layer over the whole thallus, the heterocysts constituted about 5% of the algae, whilst in species with cephalodia or internal islands of blue—green •algal cells, the heterocyst ratio was of the order of 13 to 50%. Nitrogen fixation was generally assayed by using the 15 was also acetylene reduction technique, though the heavy isotope N2 used. Fixation was only demonstrated in lichens where the phycobiont had heterocysts. No British material with non—heterocystous phycobionts was tested. Two lichens of this type from Israel were tested, using the acetylene reduction technique and gave results which were only indicative of minimal nitrogenase activity. Fixation exhibited wide inter— and intra—thallic variation. It was affected by light and dark, oxygen in the short term, CO2 in the long term and by pH. Using Peltigera polydactyla 15N2 was incorporated into the thallus, a method being evolved to keep the samples active over 37 days. see Scott (1957) 2 Immediate transfer of the fixed nitrogen occurred from the algal to the fungal zone. Each zone increased its degree of labelling consistently. In neither case did the label reach a peak. Oxygen turnover was measured using a Clark type oxygen electrode. The exchange was comparitively slow, with little increase in evolution over the intensities 5,000 — 10,000 lux. Under those conditions of augmented carbon dioxide ( HCO3 ) a "compensation point" was noted and oxygen was evolved more rapidly. DCMU inhibited photosystem II completely, but not nitrogen fixation. 3 1.2.1 GENERAL INTRODUCTION Lichens are pioneers in plant succession, where they are able to form long lived and stable communities. It is their adaptation to xeric or mesic environments that has enabled them to dominate habitats where competition is slight. They are remarkably specific to different substrates and this characteristic is used in their classification. However this is not entirely rigid and certain lichens have such a wide distribution and little substrate specificity, that they may become established in several communities. They are referred to as corticolous when growing on trees with bark, saxicoluus when on rocks and rock—like substances, terricolous when on soil, muscicolous when on mosses and phyllicolous when on leaves. These communities can be further subdivided, for example some lichens are specific to certain types of trees, which may be rough or smooth barked or have a distinct pH. Others to rocks, in which case hardness and softness, acidity and alkalinity can affect their distribution and much work has been carried out to examine the vertical zonation of lichens on rock faces. Hale (1955) has studied lichen frequency and cover on trees in relation to pH, and Harris (1971, 1971a) has examined the distribution throughout a tree canopy and related the variation of physiology to the environment. Santesson (1939) in Sweden has discussed distribution in relation to water submersion and similar zonation patterns have been reported by Hale (1950), Raup (1930), Watson (1919) and Lewis (1964) and various workers have investigated lichen colonisation of soil communities. These are most vulnerable, since they are subjected to rapid change and competition. Cladonia species often appear to form the main biomass in these areas, with gradual zonation extending backwards, from the most xeric regions, though other lichens may be more important 4 in certain habitats, where they are better adapted. This is true of the fast growing Peltigera species, but see the work of Brodo (1961), Looman (1964), Rogers, Lange and Nicholas (1966) and Ahmadjian and Hale (1973). Though lichens were first used by herbalists, they_were not studied in any detail till the eighteenth century and Acharius (1757 — 1819) a Swedish Medic first put lichenology on a sound scientific basis. With the improvement of the microscope, advances were made, and DeBary and Schwendener were the first to understand the dual nature of lichens and the first to consider them on a physiological basis, though their findings were controversial and were not accepted for many years. As Smith (1963) has said, "there has been much speculation about the nature of the association, between their constituent algae and fungi. Unfortunately this speculation has not been accompanied by much experimental investigation. A belief that they are 'difficult' material to vqe in laboratory experiments, has discouraged research into their physiology and difficulties in their taxonomy have discouraged botanists from becoming familiar with the group." He went on to say that "lichens do not present any greater problems in physiological laboratory experiments compared with:other organisms, provided that proper procedures are adopted," As Hale- (1967) has shown, Smith has been in the forefront of lichen physiology, with his work on carbon metabolism, in which he developed the technique of disc dissection. Both Quispel (1959) and Collins (1960)suggested that in order to discuss lichen physiology, they had to define the lichen association, though this was difficult due to the variability of other lichen—like associations present in nature. This leads to controversy as Ahmadjian (1967) has pointed out. A true lichen cannot be described as a fairly permanent,_ association between algae and fungi, on the same substrate, involving physical and physiological interractions, but should be reserved for associations between algae and fungi, that have formed a new morphological' unit, distinct from either of the two partners. While Quispel and Collins are justified on a comparative basis, their views do not affect the physiology of true lichens, which have been summarised' by Ahmadjian (1967a), as follows:- a) the growth of the individual symbionts is slow. b) the net rate of assimilation is low. c) the lichens exist on a nutrient-poor substrate d) their exposure, often to extreme environmental conditions, allows them only brief periods for optimum activity. e) they are frequently wetted and dried. f) they have low rates of protein synthesis. Also, not mentioned by Ahmadjiant the rate of lltliciem of substances •from external sources is very rapid. It has been assumed that the algae synthesise organic compounds autotrophically- which were obtained by the fungus by haustorial penetration (Ahmadjian, 1965; Peveling, 1973), though Quispel (1959) found that isolated algae behaved heterotrophically, but could be stimulated to autotrophic growth by the addition of ascorbic acid.