Cytokinins

Chemistry, Activity, and Function

David W. S. Mok

• Publisher: CRC Press
• Year: 2019
• ISBN:
  9781351088183
• Edition: 1
• Topic: Medicine
• Subtopic: Biochemistry in Medicine

Cytokinins are hormones involved in all aspects of plant growth and development and are essential for in vitro manipulation of plant cells and tissues. Much information has been gathered regarding the chemistry and biology of cytokinins, while recent studies have focused on the genetics and cytokinin-related genes. However, other than proceedings of symposia, no single volume on cytokinins has been written. This book is the first of its kind, homing in on the key subject areas of cytokinin-chemistry, biosynthesis, metabolism, activity, function, genetics, and analyses. These areas are comprehensively reviewed in individual chapters by experts currently active in the field. In addition, a personal history on the discovery of cytokinin is presented by Professor Folke Skoog. This volume summarizes previous findings and identifies future research directions.

Chapter 1
A Personal History of Cytokinin and Plant Hormone Research

Folke Skoog

Table of Contents

Prologue

I. Beginnings of Plant Growth Substance Research in the U.S

II. Auxin and the Regulation of Bud Growth

III. Regulation of Bud Formation

IV. The Discovery of Kinetin

V. Triacanthine and the Cytokinin Activity of Isotriacanthine

VI. The Race to Isolate a Naturally Occurring Cytokinin

VII. Chemical Studies of Cytokinins

VIII. Isolation of Cytokinins from Corynebacterium fascians

IX. Cytokinins as Constituents of RNA Molecules

X. Concepts of Hormone Action

XI. Development of the Revised Medium for Plant Tissue Culture

XII. Perspectives

References

Prologue

I was asked to. submit an account of the early research on plant hormones and especially on the work on cytokinins that took place in my laboratory at the University of Wisconsin. Considering my limited capacity to recall, to concentrate, and to write, and having seen the results of similar tasks by other octogenarians, I am convinced it would be in the best interest of all concerned (eventual readers included) for me to refrain. Instead, I am pleased to discuss and answer a set of questions, prepared by Professor Donald Armstrong, on my experience with plant hormone research. Professor Armstrong has been engaged in plant hormone research ever since he started as a graduate student with me in 1961. He continued as a research associate with me and others and is now a faculty member of the Department of Botany and Plant Pathology at Oregon State University. He has made outstanding contributions to our knowledge of cytokinins, especially those he has isolated and studied in specific tRNA species from a wide variety of organisms. He has studied the action of different types of cytokinins, their metabolism, and their degradation. He has shared in much of my work and is thoroughly familiar with all of it, and he is uniquely qualified to review it. I am deeply grateful to him for his interest in this matter

I. Beginnings of Plant Growth Substance Research in the U.S.

Dr. Skoog, you received an undergraduate degree in chemistry from the California Institute of Technology in 1932 and a Ph.D. in biology from the same institution in 1936. During this period, the Division of Biology at Cal Tech became an important initial center of plant hormone research in this country. How did this come about?

It was introduced by T. H. Morgan, the eminent geneticist, who in 1928, when he was facing mandatory retirement at Columbia University, was persuaded by Cal Tech physical scientists (Millikan and others) to come to Cal Tech to start a division of biology. Morgan was interested in establishing an institute of quantitative experimental biology. He collected a staff of about ten investigators. He brought his former students and current research associates from Columbia: A. Sturtevant, C. Bridges, Jack Schultz, two plant geneticists (Sterling Emerson and Ernest Anderson), and two biochemists, one of whom was Kenneth Thimann. He also brought two plant physiologists, Robert Emerson (fresh from Otto Warburg’s laboratory) and one plant growth specialist, Herman Dolk, from F.A.F.C. Went’s laboratory in Utrecht. He brought these men together and looked after their research interests and personal needs, with truly paternal care.

Why Dolk? Morgan at heart was a classical embryologist or developmental biologist. He had spent much time slicing earthworms to study their regeneration. He was interested in the polar regeneration of head and tail regions, and it is said he had the notion of a tail-forming and a head-forming substance. In any case he was greatly interested in polarity. Around 1910 he wrote an article on polarity in plants. When he heard that two young students of Went, his son (Frits) and Dolk, had demonstrated that a substance that promoted growth moved polarly down oat coleoptiles, he arranged for Dolk to join his group at Cal Tech. A small building with two underground rooms, superficially environmentally controlled, was constructed for Dolk’s work on the plant growth substance.

How did you personally get involved in plant hormone research?

Well, in 1929 when I was a sophomore at Cal Tech, taking physics, I met Carl Lindegren. Lindegren was a graduate student with Morgan, but he also was taking sophomore physics. Since he was an old Swede from Rhinelander (Wisconsin), I told him that I didn’t have to take German, but I had to take something else. He said, “Fine, you come to work with me.” Lindegren was working on the genetics of Neurospora, which eventually, largely through Lindegren’s influence, became a very important organism in microorganism genetics, especially as developed by Beadle, who was a young instructor at Cal Tech at that time. I worked with Lindegren while I was doing my regular undergraduate work in chemistry. My task was to study the mating types of Neurospora. This was fine, but the study of Neurospora at that time, before the availability of B-vitamins, was more or less a kitchen science. You had to cook the oatmeal and grow them, and it became routine and pretty monotonous. So, I told Lindegren that I was going to see what I could do with the biochemists. So he said, “Well, those biochemists…, I tell you, I wouldn’t go there. There’s a young Dutchman just come here to work, and he has a new thing, something that stimulates growth. I’ll talk to him, and you can work with him.” So I started to work with Dolk, which was the beginning of my work with plant hormones.

At that time the growth substance, later called auxin, had been shown to promote elongation of cells, especially in regions some distance below the tip of the coleoptile where it was form d. Frits Went suspected that a second growth factor, moving from the seed was required for auxin to be effective in promoting growth. My project was to obtain evidence for this second factor. Using time-lapse photography, I studied the distribution of growth rates along the coleoptile, to see how it might be influenced by factors coming from the seed. Unfortunately, Dolk had an accident at the Grand Canyon, in which his car went off the road. He came back from this, but his car might have been damaged. Two weeks later he took the same car out in the desert, and he blew a tire and was killed. He was a tine man and a keen, exceptionally talented experimenter.

When Dolk died, Thimann, who had started to collaborate with Dolk, wanted us to continue the work begun with Dolk. I worked with Thimann as an undergraduate (but on a different problem) and continued to work with him as a graduate student at Cal Tech, starting in 1932, until he left for Harvard University in 1935. His early guidance and my close contact with him were most beneficial. Our friendship has continued for more than 60 years.

I finished my graduate work in 1936 under the direction of Professor Frits Went, who was brought to Cal Tech by Morgan soon after Dolk’s death. Went’s broad botanical knowledge and his imaginative, enthusiastic approach to research were both beneficial and inspiring. His deep concern for my personal well-being as a lone student during the economic stress of the Depression is something I shall always remember with deep gratitude.

II. Auxin and the Regulation of Bud Growth

Much of your early career in plant hormone research appears to liave centered on the role of auxin in regulating the growth of buds. Would you trace how this work developed and comment on the ideas, events, and people that influenced your thinking during this period?

In 1925 Keeble and Snow proposed that the inhibition of lateral buds by the terminal bud in the broad bean Vicia faba was due to a factor moving polarly from the terminal bud, down the stem, to the lateral buds. The reason they concluded that the movement was polar is still not clear to me. When I began to work with Thimann, he suggested that we attempt to find evidence for this factor by procedures that had been used to demonstrate the growth substance in Avena coleoptiles. We found that material promoting growth in A vena coleop-JS could be obtained from terminal buds of beans, y diffusion into agar blocks. Furthermore, when ihe agar blocks or excised terminal buds of beans were placed directly on the cut surfaces of decapitated plants and replaced every 3 h, inhibition of the laterals could be maintained. Avena coleoptile tips could be substituted for terminal buds. Small amounts of active material could also be obtained from leaves. We found that the buds could be inhibited with extracts from Rhizopus (the bread mold) and, eventually, with a synthetic preparation of the auxin indoleacetic acid. We also showed that auxin moved polarly in beans, just as it did in coleoptiles, and promoted elongation of the stems of defoliated bean plants. Thus, we concluded that auxin was not confined to coleoptiles, but occurred generally as a growth-promoting factor in plants. This work was published in 1933 and 1934.

The fact that a growth-promoting substance from the terminal bud could also inhibit the growth of laterals was not readily accepted. We argued that the effect was exerted directly in the buds by relatively high concentrations of the hormone being supplied continuously from the terminal. Others proposed that the inhibition resulted indirectly from depletion of nutrients (Went said specific growth factors) being utilized for the growth of the main stem in the presence of the hormone.

Soon after I began work as a graduate student, I wanted to test the effect of the growth substance on buds in isolation from the plant. I went to Morgan to tell him that I wanted to do tissue culture with plant material and needed equipment (forceps, scissors, etc.) for this purpose. He told me that “you don’t need all this equipment. There is a Frenchman coming in a few months. He is bringing instruments, and I will put you to work with him.” This was Boris Ephrussi. He was an expert in animal tissue culture. He did bring beautiful equipment, and I learned a great deal about tissue culture techniques (bleeding the rooster, making embryo extracts, and growing animal tissues) from him, but this did not accomplish much with plants. Not until some years later did I grow excised pea epicotyls and show that their growth could be promoted or inhibited as a function of the applied concentration of indoleacetic acid.

I was still interested in the possibility of a second factor, and as a graduate student, I also studied the effect of deseeding on the growth of Avena coleoptiles. I was able to demonstrate that a factor from the seed was moving up the coleoptile and was converted to auxin in the tip. I also found that deseeded oat seedlings in which the synthesis of auxin was reduced were more sensitive to applied auxin and could be used not only to test for five to ten times lower auxin concentrations than the standard Avena test, but could also be used for the detection and quantitative assay of auxin precursors. For example, tryptophan and tryptamine were both shown to be slowly converted to indoleacetic acid, when applied to coleoptiles. However, this work did not reveal a second factor.

At the time of my work on bud inhibition, I saw a report by an Irish botanist, Hudson, on gemma cup formation in the liverwort Marchantia. He showed that, normally, gemma cups develop only on the basal portion of the thallus, but when the thallus is cut into segments, gemma cups are formed on the basal edge of all segments except on the segment containing the apical cell. Furthermore, if the thallus is exposed to X-rays, gemma cups appear on all parts of the thallus. It seemed possible that gemma cup formation might be inhibited by auxin in the same way that lateral buds were inhibited by auxin from the terminal bud. Also, it seemed possible that auxin might be inactivated by X-rays. I found that terminal buds of young Vicia or Pisum plants, also coleoptile tips of A vena seedlings, exposed to moderate doses of high-voltage X-rays quickly lost about 30 to 40% of their auxin content as measured by diffusion into agar blocks, and in the former two, auxin production was entirely lost in a period of about 4 to 5 days. In parallel with this, there was outgrowth of lateral buds. Similarly, indoleacetic acid in aqueous solution exposed to moderate dosages of X-rays was inactivated by 30 to 40% in the presence of air. If kept under nitrogen, the inactivation was greatly reduced. It was deduced that the inactivation was due to peroxide, which is formed on X-irradiation of water in the presence of air and to which auxin had been shown to be very sensitive. I could show that the immediate effect of X-rays on the growth could be reversed, to a slight extent, by the application of indoleacetic acid to the plants. Furthermore, X-irradiation of internodes between the terminal and lateral buds led to the development of lateral buds in the portion of the plant below the irradiated region. This outgrowth of buds could be counteracted by indoleacetic acid applied above the buds, thus providing further evidence that the action of auxin in bud inhibition is exerted directly in the buds.

On completion of my graduate work al Cal Tech, and no doubt on the basis of Morgan’s recommendation, I was awarded a National Research Council Fellowship to work with Professor Dennis Hoagland in the Division of Plant Nutrition at the University of California-Berkeley. Investigations in his laboratory indicated that incipient effects of zinc deficiency of plants grown in nutrient solution could be counteracted by addition of the auxin indoleacetic acid. It was therefore of interest to determine to what extent zinc might affect the synthesis or action of the endogenous growth substance. The results of that work demonstrated that even 1 day before visible symptoms of zinc deficiency appeared, auxin production in the terminal shoots of tomato plants was reduced 50%, and within 2 days, to practically zero, leading to complete secession of growth. This effect could be prevented by growing the plants under red light, and it was shown that deficiency of zinc was associated with striking increases in peroxidase and oxidase activity in the tissue. Although the effect of zinc deficiency on auxin was both striking and specific, the essential requirement for zinc was not due exclusively to auxin inactivation and the loss of auxin synthesis.

My experience in the Division of Plant Nutrition was exceptionally valuable to me. Hoagland was a kind, wise, and practical scientist and administrator. It is of interest that he and Truog, the two outstanding names in plant nutrition in this country, worked together under E. V. McCollum at the University of Wisconsin, where they both terminated their formal studies with the M.S. degree. Hoagland’s group of investigators included faculty members and research associates. Each member had his own area of specialty, and each one was responsible also for helping all the others with work in his area. It was a very sound and effective team. In my opinion, it was the most active and efficient organization in experimental plant biology anywhere at that time. I had the special favor of often accompanying Hoagland on his daily inspection tours of the U.C.-Berkeley greenhouses where a large number of nutrition experiments were in progress. He took time to discuss the special features and the purpose and nature of each one as it was started. I especially recall his pleasure when he observed (I believe, for the first time) and described symptoms of molybdenum deficiency. His instructions on plant behavior and his emphasis on improving methodology have been of lasting value in my work.

While at U.C.–Berkeley, I also had an opportunity to work with Professor J. P. Bennett, who had a long-term interest in the dormancy of fruit trees. He had a greenhouse full of 6-ft-tall pear trees that had been kept dormant by high temperature for 4 years. I jokingly told him that I thought I could break the dormancy of these trees with an extract, prepared from yeast, that I had used to germinate dormant bacterial spores. Two ml of an aqueous solution of this preparation, injected through the tip of a branch near the top of a tree, moved down the stem and caused the leaves to come out all the way to the base of the tree. This led to fur...