1955: Kinetin Arrives. The 50th Anniversary of a New Plant Hormone

Fifty years ago, scientists from the Botany and Biochemistry departments at the University of Wisconsin announced the isolation, crystallization, characterization, and synthesis of 6-furfurylaminopurine (Miller et al., 1955a, 1955b, 1956), a plant hormone in a class now referred to as cytokinins (Skoog et al., 1965). They proposed the trivial name kinetin for this substance, and described its ability to promote cell division in test tissues from tobacco (Nicotiana tabacum). The discovery of kinetin marked the culmination of several years of research on a variety of projects conducted by many people in the two departments. A description of the little victories, the interesting twists, and the roles of persistence, patience, and good luck in this discovery has never been presented in print. Hopefully, this account will serve as an illustration of how scientific advancement can arise from fruitful collaboration and the utilization of many odd bits of information often obtained rather fortuitously. In the early 1950s, Professor Folke Skoog and his group in the Botany Department were continuing their work on the chemical control of new shoot formation in excised pieces of tobacco stems grown in culture (Skoog fondly referred to these de novo formed shoots as “buddies”). This research effort was hindered by the fact that the stem pieces, which were obtained from plants grown in the greenhouse, were difficult to obtain in a sterile and completely healthy condition. The results obtained with the stem segments were quite variable, probably because of the harsh sterilization protocol (mercuric chloride was employed for sterilization), varying light intensities and temperatures at different times of year, as well as greenhouse management procedures. To avoid the difficulties and variability of using greenhouse-grown stem pieces, Skoog sought an alternative tissue to use in these studies. By the 1950s, there had been advances in the art of plant cell culture; in particular, several laboratories had reported that certain complex materials added to an otherwise chemically defined culture medium (consisting of minerals, a carbon source such as Suc, and often the plant hormone auxin) could result in the sustained growth of plant tissues or cells. These materials included coconut (Cocos nucifera) milk, which had first been used by van Overbeek to stimulate the growth of young Datura stramonium embryos in culture (van Overbeek et al., 1941). Skoog explored whether his group could continuously culture tobacco tissue on coconut milk-containing medium, and thereby produce “standardized” samples of tissue for their studies of shoot formation. Skoog's group found that although coconut milk sometimes promoted the proliferation of tobacco tissue, the effect was quite variable; in fact, some batches of coconut milk inhibited growth. They were using coconuts purchased at a local grocery store, and recognized that some of this variability might be due to inhibitors present in certain batches of coconuts. Skoog therefore sought advice from Professor F.C. Steward at Cornell University. Steward's laboratory was attempting to purify and identify materials in coconut milk that promote the growth of carrot (Daucus carota) cells in culture. Skoog hoped to obtain some details of Steward's coconut milk purification procedure, not with the intent to compete with Steward on the identification of the active factor(s) but rather to partially purify the coconut milk factors to an extent that would enable his group to prepare consistently healthy cell cultures for their studies of new shoot formation. Steward replied to this request by sending a letter to Skoog stating that it would be best to leave such matters to the Cornell investigators, and Steward also included a few newspaper clippings that praised his own work but provided no useful details of the purification methods. Skoog, who competed in the 1,500-meter race for Sweden in the 1932 Olympics, was not the type of person who would let such a rebuff go unchallenged. Within minutes of receiving Steward's reply, he contacted Professor Frank Strong, a natural products expert in the Department of Biochemistry at the University of Wisconsin, to initiate a collaboration to compete with Steward's group on the identification of the coconut milk factor(s). The early stages of the project moved rather quickly. Graduate students Jack Mauney (Botany), Rod Clayton (Biochemistry), and Bill Hillman (Botany) did the tough laboratory work of developing methods to purify the active factor(s) and developing a bioassay to test each step of the purification. At first, their assay employed serially cultured tobacco tissue, but later they used callus tissue derived from carrot root pieces. As a starting material for purification of the active factor(s), they shifted to an extract of ground coconut meat rather than the milk because their assays indicated the meat was a richer source of activity. In late 1952, their work was published in Physiologia Plantarum (Mauney et al., 1952). They described the preparation of a fraction that was over 4,000 times as active on a fresh weight basis as the crude coconut meat extract. The active substance(s) in this fraction was heat stable, acid and alkali labile, nonvolatile, water soluble, and organic. It was present in a Neuberg mercuric precipitate and was bound quite strongly by activated charcoal. The factor moved toward the cathode during electrodialysis and appeared to be amphoteric. The paper also reported that ion-exchangers such as Dowex 50 “held the activity so tenaciously that successful elution of the factor was never accomplished” (p. 495). Progress slowed as graduate students completed their theses or shifted emphasis, and this paper was the only publication from Wisconsin on the activity from coconut. In 1951, Carlos Miller, who had recently received a Ph.D. from Ohio State University, began postdoctoral work in Skoog's group. Two of the projects that Miller initiated were to converge, prompting a new line of investigation in the search for growth promoting activity. One project continued the investigation of ways to manipulate organ formation from tobacco stem pieces in culture. As discussed above, results with greenhouse-grown stem segments were quite variable. Miller thought that enriching the culture medium might mitigate this variability, for example, stem segments from less vigorous plants might respond better if a range of metabolites (that might otherwise be limiting) were supplied to the culture medium. To test this, he added yeast (Saccharomyces cerevisiae) extract to a medium that was known to promote root formation because it contained a certain level of the auxin indoleacetic acid. To his surprise, the yeast extract did not enhance root formation but instead caused a massive amount of undifferentiated growth and cell division! In a separate project, Miller was searching for a source of growth-promoting material that was more reliable than coconut extract in the carrot tissue assay. He found that yeast extract was quite active in promoting growth of the carrot tissue when used directly in the culture medium. In the Physiologia Plantarum paper (Mauney et al., 1952), which primarily reported on the coconut project, it was noted that a sample of yeast extract was inactive when used directly in the carrot tissue assay (p. 490), but it was also noted that some activity could be obtained from yeast extract after partial purification (p. 496). However, Miller had fortuitously chosen a particular bottle containing a sample of yeast extract that had relatively high activity when used directly in the culture medium. Miller set out to purify the active material(s) from yeast extract. There was a dwindling supply of yeast extract in the particular bottle that had relatively high activity, so several large containers of different Anheuser-Busch brewer yeasts were ordered in the hope of finding a plentiful source of factor(s). Unfortunately, none of these batches exhibited activity. Yet Miller made progress working with the limited supply from the small bottle of yeast extract that had activity. The chemical techniques available at that time for compound identification from a crude mixture were limited; one common technique was precipitation with metal ions. As previously found in the coconut work, considerable activity was present in a mercuric precipitate, and some activity was adsorbed onto activated charcoal. Every step of the purification had to be evaluated using the bioassay of cell proliferation in culture. The carrot culture assay was giving variable results and so, in May of 1953, Miller started using callus tissue from tobacco stem pieces. When tobacco stem pieces are placed on a simple medium without yeast extract, there is typically an initial burst of growth and cell division from the basal end of the stem piece (this burst of growth formed what was sometimes called “wound callus”). However, in Miller's assays there was no further proliferation (even if the tissue was transferred to fresh medium) unless the activity from yeast extract was present in the medium. Growth of tobacco cells on control medium containing auxin (left) and on the same medium with addition of the silver nitrate precipitable material. This image is from the original work of Miller in 1953. Miller continued to pursue his hunch that a purine derivative might be involved. He sampled bottles of nucleic acids from the laboratory shelf for activity in the bioassay because he thought they might contain additional purine derivatives. RNA did not promote growth, but, almost unbelievably, very good growth occurred when as little as 50 mg/L of herring sperm DNA was added to the culture medium. Moreover, as had been found with yeast extract, the active material could be precipitated from a solution of herring sperm DNA with silver nitrate. That a concentrated source of activity was commercially available was cause for great celebration! To ensure a large supply of active material for further studies, Skoog ordered a keg of herring sperm DNA that had been prepared by the same company using the same procedures by which the small active sample that Miller had first tested had been prepared. However, the new batch of herring sperm DNA did not possess any trace of growth-promoting activity. The excitement abated. Miller had serendipitously chosen samples of both yeast extract and herring sperm DNA with relatively high activity, but no activity was present in any newly purchased batches. For the next several months, work continued with the dwindling supply of active samples of yeast extract and herring sperm DNA. Miller achieved a partial purification of the activity by preparing ethanolic extracts of both sources, followed by silver precipitation, partition into diethyl ether or n-butanol, and paper chromatography. This yielded preparations that were active in the tobacco stem callus test at less than 1 mg/L. Clearly, the active materials were quite potent. The absorption spectra of the ether-soluble factors from both yeast extract and herring sperm DNA were very similar in several solvents and exhibited a similar shift in alkaline solution. In addition, both activities migrated almost identically on paper chromatograms. Thus, each of the two sources seemed to have yielded the same or closely related active substances. Moreover, if the ether-soluble fraction was subjected to chromatography on filter paper using only water as the developing solvent, the activity could be visualized as a dark quenching spot under shortwave UV light at about Rf 0.5. Miller and Skoog submitted a manuscript describing these findings to the Proceedings of the Society for Experimental Biology and Medicine. It was rejected. Part of the reviewers' concerns had to do with the rather poor quantitative nature of the assay for growth-promoting activity, but Miller and Skoog considered many other points raised by the reviewers to lack validity. They knew the project was gaining momentum, and they decided not to address the reviewers' comments or to make any additional effort to publish their progress. The rejection increased their resolve to identify the active compound(s) and caused Skoog to terminate his membership in the Society for Experimental Biology and Medicine (this latter point is from Skoog, 1994). By this time, Miller was becoming increasingly confident that the active substance was an adenine derivative. Some of this confidence came from a chemical test that he had performed earlier for the presence of indoleacetic acid and other indole compounds in the active fraction. At that time, many growth phenomena were regarded as being influenced or even caused by auxin. Therefore, Miller had tested the active material for the presence of indoles by performing paper chromatography of the material with water as the solvent and then spraying with Salkowski reagent (a mixture of sulfuric acid and ferric chloride). No color appeared after several minutes, i.e. the test for indoles was negative. However, to evaluate whether the sample might contain bound or conjugated indoles that could not react with the Salkowski reagent, Miller held the chromatogram over a hot plate. As the paper began to char, a pink spot appeared at the position of the active material. Miller then found that a spray of sulfuric acid alone was even more effective at producing the color, indicating that the color was not due to the reaction of a bound indole compound. His search of the literature revealed that a mixture of sulfuric acid with Cys (known as the Dische reagent) would form a pink product when reacted with deoxyribonucleosides. He found that the Dische reagent was quite effective at reacting with the active material to form a pink compound. As discussed below, this would turn out to provide an important clue for the determination of the structure of kinetin. In addition, when some of the purified active factor was treated with the Dische reagent (or with sulfuric acid alone) and then subjected to chromatography, a new quenching compound (a compound with a different Rf value) was detected. The new compound appeared to be adenine because of its position on the chromatogram as well as other chemical properties, i.e. acid treatment appeared to liberate adenine from the active material; thus, there was growing evidence that the factor might be a substituted adenine! Many further attempts were made to obtain active substances from the newer but inactive batch of herring sperm DNA, such as hydrolysis of the nucleic but these attempts were not However, the new batch of DNA, which had been at for many months, was and very began to activity. Some was in the DNA sample during In fact, this of activity had been noted in the Miller samples of DNA from laboratories the Wisconsin with the to test the samples for activity if any additional samples tested to whether the active samples had in DNA samples were tested for activity in the tobacco stem callus assay. of had activity. that had been at for relatively times had activity, newly purchased samples or bottles of DNA and were these in Miller a simple to a new sample of inactive he some DNA into water in a and it for he the of DNA with and the fraction for a compound with the same quenching and Dische reagent at the same Rf value) and of the active substance from the original A compound with the was that the DNA an amount of a cell activity. A for the DNA had been conducted in early of indicated that an during was for the in the initial the DNA sample had provided for activity to be Miller then prepared a large batch of the active material. The was with n-butanol, and the was by an preparation from which were by The solution was to and then a large of the Dowex 50 The bound the activity very that activity from the coconut extract had been held but not Dowex Therefore, Miller more elution than previously used in the coconut He found that the active substance could be from the Dowex by acid or more by 1 The elution by acid provided a several inactive compounds in the early as the was with the activity came in later Miller the and to the acid without of the active he the to about and Dowex 50 activity bound to the Dowex at this level of He the Dowex with water and then added 1 to the activity. the of Miller a of the with the the dark of the He the as they the the and the with water To if the might the purified Miller a small in and performed paper chromatography as described of the of the material were to of the active substance obtained from the bottle of quenching material moved to the position on a paper the of the chromatogram with the active substance pink when with the Dische reagent, and the material had the absorption The next Miller the to Skoog, to be the after to provide a further Miller set a of tobacco stem callus using several of the He then to Ohio to the with his the Skoog a letter to Ohio that the factor was giving The next of the research was an effort to the structure of the compound. Frank and his group in the Biochemistry which included in an group found that the compound had an weight of by an of They also the and and noted two that the compound was finding was that the compound could not be In of 1 after the first appeared in the Dowex a paper was submitted to the of the Society their progress on the chemical of the active compound and that it be kinetin. This paper appeared in the (Miller et al., A very important was the structure of that kinetin was a substituted adenine and that the and were the the group a of That kinetin was precipitated by silver nitrate indicated the group was not on the of are typically on and the to obtain raised the that the group was on the group of what was the of the The of and a on Miller's Frank and 6-furfurylaminopurine by in As was for work from the initial was protocol a of kinetin. of the chemical and of the compound were to of the factor obtained from DNA, the proposed The luck of bottles of DNA and yeast extract, and the of the particular and of the in the Biochemistry and Botany had to the discovery of the first active of the class of plant In of the paper describing the structure and of kinetin was submitted to the of the and it appeared in the May of the (Miller et al., a more description with appeared Miller et al., The of Miller et Carlos Miller is on the the to are Frank Strong, Folke Skoog, and the laboratory work In new purine related to kinetin were and their on plant cells were tested in now a used was the first of several active cytokinins to be by Skoog and and their quite after the discovery of kinetin. The effect of kinetin on that were also by such as and was other that could be influenced by such as and In the same that the identification of kinetin was Steward's group published a paper that was a compound from coconut milk active in promoting cell division and In their studies, Steward and his group had a large batch of coconuts that had available after a in To prepare this large from a plant was used Steward's group did not that the had been previously used for preparing It was that they with coconut milk only to it as an active but this to the discovery that also have activity. it has been that a can a similar to cytokinins et al., et al., Therefore, active be considered as It be that there is also evidence that can to the of cytokinins by and In the was proposed for substances that promote the partition of a cell into new and therefore growth of plant tissues in (Miller et al., By this time, the ability of to promote cell division had been thus, kinetin and were the first to be In their Miller and Skoog and kinetin as such and as a in (p. it was not (Miller et al., kinetin can be formed by adenine and at a and The can to a group and to the group of However, as noted by Skoog it be out that some kinetin could be formed in plants under certain such as in kinetin was the first active of the class of plant to be whether kinetin was the first of a chemically defined is As noted in Miller Skoog and had earlier in their studies of growth and an of from Miller, when adenine was added to the culture medium of tobacco stem segments (Skoog and promoted new shoot formation without auxin or in the presence of of with of adenine promoted some cell The cell of adenine had been in the Physiologia Plantarum paper on coconut milk (Mauney et al., 1952). In a later the ability of adenine to promote the growth of both and tobacco tissues was was effective in a very range of and the relatively high of adenine than kinetin was effective at than Miller, That the effect was due to adenine and not a was by several different a sample of and the activity in paper chromatography that adenine was a came from the of the cell activity of a of and As the of the group so did the activity, and adenine into this as the As noted in the other be for example, that a relatively high level of adenine to the culture medium some cytokinins to be from Skoog and Miller the effect of the auxin to on the of to University, Miller the of In he reported the partial purification from of a that was a purine derivative from kinetin Miller's work and that of and his group came first of a and Miller, 1965). The used for this class of plant was proposed in to avoid with the referred to in studies of (Skoog et al., 1965). This account of the discovery of kinetin was from published the laboratory of Carlos Miller, and on his of this this is an interesting and one that the to scientific many over many have Carlos Miller about the details of the discovery of kinetin. for this with