Dr Cyril Appleby, plant biologist

Dr Cyril ApplybyPlant biologist

Cyril Angus Appleby was born in 1928 in the seaside country town of Victor Harbor, South Australia. After completing his schooling at Victor Harbor High School, Appleby received a State Government scholarship in 1945 to study at Adelaide High School and sit for the Leaving Honours Certificate. In 1946 he was awarded a Commonwealth Government scholarship which led him to study Science at the University of Adelaide.

Appleby went on to obtain a BSc Hons in Biochemistry from the University of Adelaide in 1950. After briefly working as a biochemistry demonstrator and medical laboratory technician, Appleby obtained at PhD at the Department of Biochemistry at the University of Melbourne for a thesis entitled: “The Cytochrome-linked Dehydrogenase Systems of Yeasts and Higher Plants”. His PhD research achievements included the first-ever crystallisation of a complex cytochrome. In 1956 Appleby became a Research Scientist in the Biochemistry Section of the Division of Plant Industry at CSIRO, Canberra. There he researched the structure, genetic origin and biological function of plant-kingdom and microbial haemoglobins and cytochromes, particularly within the nitrogen-fixing symbioses of legume and non-legume plants. His pioneer work demonstrated that haemoglobins were present throughout the plant kingdom and that plant and animal haemoglobin had a common genetic origin.

Whilst at CSIRO Appleby fostered several international partnerships with overseas laboratories. In 1959 he travelled to Boston as a Rockefeller Foundation Fellow at Brandeis University. In 1971 and many times later he worked at the Department of Physiology and Biophysics at the Albert Einstein College of Medicine, New York. Other international laboratory visits included the Institute of Organic Chemistry at the Bulgarian Academy of Science, Sofia (1978 and 1988); the Alberta Heritage Foundation for Medical Research and the Department of Chemistry at the University of Alberta (1983); Kings College, London and University College, Cardiff (1983); the Scripps Institute of Molecular Biology (1984 and 1986); the Department of Biochemistry of Cornell University (1984 and 1986); Carlsberg Laboratory, Copenhagen (1987); Swiss Federal Institute of Technology (E.T.H.), Zurich (1989).

Appleby was awarded the LKB Medal of the Australian Biochemical Society in 1979. He retired from CSIRO as Chief Research Scientist in November 1988 after which he continued as an Honorary CSIRO Research Fellow.

Interviewed by Dr Jim Peacock in 2011.


Early beginnings - sermons at the seaside

My name is Jim Peacock. I have been asked by the Academy to interview one of its illustrious fellows, Dr Cyril Appleby, about his research career and I am very pleased to be here to do that.
Good morning, Cyril. You’ve been known for most of your scientific life around the world as ‘Mr Leghaemoglobin’ but, in the latter part of your career, you became even more famous and were known as ‘Mr Plant Kingdom Haemoglobin’—and I feel privileged to have known you over both those times.

I want to go right back to your beginnings and ask you some questions about where you grew up and what the circumstances were that drew you into research.

I was born in the seaside country town of Victor Harbor in South Australia in 1928. My father was the carpenter. That is how big the town was; one carpenter was plenty. My mother looked after four fractious children, of which I am a reasonable example. She was also a magnificent soprano, head of the large Congregational Church choir, and also a leading singer at town hall concerts. That made me very proud. My father, besides being the carpenter, was also a non-conformist lay preacher. There were smaller churches within the parish and on Sunday mornings, if no ministers were available, he would get on his big bicycle, and ride out to such little churches and deliver fundamentalists sermons. ‘Fundamentalist sermon’ is the bit to remember here.

I should give some background to my heritage here. My paternal great grandmother had come from the Orkney Islands and married an English migrant in Adelaide. Their marriage certificate had been signed by crosses, so there was no suggestion of silver tailed background there; nor, indeed, had there been for the Wilkins family. My maternal great grandfather Harry Wilkins was the first European child to be born in the South Australian colony on 1 January 1837, the colony having been proclaimed by Governor Hindmarsh the previous week. Harry’s own youngest son was Captain Sir George Hubert Wilkins, who had been a World War I official photographer. He had twice been awarded the Military Cross for gallantry. After the war he had been the first person to fly an aeroplane over the North Pole and he had also been the first person to try, unsuccessfully, to get under the ice to the North Pole with a refurbished World War I submarine. He was also married to an actress. So just imagine what this sort of person was for me to look up to from my rather dour, non-conformist religious background.

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Receiving insight from burning leaves

When you were growing up and going through school, was there a particular event or a particular bit of excitement that really first planted in your mind that perhaps you would look to a career in science?

Yes, I was eight. I was looking at a neighbour burning leaves in the gutter and wondered, ‘Now, what is smoke all about; what is that magic stuff escaping from the leaves?’ So I thought, ‘Prayers tonight,’ and I asked God. There was no response and I thought again, ‘It is about time that I started to work a few things out for myself.’

Then, at the age of 11, I was reading this sixpenny magazine, a weekly called Modern Wonder, and there was an article about evolution in it. It was fascinating. I had never known anything about evolution. My father came up and said, ‘Son, what is that you are reading?’ I said, ‘Oh, look at this, Dad, an article about evolution.’ He replied, ‘My boy, that is the work of the devil. Have none of it!’ I guess it was at that point that I decided I was going to have to find things out for myself. I was not going to accept such stuff anymore.

Then at high school, for my science prize in the second or third year, instead of an English poetry book, which the headmaster thought I needed, I chose James Stokley’s recent book, Science remakes the world. In it was the description of sulphanilamide, the new wonder drug, of synthetic plastics that would last forever, and of the wonderful insecticide DDT.

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A ‘precocious’ student

Were you a Victor Harbor High School student, or did you attend another high school?

I went to primary school in Victor Harbor for seven years. The first five years were very pleasant, with gentle and polite teachers. For the sixth and seventh years, a merged class was run by the sadistic head master; AVG I will call him. Rote learning — this is even in the last year of primary school — was it. You questioned him and you were in big trouble. In fact, one day he said to me, ‘Appleby, go to the library, get the dictionary and look up the definition of “precocious” and read that word out to the assembled sixth and seventh classes’. I hope he rots in hell still!

So, at the end of your high school days, you were on your way to a university. Which university was that?

I went to Adelaide University as an undergraduate. Following my leaving certificate pass at the Victor Harbour High School, I had been awarded one of 24 scholarships — these were quite new and 12 were reserved for country students. This was very good and meant that I could proceed to leaving honours, the better matriculation year. One could go on from a leaving certificate pass to university. But the head master came to my father and said, ‘Mr Appleby, Cyril is not ready for university. He is not mature enough’.

He’s precocious?

Exactly. At primary or high school I was not a sporting hero; far from it. I was never the captain of a school team. In fact, I never found myself with any sort of team spirit, but I quite enjoyed going after something and understanding it. I did not want to be the boss of anything. But one had to sit on oneself. Here was this smart arse, no good at sport and not well tolerated.

I had to dumb down. But then I went to Adelaide High School which in 1945 was the only state school where one could study for the leaving honours exam. I found myself in the same classes as most of the other 24 scholarship winners at this first-rate school. It was a compact place, much bitumen and no fancy stuff. In lunchtime and recess discussions, the sky was the limit for questioning; indeed there were no limits. You could push it, which was fantastic — I was 15th out of 37 in my home class — and one could be commenting and asking questions the whole time. You could discuss anything with your teachers, although politely. This was 1945, still.

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The good and bad of university

That really put you on the road ready to go to university.

Well, for the first year I made a few wrong choices, having thought earlier that I wanted to be a medical bacteriologist. Incidentally, my father thought that I should be a medical missionary, but I decided that was not on. It was something that I disagreed with. So at university I chose fairly easy subjects—well, useful ones — biology, organic chemistry, and bacteriology, which in Adelaide was taught by Nancy Atkinson, a genuine working microbiologist. For my first two years most of the other lecturers were of rather stodgy 1930s mindset; they maybe had been somehow famous then. But in my third year — that was 1948 — things looked up magnificently. Ken Pausacker came back from Oxford with a newly-awarded Doctorate in Philosophy. He had worked with Sir Robert Robinson on the structure of strychnine and he brought with him a new book called Remick: Electronic interpretations of organic chemistry. Suddenly we were into a middle-century mindset. Also, Nancy Atkinson, besides her formal medical bacteriology lectures, taught us about membranes, physical chemistry and charge separation — stuff that we should have heard about in Professor Mitchell’s second year biochemistry lectures — and, indeed, the fact that DNA, not protein, was the source of genetic information.

Yes, that was just exactly that time.

Also in third year we had a few lectures from a new appointee to the biochemistry department, Peter Nossal. He had just finished his Masters of Science degree in Sydney. He was, of course, the older brother of our sometime Academy chairman, Sir Gustav Nossal. Peter’s weekly lecture on biological organic chemistry to final-year chemistry students was so good that five of us sneaked in twice a week to Nossal’s regular biochemistry lectures. In turn, these were so good we decided that we wanted to do honours in biochemistry.

When did you sign up with Peter Nossal for honours study?

In 1949, on the first Monday of February, five of us assembled with great excitement in his lab. I think his brain — he died young — would have developed even better than Sir Gustav’s.

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Lively, intelligent Judy

Cyril, I would like to interrupt with a question about your family, and that concerns Judy.

You met your future wife at the end of your degree in Adelaide, I think — and, just as in your research, things moved along rather quickly.

Well, it was at the end of the second term, the second-term vacation. I had been a good boy, not running around with girls very much. I had finished my organic chemistry practical schedule and Pausacker, who I just talked about, said 'Appleby, you can do what you like for the third term in organic prac'.

So I rode my bicycle from my home at Victor Harbor to the beach at Middleton, where there were these wonderful pink shells. I hoped to extract and identify their organic pigments. But, as I rode past Bradwell dairy farm gate near Port Elliot, out came Judith Basham on her bicycle. We had known each other at Victor Harbor High School but then had gone to separate schools in Adelaide for matriculation study. We stopped and talked — for two hours. We found out how much we had in common. We both liked reading books and beach walking. Neither of us could catch, hit or kick a ball, and we didn't like being the captain of anything or being captained. The courtship developed and we were married at St Jude's Church in Port Elliot on April 19, 1952. We had four wonderful daughters, with very different personalities.

We had a very, very strong family interaction, which Judy had influenced; my own background had been

somewhat fragmented. One day at the Port Elliot beach, with only the first three daughters, a former schoolteacher saw us together and said, 'My dear Judith, I had always pictured you as the perfect homebody'. This made Judy very angry because she was so much more than that. She was a lively, intelligent person.

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Upsets during honours year

When you were in honours did you have any idea of what you wanted to do after that?

By this stage, I thought that I might like to be a research biochemist discovering new drugs, even better than sulphanilamide or penicillin. Earlier, I was going to be happy as a diagnostic bacteriologist. But that early 1949 period, as one of Nossal's research apprentices, was very exciting. Although he had come from a fairly humdrum lab in Sydney, Peter had done some brilliant work on the Krebs tricarboxylic acid cycle.

But things didn't work out exactly as you might have planned and you went from Adelaide up to Brisbane.

In the middle of the honours year, something upsetting happened. We heard that Nossal had been accepted to go to Sheffield for PhD study with future Nobel Laureate Hans Krebs and that Professor Mitchell with his 1930s mindset was going to look after us for the rest of the year. We thought, 'Oh, it will work out,' but it didn't. I don't think any of us had a research discussion with the professor for that half year and, indeed, I misunderstood what he was going to require of us in one of his formal examination papers. I graduated with second-class honours, and this upset me a fair bit. Mind you, I talked to Hal Hatch, our former CSIRO colleague and eminent fellow of many academies sometime later, and found out that he, also, had been awarded second-class honours in Sydney. That made me feel quite a lot better.

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Medical research, malaria and more upsets

So you went up to Brisbane as a technician?

Well, I had accepted a job as demonstrator in biochemistry back in Adelaide, and this worked for a while.I was looking after the animal house, getting class preparations ready then supervising such classes, and I invented for myself a little research project looking at nucleic acid metabolism of nucleated erythrocytes — I don't remember quite why — and the work went nowhere. I thought, 'I've had enough of this,' and I think Mitchell had had enough of me as well, although we never talked about my research program. It was a very odd place. I went to Brisbane and was accepted as a technical officer with John Callaghan, who had been appointed at the Queensland Institute of Medical Research — headed by a first rate person Ian Mackerras — to investigate the breakdown of haemoglobin in the parasitised erythrocyte.

Was it malaria?

Malaria, yes. I said to Callaghan, 'I don't care for this much; I don't like malaria, oh no'. But he explained that the mouse parasite Plasmodium berghei, with which he worked, does not infect humans and it has the considerable advantage that infections are synchronous. So, a couple of days after the mice were infected, one could do a heart puncture and begin work. Things were starting to look good. I thought, 'I'm enjoying being a research assistant in an interesting medical research institute'. But Callaghan, whom I knew already as a dedicated member of the Australian Communist Party, went with others on a junk from a north Queensland port to a peace conference in Shanghai in mid-June. This was the last year of the Korean war, 1952. He may have asked for a leave pass but didn't receive one. So he was absent without leave and this was only two months after I had arrived at the institute to start a new scientific life. At its annual general meeting in July, the Institute of Medical Research Council — dominated by heavies from a very, very right-wing state government — dismissed Callaghan and decided to shut down the biochemistry group. So, after escaping from a bad start in Adelaide, I suddenly found that all I had left were ten months of an agreed probationary year.

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Saved by a visitor

But you were saved in a way by a visitor; is that right?

Well, Ian Mackerras, the director, had been an entomologist in New Guinea during the Second World War. He and long-time colleague Doug Waterhouse, who by 1952 had become assistant chief of the CSIRO Division of Entomology in Canberra, used to meet every year. Mackerras came to Canberra in January 1953 and said, 'Doug, I have got this nice young man in my institute who is going to be without a job in May, through no fault of his own. Have you got a spot for him?' Doug — he and Mackerras being good mates — said, 'Well, Ian, as a matter of fact, I have snaffled all the available money from under the nose of my own ageing chief, Nicholson, and there would be none left for your boy'. Waterhouse said then to Mackerras, 'Come down the corridor to see Otto Frankel, recently appointed Chief of Plant Industry. Dead wood has been swept away there and he is making new appointments'. Mackerras was introduced to Frankel and I was perhaps boosted up to whatever quality I didn't quite have.

It was fantastic timing because the week before, Victor Trikojus, Professor of Biochemistry in Melbourne and his new Senior Lecturer in Plant Biochemistry, Bob Morton — just arrived from Cambridge — had come to Canberra and said to Frankel, 'We want to revive our moribund plant biochemistry laboratory. Could you endow a three-year fellowship, with the graduate then returning to CSIRO Canberra?'
From Frankel: 'Maybe Appleby would be interested'. Would I? I was on the next possible flight from Brisbane to Melbourne for an interview.

And you sort of had a promised role back in Canberra in Plant Industry.

I didn't yet know that. I was ushered into the professor's office in the old Tin-Alley Biochemistry Department — there have been two more biochemistry buildings, bigger and bigger, since then — and here was the grand Professor Trikojus, as well as a smaller, intense person. I realised, soon enough, that it was Morton.

He was an Australian who during the recent world war had become First Lieutenant on a frigate as a Royal Navy volunteer reservist — very crisp and eager — and I thought, ‘I don’t think I am caring for this. When they find out about my background of two wasted opportunities, I will be nowhere.’

But Morton didn’t think that was such a trouble?

Morton, in discussion, found out that in Brisbane I had learnt how to use a hand spectroscope to look at haemoglobin breakdown by the malarial parasite. This pleased him; I didn’t quite realise why. He said, ‘Well, now, I am interested in having my new appointee show that something called cytochrome b2 (which has been found in bakers’ yeast) is not related to an oxidase called yeast lactic acid dehydrogenase, because other such dehydrogenases are flavoproteins and I am determined to establish the situation for the yeast enzyme'. Then he said to me, 'Mr Appleby, you told me that your wife is pregnant,' and I said, 'Yes, Dr Morton'. 'When is the baby due?' 'Oh, the end of September.' 'Very well then, Mr Appleby, I challenge you to crystallise cytochrome b2, showing that it is not the dehydrogenase, as a present for your first born child.' I realised then that the job was mine.

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Yeasts and bucket chemistry

I would like to cut to the crunch about this possible cytochrome.

Oh, yes, the story. At Cambridge in the early 1940s cytochrome em>b2, having been discovered in Delft (Dutch) bakers yeast, seemed to be associated with a lactic dehydrogenase enzyme. In the same Cambridge laboratory, after the war, Morton had found that an animal succinate dehydrogenase, supposed earlier to be a very similar cytochrome, was in fact a flavoprotein. Obviously he wanted the same to happen for the cytochrome b2 of yeast. So he said, 'Go and find every possible sample of our wild colonial yeasts', because Australian yeasts were still red, not like anaemic post-war English yeasts. My first samples looked good, their spectra indicating lots of cytochrome. 'Find out which one of them gives also the best yield of lactic dehydrogenase.'

So, to cut the story short, these yeasts used to come in one-pound packets, like butter, and I located 18 different brands. I would grind them one at a time and look at fresh and dried yeast extracts to see what their cytochrome and dehydrogenase contents were. Morton would watch every day as I used his microspectroscope and I would see the absorption bands of cytochromes a, b and c, which Cambridge Professor David Keilin had discovered in 1925 in yeast and insect muscle. But I could never see the minor band of cytochrome b2 although Morton could spot it every time. He anticipated, without letting me know, which of the yeasts might be any good. Finally I saw a trace of this cytochrome b2 in Barretts, a yeast manufactured in Melbourne. On hearing of our work the company owner, Mr Barrett said, 'All right, we will make and give you a whole seed yeast batch of the size we use for regular commercial production'.

We already knew that dried Barretts yeast was better than fresh yeast for our work so very generously the company made and air-dried this seed yeast batch and sealed it into metal drums which we then stored in our cold room. It lasted for two years.

I would extract these yeast samples using every available procedure. In the end I used as a first step the lipid solvent n-butanol. I would suspend pulverised dried yeast in smelly butanol at ambient temperature, and in a large, noisy, 1930s vintage Jouan centrifuge,

spin off a copious lipid-containing supernatant. This made the butanol-saturated yeast cells permeable to aqueous solutions, enabling lactic dehydrogenase and cytochrome b2 extraction. Again, to cut that story short, I found by large-scale acetone fractionation of such extracts at low temperature — this was in the 1950s, mind you, before invention of those compact procedures of ion-exchange and molecular-exclusion column chromatography –– that substantial purification of cytochrome b2 and of lactate dehydrogenase could be achieved.

It sounds a bit like bucket biochemistry.

It was real bucket biochemistry, involving small, then larger stainless steel beakers suspended in an ice-salt bath then later a large refrigerated ethanol-water bath kindly created and donated by a Melbourne domestic refrigerator company.

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Hard work and carelessness lead to a world first

As I understand it, Cyril, when you were trying to crystallise this flavonoid cytochrome, which was something that was going to be very hard to do, you were a little bit naughty in terms of your lab duties and went off on a picnic; is that right?

Well, I had found that by acetone fractionation, first stirring in cold acetone to 30% in the steel beaker instead of having to centrifuge the curdy suspension (we did not have a large, refrigerated centrifuge), the precipitate would stick to the sides of the beaker. I would discard this first precipitate because the assay showed no cytochrome b2 and little enzyme activity. Then, at 35% acetone a lovely red oil would separate out. You didn't even have to centrifuge it; you just had to carefully decant the clear paler red supernatant. This lovely red oil could then be dissolved in a dilute lactate buffer. Lactate proved to be a very important protectant; in general terms, if one can keep a dehydrogenase enzyme reduced, it’s more stable.

I was getting on and on and the purification was getting better and better and already I could see that the lactic dehydrogenase, which was assayed using ferricyanide or cytochrome c as the electron acceptor, was being purified in exact parallel to cytochrome b2 which I was detecting with a microspectroscope or spectrophotometer. I thought, ‘I have got Morton; this dehydrogenase is a cytochrome’.

But one Wednesday I had finished some work trying to adsorb the enzyme on calcium phosphate gel and, by nightfall I’d had enough. I thought, ‘I really am going to go to the staff picnic in the Dandenongs tomorrow’. So I left the rest of the preparation, which had been dialysed to a very low salt concentration with lactate present, under nitrogen in a small tube in the cold room for a day and a half. I came back on the Friday morning and became worried because there was a turbid precipitate in the tube. I thought, ‘This is bad’. But then, when I looked carefully with a hand spectroscope, by moving down the tube I could see a lot of cytochrome c at the top, and down the bottom a different, intense red-pink band which was indicative of cytochrome b2. I thought, ‘My God, I have either done it or I have stuffed it’.

You had the tube with the apparent crystals and Dr. Morton became very excited. When did he actually see that?

Morton came in at about five past nine, just before his first lecture. He said ‘Mr Appleby, why are you looking so upset?’ Then I said, ‘Well, I think I have managed to precipitate all of the cytochrome b2’. ‘Mr Appleby, how careless of you. Instead of working yesterday, you went off to a staff picnic’. So he grabbed the tube out of my hand and flicked it and in doing so generated a schlieren, a reflectance pattern characteristic of a crystalline protein. This was something I had not seen before. ‘Mr Appleby, Mr Appleby, crystals, crystals!’ he cried. He grabbed the tube and raced down the corridor to the professor's office. ‘Professor, professor, I have crystallised — ah, ah — we have crystallised the first ever cytochrome’.

Later on I said to him: ‘There, Dr Morton, is your crystalline cytochrome b2 as the dehydrogenase’. But he said, ‘In a minute, Mr Appleby’. He took some of my suspension, shook the crystals up and added a few drops of hydrochloric acid, producing a red precipitate of denatured cytochrome b2 and a slightly fluorescent yellow supernatant. ‘There, Mr Appleby, is the second, flavin prosthetic group of lactate dehydrogenase. It remains for you to identify it as riboflavin phosphate or flavin-adenine dinucleotide’. I don’t know if or when he had already worked out that the crystal might be a double-headed enzyme.

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Two groups, one enzyme an important start

So was this the first crystalline enzyme shown to have two prosthetic groups?

Exactly. A haem and a flavin. So, Morton profoundly stated: ‘Well, Mr Appleby, we should write a note to Nature’. This was in December 1953.

Was that your first published paper?

It was indeed my first paper.

Quite an important start.

Yes, in the Nature paper we described this crystalline enzyme with its two prosthetic groups in equal proportions. Twenty years later, when the protein crystal and gene structures had been determined by others, it was found that there was indeed only one gene involved, with a recognisable intervening sequence. It could be seen that the left side was where the flavoprotein resided. It was recognisable from other existing flavoprotein structures. The right side was recognisable as a classical cytochrome b fused to the flavoprotein.

So ultimately the gene story cemented a wonderful finding: that this enzyme probably came from two other existing molecules and was put together in a particular way.


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Off to Canberra to research haemoglobin in plants

Now, am I right that you then went back to Canberra, having finished your studies with Morton, and took up a job in CSIRO Plant Industry under Frankel?

That is right, yes.

And you were a employed as a research scientist?

Yes. Morton and Trikojus had not told me that they would wait until the end of my first year to see how I was progressing. Then at the end of the year they said, ‘Well, Mr Appleby, we have been considering having you accepted as a PhD student.’ In truth, they had been recording everything so that, if it worked, I was enrolled, and eventually I was accepted back at Plant Industry as a research scientist with a nascent PhD.

It has always interested me, Cyril: when you were appointed into Plant Industry as a scientist, how was it that you initiated your work on plant haemoglobins?

I knew already that legume root nodules showed a red haemoglobin colour when they were cut open; that was all.

I guess that in those days the concept was that only legumes had haemoglobin in their nodules; and there was quite a world famous group in Plant Industry, working on nodule biology.

Well, yes. When Frankel became chief he appointed John Falk in 1956, a porphyrin chemist, as the head of the biochemistry section, which until then had a plant physiological bent. Also, Frankel had enticed Phillip Nutman, a senior scientist at the Rothamsted Experiment Station, UK, to come on a three­year fellowship to get the microbiology group out of a mindset from way back.

Soon after my arrival in Canberra, Falk and Nutman asked if I would consider looking at the function of leghaemoglobin, known to be present in nitrogen-fixing legume root nodules but nowhere else in the plant kingdom — and this was indeed a magic opportunity. I already knew about haemoproteins, spectroscopy and spectrophotometry so was ready to go.

So did you try to decide why it’s there? Is that the question you asked?

Oh, sure. Another recent appointee was microbiologist Fraser Bergersen, with whom I established a very successful, although wary, long-term collaboration — we had very different personalities.

That particular colleague, I think, worked in association with Phillip Nutman, the prominent scientist from the UK, who was visiting Plant Industry and the nodule group at that time.

Yes, Bergersen was a ‘Nutman acolyte’; an acolyte in the best sense. Not a dumb follower, but a brilliant young man destined to be a high priest himself one day.

I’ve often wondered about your thoughts concerning haemoglobin in plants. I mean, this red stuff, which is like our red stuff in blood: why was it there in the nodules of legumes?

Bergersen showed me how to grow nodulated soy beans with roots inoculated by a specific Rhizobium strain. Then we would just crush the harvested nodules in a buffer and centrifuge them at low speed to separate the nitrogen-fixing Rhizobium ‘bacteroids’ from crude leghaemoglobin, which remained in the supernatant.

But when you isolated the bacteria, were they red?

No, no. The washed bacteroids were an ordinary tan colour. Then I found that, if I remixed these putative nitrogen-fixing bacteria with oxygenated leghaemoglobin, as the oxygen supply was cut off the respiration of the bacteria was enough to deoxygenate the leghaemoglobin. Whereas, if I had grown that same Rhizobium strain in a pure culture in air, it could not deoxygenate the oxyleghaemoglobin.

But, when the haemoglobin was deoxygenated, it still didn’t get into the bacterium.

Oh, no! Then I found that both the nodule bacteria (the so-called bacteroids) and free-living air-grown bacteria (which could not reduce nitrogen) could deoxygenate myoglobin. Now, myoglobin is half oxygenated at about 800 nanomolar dissolved oxygen. So, I thought ‘Ah! Maybe there’s a different oxidase in the bacteroids, able to grab the oxygen from oxyleghaemoglobin’.

What did I know about leghaemoglobin? I knew that Professor Keilin, my demigod from Cambridge, had taken a crude oxyleghaemoglobin solution and used a rotary vacuum pump in a procedure that would deoxygenate myoglobin or haemoglobin immediately. But the procedure could not deoxygenate the leghaemoglobin. So, leghaemoglobin had to have a much higher oxygen affinity. I decided then that I was going to find out the nature of the oxidase present in the nitrogen-fixing bacteroids and how it was different from the oxidase present in the free­living bacteria. Also, I was going to measure the oxygen affinity of the leghaemoglobin.

Did you wonder if the action of the bacteroid oxidase enabled nitrogen fixation to proceed? Was that in your mind?

Yes; what might be the nature of such oxidase? People knew already that nitrogenase, the nitrogen-reducing enzyme complex, was extremely sensitive to oxygen.

So, in searching for the reason for leghaemoglobin, I guess a seminal point was that a high oxygen concentration killed the activity of the nitrogenase.

High oxygen for a minute or two does inactivate it. But there had to be a situation whereby enough oxygen got into the bacteroids at a low tension to help make all the ATP needed for nitrogen reduction. So I had several objectives in mind: to purify and characterise leghaemoglobin; to determine its oxygen affinity; to understand the nature of the bacteroid oxidase; and I think, most importantly, to understand how this ‘animal’ haemoglobin came to be present in the plant kingdom.

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Fun with scientific gadgets

I think that was a very interesting story and things were beginning to fit together in the plant haemoglobin jigsaw puzzle. Then, I guess of particular importance was a trip that you made to New York — some time in the seventies, wasn’t it — which really helped to put some key pieces in place.

I had managed to measure for the first time the oxygen affinity of leghaemoglobin using an equilibrium procedure. This was easy with proteins such as vertebrate haemoglobin and myoglobin. One would use an instrument called a tonometer, which would have a little observation cuvette about this big (indicates) and a gas chamber maybe this big (indicates), and there would be a known concentration of oxygen in the gas chamber. You would roll the apparatus over to achieve equilibrium and, using a spectroscope or spectrophotometer, measure the degree of oxygenation of the haemoglobin. Then you would squirt in a bit more oxygen, roll the cuvette assembly around again and re-determine the spectrum. This is easy for something that is half saturated at high oxygen concentration. For instance, myoglobin is half saturated at about 800 nanomolar dissolved oxygen and vertebrate haemoglobin is half saturated at about 30 micromolar dissolved oxygen. But to do the same for leghaemoglobin, which we knew already had a high affinity, one would have needed a 100-litre or larger flask. So I developed a gas flow procedure to measure leghaemoglobin oxygen affinity. It was hard work, begun in Canberra over 50 years ago.

You were pretty good at scientific gadgets.

Well, okay. Indeed, in the final operation with half a year spent in Canberra using a hand spectroscope, then part of a year at Brandeis University in Boston using a ‘Rolls-Royce’ Cary 14 recording spectrophotometer to observe leghaemoglobin partial oxygenation while changing oxygen concentration in the gas stream, I achieved one part per million of gas accuracy. I could not tolerate one part in a million oxygen leakage. I found that leghaemoglobin was half saturated at about — I thought then — 80 nanomolar dissolved oxygen. No­one else in the world could manage this.

But then I was bypassed within two or three years when Quentin Gibson, whom I revered as the high priest of haemoglobin kinetics, developed a stopped-flow procedure. In one syringe there would be a solution of haemoglobin and in a second syringe a solution containing oxygen or other challenging ligand.

In the stopped-flow device, one could push the contents of these two syringes through a mixing chamber into a spectrophotometer cuvette. Then an exhaust syringe filled up and hit a stopping device with trigger — making a magnificent bang — causing a storage oscilloscope trace to begin.

Friendship and inspiration in New York

I know that I’ve talked to you about your association with other haemoglobin aficionados around the world but of particular importance, I think, was your association with the two Wittenbergs in New York. Why was that so important and how come you hit it off so well with them?

When I had found that this legume haemoglobin had such a high oxygen affinity — I thought it was then about 80 nanomolar dissolved oxygen for half saturation — I realised that it might function in a process of facilitated oxygen diffusion. This had been proposed by Jonathan Wittenberg several years before for myoglobin in muscle. If there were a carrier protein present at a high concentration, such that on the oxygen loading site it could be substantially oxygenated and on the oxygen unloading site it could be substantially deoxygenated, you would have a magnificent oxygen supply system. You could call it an oxygen buffer system, or a facilitated diffusion system. You could get a continuing transfer of oxygen delivered at an instantaneous concentration that, in my imagined situation for the legume root nodule, would be low enough to not inactivate the bacteriod nitrogenase.

So you imagined that the leghaemoglobin soaked up the oxygen —

Yes, and held it like a buffer.

and held but then delivered it —

Yes — dribble, dribble, dribble —

—at a stable, low level into the bacterium.

Exactly. In 1962 I published a paper in Biochimica et Biophysica Acta, where I proposed that leghaemoglobin acted in this way. I forgot all about this — I was busy worrying about the nature of the bacteroid oxidases — until in 1967, during a meeting in Japan, a wild, long-haired American came up and said: 'So you're Appleby. You must be stupid proposing that your leghaemoglobin can function in the process of facilitated oxygen diffusion. Everybody knows that such high-affinity haemoglobins, like the one from Ascaris, the intestinal worm, have this high affinity because their oxygen off rate is so slow that they couldnt facilitate anything'. So I said, 'Well, look, Wittenberg, they wouldnt know, because the only result on leghaemoglobin oxygen affinity is mine, determined by an equilibrium procedure. Why dont I send my pure leghaemoglobin to you in New York so that you can determine by your kinetics procedures if it is suitable for a facilitated diffusion process?' Jonathans response was: 'No, no; come yourself. Call in when you are on your way back to Australia'. So I thought, 'All right; this looks like a serious contest'. But, in fact, for the rest of that meeting, Wittenberg and I had a delightful time. I thought then and now, if I am an oddball, he would be a super oddball.

Yes, I would say so.

Indeed you could say so.

So I stopped over in New York during September 1967 and met Beatrice Wittenberg, then gloriously pregnant with her youngest child and hence unable to be at the Japan meeting. I could tell straight away that it was not going to be another dose of Jonathan, which was good, because one of him is plenty! Bea is another fantastic person. So Jonathan went over the essentials of facilitated diffusion with me again—high free oxygen at one end and low free oxygen at the other. Then Bea said, 'Now, Cyril, this is what it is all about. I am looking in heart muscle tissue at the myocytes where we think the facilitated flux of oxygen via myoglobin produces this steady supply of oxygen to the myocytes so that they can contract'. She then said, 'We have no way of measuring energy production and utilisation in the system, but your colleague Fraser Bergersen in Canberra, earlier this year, has done a magnificent thing. He has shown for the first time that, if one isolates the nodule bacteria anaerobically, washes them free of leghaemoglobin and of other nodule constituents, they would slowly reduce nitrogen to ammonia. Might all of us, together, set up the situation whereby we prove the process of facilitated diffusion by demonstrating an increased efficiency of nitrogen fixation by these bacteria in the presence of part-oxygenated leghaemoglobin'.

So that was the assay.

Yes. But then I said, 'Well, that is going to be difficult,' because Bergersen used to think, following a postdoc year in Wisconsin — then the Mecca for symbiotic nitrogen fixation research — that leghaemoglobin, being outside the bacteria, was acting as a reductant and perhaps it supplied electrons to the nitrogenase while itself going from a ferrous to a ferric state. I said also, 'When Bergersen finally got his washed bacteroids to reduce a little nitrogen, he added back some crude leghaemoglobin — his own preparation and not mine — which killed the reaction. So, it may take a little while to get him on board'.

To cut that story short, I went back to Australia, made very pure leghaemoglobin and measured its spectral properties.It was as well behaved as was myoglobin. It was nice stable stuff, so long as cupric ions were kept out of the way. They could degrade it via autoxidation processes.

I returned to New York in 1971 and the Wittenbergs and I kinetically measured the oxygen affinity of soybean leghaemoglobin using a stopped-flow apparatus.

The on rate for oxygen, reported as a second order rate constant, was 120µM-1.s-1.

And did you determine the off rate with them?

The off rate was easy. You squirt oxyhaemoglobin into the observation chamber from one syringe of the stopped-flow device and dithionite as oxygen reductant from the second syringe. The off rate for soybean leghaemoglobin was measured as 5.6.s-1, just right for oxygen transfer via facilitated diffusion. Division of the off rate constant by the on rate constant enabled us to calculate that soybean leghaemoglobin would be half oxygenated at only 48 nanomolars of dissolved oxygen — an ideal concentration for delivery to the Rhizobium bacteroid oxidase.

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Doing experiments around the world

At any time that I have talked to you about plant haemoglobins or rather listened to you, which is often, you've inevitably mentioned Quentin Gibson. Why is that? Who was he really?

When the Wittenbergs and I measured the oxygenation kinetics for soybean leghaemoglobin, their stopped-flow apparatus was only just with it. The dead time between the last mixing in the reaction chamber and the oscilloscope trace trigger was three milliseconds and we were just on the edge of failure. The trace would start halfway down the oscilloscope screen, which made rate calculation difficult.

For lupin leghaemoglobin, the oxygen on rate was about three or four times as fast again, and the Wittenberg stopped flow machine — developed by Quentin Gibson many years beforehand — was useless. But by the early 1980s Gibson had developed a flash photolysis apparatus in which a 10 nanosecond pulse of laser light produced enough actinic energy to cause photodissociation of oxygen from the haem of oxygenated haemoglobins. Then in the dark at relative leisure an observation photomultiplier coupled to a computer could measure oxygen recombination at microsecond speeds. So, from Canberra I would take frozen lupin, soy and later many other plant haemoglobins to Cornell for Quentin's and my measurement of oxygen recombination rates; then on to New York for measurement of off rates. Notably, we found an oxygen on rate of ~500mM-1.s-1 for lupin leghaemoglobin, about 50 times faster than for vertebrate myoglobins and four times faster than for soybean leghaemoglobin.

During this time I had become something of a goodwill ambassador. Earlier there had been trouble between Quentin and Jonathan. Jonathan had resubmitted a rejected joint paper to a new journal without telling Quentin. Quentin himself (at the time a senior editor of the prestigious Journal of Biological Chemistry) had resubmitted the corrected paper to the original journal. Both versions were published. I was the person who brought these two together again, so I do have some tact.

That really was a wonderful partnership, wasn't it?

Yes, the interaction between Bea and Jonathan Wittenberg, Quentin Gibson and me, as much anything else, has led to the overall understanding of plant haemoglobin properties and function. But, to return to earlier discussion. How were the Wittenbergs and I going to get Fraser Bergersen on board for our planned investigation of leghaemoglobin involvement in facilitated oxygen diffusion? It happened that in the middle of our 1971 kinetics measurements there was a nitrogen fixation symposium being held at the Rockefeller Foundation in downtown New York. I, a former Rockefeller Fellow, attended this symposium and one night came with Bergersen and Mike Dilworth from Perth to the Wittenberg home for dinner. Bergersen and Dilworth got into characteristic heated discussion, and suddenly Bergersen found himself supporting the concept of a role for leghaemoglobin in nodule oxygen transfer. By the end of the night he realised how important facilitated diffusion might be, and effectively abandoned his earlier concept of leghaemoglobin as a direct nitrogen reductant. So he and I, after our separate returns to Canberra, set up a stoppered round-bottom flask with a magnetic stirrer, a strong light underneath and a hand spectroscope on top.

The flask contained a suspension of nitrogen-fixing bacteroids and a gas phase with variable oxygen concentration. Bergersen would perform nitrogen fixation assays by withdrawing gas samples via a porthole and measuring reduction of the artificial substrate ethylene — a recent Dilworth discovery — while I would use the spectroscope to estimate the degree of leghaemoglobin oxygenation as stirring rate or gas phase oxygen concentration were varied. The result was magnificent. As the leghaemoglobin became partly oxygenated, nitrogen reduction rate increased dramatically.

It was good that your two laboratories in Canberra came together.

Sure. But one point of that story is that it did not show us whether the leghaemoglobin was specifically reacting with some binding site on the bacterial surface or whether it was delivering free oxygen. On hearing this result Wittenberg said in grand style 'I will come with every possible oxygen-carrying protein and we will see whether they all do it; because, if so, it has to be facilitated diffusion'.

Of free oxygen, yes.

If just leghaemoglobin proved to be active this might have been at a specific binding site. I think we had

better cut that story short by saying that we tested a range of exotic oxygen carriers. We got them from crayfish — free from the Sydney Market — by bleeding them and purifying the copper protein haemocyanin. Bergersen dug earthworms from his compost pit for us to make Lumbricus haemoglobin. I got Ascaris haemoglobin from pig gut tapeworms. Jonathan and I went to Brisbane to get Gasterophilus haemoglobin from botfly larvae which were present in wild horse stomachs. Jonathan had brought from New York other vertebrate and invertebrate haemoglobins. They all functioned — some more efficiently than others — in the stimulation of bacteroid nitrogen fixation. So we proclaimed, in a complex Journal of Biological Chemistry paper, that the natural function of leghaemoglobin was the facilitated diffusion of free oxygen.

Plant haemoglobin origins revealed

So, Cyril, I think it was at about that time that you first came, probably with some doubting steps—I don't know—to Liz Dennis and me to ask whether we would be interested in taking a look at plant haemoglobin genes and seeing if we could help solve some of the elements of the puzzle.

I tried as far back as 1978 — you might not remember this — and I said, 'Look, I have just isolated four different soybean haemoglobins: a, c1, c2 and c3. What about you working on their gene structures?' You said, 'No, no, no' — and just as well because already, at Aarhus in Denmark, Kjeld Marcker was doing just that. In 1981 he published a wonderful paper in Nature to show that the leghaemoglobin gene had three introns — three intervening sequences — whereas all of the known animal haemoglobin genes only had two. Here was another one in the middle. Knowing this, in my simple minded way I thought, 'Whoopee,' because introns for genes were supposed then to have arisen very early in the assembly of proteins as a glue, and sometimes they got lost as evolution proceeded. Here was leghaemoglobin with three introns; better than all those animal haemoglobins; so maybe plant haemoglobins had arisen first?

But the fewer animal haemoglobin introns were in exactly the same position as they were in plants.

The other two were in the same places, yes. These poor animals found that they could do without the third central intron — it was missing from animal haemoglobin genes — and in my mind this meant that animal haemoglobin might have evolved after plant haemoglobin. Well of course my dream was shattered eventually as the introns-early hypothesis was gradually abandoned because of many others work.

Now you became interested in another plant that wasn't a legume but it had nodules and haemoglobin.

Yes. Mike Trinick, a Perth then Canberra CSIRO colleague, had work in New Guinea during the 1960's. One day his technician said, 'Dr Trinick, I have found that this plant Parasponia' — which was an opportunistic Ulmaceae species growing between rows in a new coffee plantation — 'has nodulated roots'. Trinick established that the nodules contained Rhizobium and that they were involved in symbiotic nitrogen fixation. Today, it remains the only situation where a non-legume plant has been found to be associated with Rhizobium in a nitrogen-fixing symbiosis.

Yes; because others like Casuarina have a different bacterium symbiont.

Yes. Before Trinick's discovery of the Parasponia symbiosis, and with haemoglobin known to be widespread in animals but only once in plants (in legume nodules), it was thought that maybe it had got there by a unique act of horizontal gene transmission. Perhaps an insect, via a viral vector, had managed to stick the gene into the plant. I thought, 'Now, if I could get hold of Trinick's Parasponia nodules and show that they contained haemoglobin, we could purify and characterise it and show that it had the proper gene structure'.

So that is when you came to us.

Yes, if that supposed unique act of horizontal gene transmission to a primitive legume could be ruled out, then one had to start thinking about vertical descent of haemoglobin genes between the plant and animal kingdoms.

And we did it!

We did it, yes.

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Puzzling Parasponia

But not everything has been fully elucidated and I guess that we still have the mystery around the single haemoglobin in Parasponia and what it does. Do you want to say something about that?

Yes. Trinick, John Tjepkema from Maine and I extracted, identified and purified Parasponia haemoglobin. Its spectra were beautiful, just like those of leghaemoglobin and the myoglobins. I went to the Wittenberg's laboratory again and we found the oxygenation kinetics for the Parasponia to be just right for a function in facilitated oxygen diffusion.

We found the same thing in another nitrogen fixing genus, Casuarina, where the endophyte is not Rhizobium but the actinomycete Frankia. Research student Tony Fleming and I identified a membrane-bound haemoglobin in Casuarina glauca nodules, then extracted and purified it.

This Casuarina symbiotic haemoglobin had spectra and oxygenation kinetics much like others I had studied. So here were three very different situations: legumes with a Rhizobium endophyte; Parasponia, a non-legume with a Rhizobium endophyte, and Casuarina, a non-legume with an actinomycete entophyte, all with the same sort of haemoglobin. This is where I had to come to terms with you geneticists in the adjacent building.

Yes. We were able to look at the gene sequence of the Parasponia haemoglobin using a soybean leghaemoglobin gene as our marker.

You tried to.

Well, we could not find it at first.

You could not find it, because a gene probe made from the soybean symbiotic haemoglobin would not hybridise with extracts from nodules or other Parasponia tissues. Meanwhile in the early 1970s I had been working with an organic chemist in CSIRO Melbourne called Alex Kortt. Alex and I had worked halfway through the amino acid sequence of the Parasponia leghaemoglobin. When he came to Canberra on one of his quarterly visits he said, 'Cyril, I think I have got something. Look at this. Here are six adjacent amino acids near the end of the Parasponia haemoglobin protein chain that could be used to make a unique 18 mer'. I asked Alex what that that meant and he explained.

He was talking about the DNA triplet code for amino acids. We were able to make that oligomer, as it is called.

Oh, yes. But there is a little bit coming before this. I had said to Alex, 'I think I understand; one could make a unique 18 mer, which might capture that gene in a way that the Peacock group could not using probes modelled on soybean leghaemoglobin. I will bet you, when you tell this to Jim Peacock tonight at our 5 pm appointment, he will be on the phone immediately to Marcker in Denmark, and my prize will be a bottle of Coonawarra cabernet for dinner'. I won the bet!

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Frustration and friends lead to an exciting discovery

Part of the complexity around Parasponia was that a related plant, Trema, which did not form nodules in association with bacteria, had a haemoglobin gene. What did you make of that?

Well, my version of that story is that Jörg Landsman from Germany who was first author on our Parasponia haemoglobin gene paper said to me one day when it was all over, 'Cyril, what is the closest plant to Parasponia which does not nodulate?' and I said, 'Oh, that is Trema'. How on earth would we get Trema? I said, 'It just happens that in his glasshouse, Mike Trinick has been trying desperately, with almost 100 different Rhizobium strains, to achieve nodulation of Trema'. 'Very well,' said Jörg in his slightly Teutonic mindset, 'I shall go to Trinick and ask him to make a great lawn of sprouted Trema. Landsman had seen me doing the same for Casuarina when we were at the beginning of the Casuarina haemoglobin gene work'. Well, knowing Trinick, this was not the way that one got anything from him. But calm was restored. Trinick, in fact, had many Trema seedlings growing already and also seedlings of Celtis, another member of the Ulmaceae. With heroic effort Trinick was able to germinate and grow for Jörg's successor Didier Bogusz, many young plants of Trema, Celtis and related members of the Ulmaceae. Also, wife Judy and I happened to own 11 hectares of rural property near Moruya — where we now live — with Trema growing as an opportunistic species after a 1980 bushfire. I would drive down with a liquid-nitrogen Dewar flask in the back of my car and, with a leather welder's apron on here (indicates) and a little flask here (indicates), I would be picking and freezing these little tiny Trema leaves. But progress was poor because every procedure we tried for extraction of leaflet genomic DNA or messenger RNA was wrecked because of very high polyphenol oxidase activity. None of the usual tricks worked. So the whole team was in your office one afternoon, when you said — in typical fashion — 'I have had enough of all this. Let's come to the glasshouse. Mike, show us your best young plants so that we can see what the roots look like'.

I can still remember that.

Here were these precious young plants from which we had been harvesting succulent leaflets from time to time, and with Trinick practically crying with rage as most were ripped out of their vermiculite beds for root harvest.

Well, we were able to isolate a clean preparation of the DNA then identify and determine the sequence of a Trema haemoglobin gene. That was it. There it was in the roots of a plant that was not able to nodulate. So it really cemented home the fact that all plants probably have haemoglobin and that the similarity in sequence and structure meant that animal and plant haemoglobins probably had a single origin back in a protoorganism.

That, indeed, is what we thought and has now been shown to be the case.

We all were involved in publication of that particular concept. That was really a very exciting time, I think. The protein chemist had been important, the haemoglobin biochemist was critical as was the microbiologist Trinick, and we molecular biologists concerned with nucleotide sequencing.

Of course.

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Class 1 and 2 haemoglobins and that little weed Arabidopsis

So, Cyril, apart from having to let nucleic acids and genes come into your life, you also had to admit us, Liz and myself, even though we worked on that little weed Arabidopsis.

I already knew the virtues of that plant. When your predecessor Lloyd Evans was chief and his daughter and one of my own daughters, Diane, were in the same class at school, they had a project: who could be the first person to start with a seed and get a mature plant to make more seed? I knew that this little plant Arabidopsis had a very short life cycle. So I smuggled some Arabidopsis seeds home but, for some reason, Catherine Evans didn't. So, Diane won the contest.

I think you will agree that Arabidopsis was again important because we were able to define more than one haemoglobin gene in its genome and those genes fitted into class 1 and class 2 haemoglobins that had been postulated before.

These class 1 haemoglobins all seemed to be non-symbiotic, and with very high oxygen affinity and hexacoordinate structure.

In them you have an flattish centre with its active haem, and with distal and proximal histidines bound tightly to the haem iron. This makes it very hard for oxygen to get into and especially out of its haem binding site. The net effect is that they have enormously high oxygen affinities. Thus, such class 1 non-symbiotic haemoglobins, present in cereals, legumes and many other plants really could not function as oxygen carriers because they would not be able to manage the facilitated diffusion of oxygen. Well, they could transfer oxygen, if there was something adjacent to grab it. But no oxidase, even the high-affinity cytochrome cbb3 complex I helped to characterise in Rhizobium bacteroids, could be working at sub-nanomolar dissolved oxygen.

Yes; and the class 2 haemoglobin had very different kinetics again, didn't they?

Well, that was all very well and it was a nice little scheme that your group helped to create, which had something called presymbiotic haemoglobins in class 2 and the non-symbiotic haemoglobins in class 1. But, within class 1, there was just one symbiotic haemoglobin, which I had found in Parasponia root nodules. I knew very well that it was a symbiotic one: it had the right oxygenation traits. But most plant gene phylogenists would put all class 1 haemoglobins into a non-symbiotic box, including my Parasponia haemoglobin — very, very definitely a symbiotic one. That used to make me very cross. Even you in your papers used to say, 'Well, perhaps the Parasponia haemoglobin is a dual function haemoglobin,' and I used to think to myself, 'This is nonsense'.

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Oxygenation kinetics and clever plant haemoglobin

You always thought — and I suspect that you still do — that Liz and I had missed another haemoglobin gene in Parasponia; we haven't, you know.

Some years later I thought to myself, 'I am going to get Peacock and his mob'. I had had a continuing long-distance interaction with USA colleagues Mark Hargrove and John Olson — the latter being a former student of my hero Quentin Gibson. One day I said: 'Mark, please could you make overexpressed Trema haemoglobin,' which had not been done. 'I will harvest and send seed for plant growth, and organise the probes that you might need from my former colleagues at CSIRO in Canberra'. Jean Finnegan was involved in this, incidentally.

Yes. I remember that this interaction led to the making large amounts of Trema and Parasponia haemoglobins using bacterial cultures to express the proteins.

Yes, Mark purified both and determined X-ray crystal structures as well as oxygenation kinetics.

His overexpressed Parasponia haemoglobin had oxygenation kinetics and oxygen affinity comparable with those symbiotic haemoglobins I had isolated naturally from Parasponia, Casuarina and legume root nodules.In contrast he found Trema haemoglobin to have the oxygenation kinetics and extreme oxygen affinity of other non-symbiotic haemoglobins. To me, this negated the idea of Parasponia as a dual-function haemoglobin.

Yet there was hardly any difference in amino acids.

There were only a few. By comparing the haemoglobin gene sequences of all identified Trema species with that of Parasponia symbiotic haemoglobin one could identify only seven critical base changes. The mutations of seven amino acid residues were enough to slide Trema nonsymbiotic haemoglobin into symbiotic Parasponia haemoglobin, and Mark Hargrove, by comparison of his X-ray crystal structures, thought that he could see the E and the EF helices sliding a little bit alongside each other, which was enough to change oxygen affinity dramatically.

So haemoglobin is really a fantastic protein. In animals, there are many special purpose haemoglobins and they are converted from one form to another with very few changes in sequence. It looks as though the plant haemoglobin is just as clever.

Yes, and Mark Hargrove with his delectable crystal structures and sequence analyses of our Parasponia and Trema showing just a little slip between these two helices to be enough — fantastic!

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The mysteries that remain

So I guess we felt — you and us, and our colleagues in different parts of the world that worked with us — that we had worked out a rather exciting new area of plant biochemistry. Then along came your retirement and along came other scientific interests for us. But, in fact, you had not really retired and we had not really heard the last of haemoglobin. But still it has been exciting, even until the present time. But, say, for the non-symbiotic haemoglobin: we dont really know what it does. It is in the roots of plants. It does not respond in the same way as the symbiotic haemoglobin, and you had suggested that it might be a sensor molecule for oxygen.

I had suggested that it might be a sensor molecule for oxygen because I had found in Trinick-grown Trema roots a tiny, tiny amount — maybe a nanomolar concentration — of haemoglobin, not nearly enough to be an oxygen carrier but certainly enough to be an oxygen sensor. But then, in a glorious slide that you showed at an Australian Academy of Science meeting in May 1989, you showed the result of smuggling artificial genes containing the promoter (a region of DNA that initiates transcription of a closely-following gene) for either Parasponia and Trema haemoglobin into tobacco. These artificial gene constructs, known as Gus reporters, contained also a specific glucuronidase gene, which meant that when tobacco tissues containing the expressed gene were exposed to a suitable dye precursor...

Yes, to make a blue colour.

…such blue colour would reveal where the doctored gene was being expressed. Here in the tobacco root the GUS reporter gene is being expressed in the very tip, where one might suspect from others evidence of high respiration rate, that there might be functioning haemoglobin.

So we took the promoter, the driving signal part of the gene, and hooked it on to the reporter gene.

Yes. I had thought previously that these non-symbiotic class 1 haemoglobins, if present in minute amounts, could be functioning as oxygen sensors. But when I saw that tobacco root with a reasonable amount of expression of a plant haemoglobin gene promoter region I realised that it probably had other functions.

It may also have a sensor function?

Well, maybe.

It might be a scavenger of nitric oxide.

Well, it might be. A believable concept is that these non-symbiotic, high-affinity haemoglobins can capture oxygen so tightly that it cannot get out fast enough to do anything useful at an external site. But then a molecule other than oxygen could perhaps come in through a different entrance channel and that transformation might occur inside the protein. The oxygen of the oxyhaemoglobin might combine with, say, nitric oxide, a potential toxin, and inactivate it. In fact, others are now showing that many non-symbiotic, high-affinity haemoglobins are concerned, in one way or another, with nitric oxide transformation. As far as I am concerned, that is all I know. Here again (indicates) is that picture of a tobacco root showing the relatively large amount of haemoglobin gene-promoter expression in the rapidly-developing tip, where there might well be an oxygen-stress situation. Mind you, that is not necessarily indicative of a need for nitric oxide or nitrite detoxification. But it was enough to suggest to me that plant nonsymbiotic haemoglobins might be something other than simple oxygen-sensing molecules.

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The lows and highs of haemoglobin research

So, Cyril, I suppose that you feel happy and satisfied with a lifetime of research centred around haemoglobin. One really important thing was to have demonstrated that it had a vertical evolution rather than a lateral transfer from some animal et cetera. Then, even though we don't know in detail perhaps all the roles of each of the different classes of plant haemoglobin, we have made progress in demonstrating at least some of their major purposes. Since you have been retired, can you comment on what you might regard as the high points and the low points?

Well, to make it very short, the nadir was that dreadful period after my honours degree supervisor disappeared to Sheffield and I drifted for several years. A late high point came when wife Judy and I — she from her school library job and I from CSIRO — decided to quit formal employment at 60 and have some fun. We went twice around the world. We ended up at the utmost point of the Butt of Lewis on the northernmost island of Outer Hebrides. Later we visited and sipped at every malt whisky distillery on Islay. We visited laboratories around the world where I interacted with former and future colleagues, urging them into collaborative studies on plant haemoglobins, some of which persist. Then we came back to Moruya where we now live, and we are still enjoying life very much.

Here we are, four years ago, during celebration of our 80th birthdays and our daughter Diane's 50th.

Cyril, thank you very much for agreeing to be interviewed today. I have really enjoyed it and I have felt privileged to do so.

Thank you, Jim. It is indeed an honour to have worked with somebody as stimulating and exciting as you, especially one who could see through my oddball style and realise that I had made useful contributions to knowledge.

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Leghaemoglobin is an iron-containing porphyrin-protein that forms in the root nodules of leguminous plants infected with the nitrogen-fixing bacterium Rhizobium. (The word comes from LEGuminous + HAEMOGLOBIN.) It is the product of the symbiosis of two organisms: the legume plant itself and the Rhizobium bacteria present in the plant's root nodules. Leghaemoglobin has a very high affinity for oxygen. Its function is thought to be to transport oxygen to the bacterium (which respires aerobically) in such a way that the nitrogen-fixing enzyme, nitrogenase (which is destroyed by exposure to high oxygen), remains unaffected. Leghaemoglobin is chemically and structurally similar to animal haemoglobin and myoglobin, and like them is red in colour.

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