M.D. (Hal) Hatch
Photosynthesis Research 33: 1-14, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands.
I can't believe my luck* M.D. (Hal) Hatch Division of Plant Industry, CSIRO, GPO Box 1600 Canberra, A C T 2601, Australia Received 7 April 1992; accepted 7 April 1992
C 4 photosynthesis, photosynthetic carbon fixation, plant biochemistry
Abstract The author gives an account of his life and work in scientific research. At the prompting of Govindjee, this is a quite personal account and, I hope, not too serious. The circumstances surrounding the discovery of C 4 photosynthesis are mentioned together with some aspects of the subsequent development of this field. Since it seems to be expected on such occasions, there is also some reminiscences and some unsolicited advice. I hope there are no gross or libelous inaccuracies.
Abbreviations: 3-PGA - 3-phosphoglycerate; PEP - phosphoenolpyruvate;
orthophosphate; PP, -
Early years It would be nice to say that in my formative years I excelled academically and wondered incessantly about how living organisms worked. Instead, the truth is that I was a very ordinary student and thought little about biology other than having some curiosity about girls. In primary school in Perth (Western Australia) I never did better than 12th in the class and there were comments on my report cards such as 'behaviour not as good as geography' which I didn't do specially well at, but happened to enjoy. A move across Australia to Sydney in 1947 at the age of 14 generated some emotional trauma as I recall, but it probably saved me academically. The consequent lack of significant social distraction for a few years provided the time to apply myself to my studies. I suppose I learned at that time that there was a reasonable relationship between effort and outcome or reward. Although I did well enough academically, the * Written at the invitation of Govindjee.
highlights of this high school period were making the First XV Rugby Football team and winning the State under 17 years mile championship. In those days it was common to be assessed by a vocational guidance officer during the last year of high school. I was advised that I shouldn't study to be a medical practitioner because I would worry too much about my failures (patients?). As a result I enrolled in science at Sydney University with a view to becoming an industrial chemist. I must say I am still not sure what an industrial chemist is. With chemistry as a major, and with my first exposure to biology, having done botany in first year, I elected to do biochemistry in the second year. But this was only after I had seriously considered switching to pharmaceutical science largely because it appeared to be a much better option, at least financially. Fortunately my dear father skilfutly maneuvered me away from this i d e a - o n e of those classic turning points in life that one looks back on and shudders! My chance selection of biochemistry to supplement my mainstream chemistry course was
another turning point. Even though biochemistry was in its infancy at that time I found it fascinating- perhaps partly because there was so much speculation and there was obviously so much to be discovered. This appeal was heightened by the lectures in plant biochemistry given at Sydney University in 1953 by F.R. (Bob) Whatley. He was later to distinguish himself in studies on photosynthesis in the University of California, Berkeley laboratory of D.I. (Dan) Arnon and subsequently was appointed to a Chair at Oxford. Dr Whatley managed to transmit his own fascination for the subject and a real curiosity about how things work. In subsequent years when science got too serious and appeared to be weighing heavily on me, I could sometimes regain perspective by thinking back to how Bob Whatley might have looked at the situation. My average examination performance only just got me approval for an extra 'Honours' year but, having encouraged me to 'give it a try', Whatley promptly departed for Berkeley. Needless to say, I chose to do an Honours research project in the area of plant biochemistry. Dr Whatley's replacement was Adele Millerd who had just returned from the United States where she had been involved in some of the very early studies on plant mitochondria. She was also a great enthusiast and so consolidated my dedication and commitment to plant biochemistry in particular. My research project concerned aspects of respiration including the cyanide-insensitive respiration of the spadix of what I thought to be the lily, Arum maculatum. After four months of work, and some odd results, I got my material classified - Zantadeschia aethiopica - a useful lesson. With my shiny new BSc (with only 2nd class honours) I joined a government research laboratory (CSIRO, Commonwealth Scientific and Industrial Research Organization) situated within the University of Sydney and dedicated to aspects of plant biochemistry related to the food industry. Here, I was allowed to undertake studies for a PhD while on my normal salary. Consequently, I was denied the experience of being a poor (financially destitute) PhD student something I have always regretted because it makes a much better story. The head of this laboratory was R.N. (Bob)
Robertson; he was one of the architects of the great era of plant physiology research that was to follow in Australia. I owe him a great deal for his advice and encouragement over many years. My PhD involved the resolution of the mechanism of a 'Pasteur Effect' seen in a pea seed extract. This problem was introduced to me by J.F. (John) Turner who also taught me the value of precision and taking time to get things right experimentally and in publication. We resolved the mechanism of this particular Pasteur Effect and in so doing discovered a unique NADHdependent protein disulphide reductase (Hatch and Turner 1960). This was the first of several of enzyme discoveries that 1 was to be associated with in the years to follow. In 1959 I was fortunate enough to land a post-doctoral fellowship with P.K. (Paul) Stumpf in the newly formed Department of Biochemistry and Biophysics in the University of California at Davis. This allowed me to expand my biochemical horizons enormously, and also my skiing horizons. Here, I worked mostly on acetylCoA carboxylase, and discovered its transcarboxylating activity (Hatch and Stumpf 1961). This was the second enzyme I was involved with that contained SH groups critical for activity. Oddly enough, all three of the novel enzymes that we discovered during the course of our later studies on C 4 photosynthesis had this sulphydryl feature in common. Paul Stumpf taught me a great deal including the value of perseverance in research and the reward of staying with one research field and following it right through; both lessons of great value later. I also recall dissipating a lot of time in Davis (and learning another lesson) trying to identify a strange radioactive compound formed from acetyl-CoA plus H14C03 in the presence of acetyl-CoA carboxylase. When reactions were stopped with alkali one would expect the product malonyl-CoA to be hydrolysed to malonate. The trouble was that I substituted N a 0 H with N H 4 0 H because the resulting mixtures were then easier to reneutralize later. I had not appreciated that with NH4OH, malonyl-CoA undergoes aminolysis to give the monoamide of malonic acid leading me to believe that I had discovered some strange new reaction between H U C 0 3 and acetyl-CoA.
After two years in California I agonised over whether to return to CSIRO in Sydney or take up an offer of a position in Brisbane at the new David North Plant Research Centre set up by the Colonial Sugar Refining Company. This laboratory was headed by K.T. (Ken) Glasziou, a colleague from undergraduate days. He showed me that anything was possible by studying for entrance to Sydney University at the age of about thirty with an expanding family to look after, having spent the previous decade flying bombers during the second world war and then dairy farming in New Zealand. The decision to go to this Brisbane laboratory was another one of those critical (and fortunate) turning points in my scientific career. The main purpose of the David North Plant Research Centre was to investigate the basic biochemistry and physiology of sugarcane and the sugar storage process. Our research group headed by Ken Glasziou did a good deal of pioneering work on sugar transport and metabolism. During follow-up studies on the mechanism of sugar storage, John Hawker and I discovered the enzyme sucrose-P phosphatase (Hawker and Hatch 1966) which was later to prove to be also important in sucrose formation in leaves.
At that time in the early 1960s, our laboratory was in regular contact with workers including Hugo Kortschak at the Hawaiian Sugars Planters Research laboratory in Honolulu. Through that contact we were aware that, as early as the late 1950s, Kortschak and collaborators had seen some unusual patterns of labelling in products formed when sugarcane leaves were allowed to assimilate ~4CO2 in the light. I say unusual because by this time Melvin Calvin with his colleagues Andy Benson, AI Bassham and others had resolved the key elements of the photosynthetic process operating in the green alga Chlorella, and this was reasonably assumed to account for all photosynthesis (Calvin 1989). In this process the first product, of course, is 3phosphoglycerate formed when CO 2 is assimilated via the enzyme ribulose 1,5-bisphosphate carboxylase. The puzzle was that the Hawaiian
workers did not find a great deal of label in 3-PGA (a 3 carbon acid) and later they showed that much of the early label was located in malate and aspartate (4 carbon decarboxylic acids). Nothing of this was published for several years. If I recall rightly it was published along with other interesting results at the prompting of a new head of that laboratory, Dr Lou Nickel. Amongst our group in Brisbane at that time was C.R. (Roger) Slack, a young scientist from Britain. Roger Slack and I were intrigued by this data from the Hawaiian laboratory and had often discussed the possible interpretations of these results. So when Kortschak and his colleagues finally published their data in 1965 (Kortschak et al. 1965) we set about repeating and extending their observations to see if we could resolve the question of what it all meant. I will return briefly to those studies, but first it is worth recounting one other interesting twist to this story. In the late 1960s, that is 3 or 4 years after we had begun studying the C 4 photosynthetic process, we became aware of a report published about ten years earlier, in the 1960 Annual Report of a Russian agricultural research institute. This report by a young Russian scientist, Y. Karpilov, showed that when maize (Zea mays) leaves were allowed to assimilate ~4CO2 in the light for short periods, most of the fixed label appeared in malate plus aspartate (Karpilov 1960). Only a small percentage was found in 3-PGA. In a publication about three years later, Karpilov and a colleague speculated that such results may be related to faulty killing or extraction procedures, and there the matter rested. Later, after our early papers on sugarcane photosynthesis were published, Karpilov resumed an interest in C 4 photosynthesis. I met him at the International Botanical Congress in Leningrad in 1975, where he invited me to dine with him and we consumed a good deal of vodka. Later, I remember standing in the balcony of my Leningrad hotel in the late night twilight thinking how strange life was, the fickleness of fate and those kinds of things. Unfortunately, I was never given a chance to return this hospitality because, soon after, Karpilov was killed in a bicycle accident in Moscow. I kept in contact with Hugo Kortschak during
the late 60s and early 70s through occasional visits to Hawaii. He was a gentle person who was always interested in how things were turning out with the C 4 pathway. I recall that amongst other things, the Kortschak's had over a number of years cared for several orphaned or delinquent children, a brave undertaking. We last met in Britain in 1981. The occasion was the awarding of the Rank Prize jointly to Hugo Kortschak, Roger Slack and myself for the work surrounding the discovery of the C a photosynthetic process. Our initial experiments (Hatch and Slack 1966) with sugarcane leaves were designed to trace the exact fate of carbon assimilated by photosynthesis using [4CO2 and the procedures originally developed by Calvin and colleagues. We confirmed the results of the Hawaiian group that most of the radioactivity incorporated after short periods in 14CO2 was located in malate and aspartate and that these C a acids were rapidly labelled from zero time whereas steady rates of labelling of 3-PGA, sugar phosphates and sucrose occurred only after increasing lag periods. More revealing and critical information was provided by our so-called 'pulse-chase' experiments where, after a period of labelling in 14CO2, leaves are transferred back to air containing unlabelled CO 2 and the movement of fixed radioactivity between compounds is followed. These experiments clearly showed the rapid movement of radioactivity from malate into 3PGA and then later into hexose phosphates and other intermediates and finally into sucrose and starch. Other critical results reported in these initial studies (Hatch and Slack 1966, Hatch et al. 1967) included the findings that (i) the chemically unstable dicarboxylic acid, oxaloacetate, was rapidly labelled as well as malate and aspartate and that oxaloacetate was almost certainly the first product formed, (ii) the C 4 acids were initially labelled almost entirely in the C-4 carboxyl, (iii) this C-4 carboxyl carbon gave rise to the C-1 carboxyl of 3-PGA, (iv) 3-PGA was converted to hexose phosphates apparently by the same path as normal Calvin cycle photosynthesis, and (v) a survey of a large number of plants showed labelling patterns similar to sugarcane in 14 grass species from 10 genera as well as a sedge species.
As a result of these initial studies, Roger Slack and I developed a simple working model to explain photosynthesis in sugarcane (Hatch and Slack 1966). In this model, reproduced in Fig. 1, we proposed that in the initial reaction, a 3carbon compound, probably PEP or pyruvate, was carboxylated to give a C 4 dicarboxylic acid with the C-4 carboxyl derived from CO 2. After reactions interconverting the C4 acids, oxaloacetate, malate and aspartate, the 4-carboxyl of one of these acids is transferred to become the carboxyl group of 3-PGA and we speculated that the remaining 3-carbons of the dicarboxylic acid might serve as a precursor to regenerate pyruvate or PEP. Soon after, we named this process the C 4 dicarboxylic acid pathway of photosynthesis and this was later abbreviated to C 4 pathway or C a photosynthesis. This is not the place to belabour the details of C 4 photosynthesis. However, the following comments may be more comprehensible if I include a detailed scheme showing my view of the current status of C 4 photosynthesis (Fig. 2). This scheme also demonstrates how far we have come since that first model shown in Fig. 1. After proposing the model shown in Fig. 1, we conducted a series of studies where predictions based on existing information led to discoveries about the enzymes involved which in turn allowed further predictions. For instance, radiotracer studies predicted a primary carboxylation Sucrose
Hexose phosphates .4, [ "
-'~Ribulose diphosphate?) Carboxyl acceptor
3-Phospho-'~ 3 C glycerate ~" 2C -J 1C*
"¢ ~ Phosphopyruvate -] 3C
41 kPyruvate " _
Aspartate \ 4C* -+-
Fig. 1. A working model published in 1966 (Hatch and Slack 1966) to explain patterns of labelling observed when sugarcane leaves assimilated z4CO2 in the light.
- k - ~ ~'"°"°"~°''" ~-~
I ~ !
o.~ ~I otr ~
= = - .~ ~ ~ '~ ~ = ~