This post touches on a lot of things. I wasn’t aware of just how many things interlinked in this story. So I hope you enjoy the story of the discovery of cytokinins, and the connections made herein. This year is the 60th anniversary of the discovery of the first cytokinin, kinetin. And this one is a two-part post. Part 1 covers the story of the discovery. In part two, next week, I’ll cover the biology of cytokinin and what it does in plants.
Plant hormones: cytokinins, part 1.
The discovery of cytokinins made possible regeneration whole plants from plant cells. This may not seem like a big deal. However, it enables a lot of modern plant biotechnology. And cytokinins have shed light on the growth and development of plants. And along with auxin was an early example of plant hormone interactions and crosstalk. I got much of this story from Rick Amasino’s (of UW-Madison) excellent write up of the discovery of Kinetin here. Go read it for fuller details of the story and the people involved.
In the early 1950s, Rosalind Franklin, Maurice Wilkins, James Watson, and Francis Crick were working in England. They independently published evidence that the structure of DNA is the now iconic right-handed double helix. Many previous scientists, including Barbara McClintock, had shown that DNA, and not proteins, were the molecules of heredity. And people knew that discrete ‘factors’ on that DNA conferred traits on the next generation. Even with the structure of DNA, it was still not clear how DNA, the template, translated into a physical organism.
At the same time of the proposed double-helix structure of DNA, another big event in the history of biology took place. In Baltimore, Maryland, Henrietta Lacks, was suffering from cervical cancer. She had a biopsy done. And then, without her consent her tumor cells were taken to try and grow them in a culture medium. This was before the era of informed consent in research. A scientist named George Gey grew her tumor cells in a culture medium. And for whatever reason, her cancer cells grew where previous attempts at culturing human cells had failed. If you want an in depth telling of this story, I recommend Rebecca Skloot’s book “The Immortal Life of Henrietta Lacks”. Getting cells from a multicellular and complex eukaryote (an animal or plant) was a big deal. Some bacteria and fungi (E. Coli and baker’s yeast, for instance) were easily cultured at the time. The ability to culture human cells would permit studying lots of things scientists could not do on whole human beings. Part of developing the polio vaccine depended on HeLa cells, for instance. In fact, The HeLa cells from Ms. Lacks underpin a lot of the life science revolution we are experiencing today. The 1950’s was when biology started to get big.
Our story also takes place in the 1950’s at the University of Wisconsin, Madison Botany department. Folke Skoog had a lab that was trying to grow plant cells in culture to to study how shoot tissues formed (shoots being the parts of plants we see most often). I’ve already stated above some reasons why culturing cells was important, but Skoog was curious about just how a plant formed a shoot. In fact culturing plant cells had had more successful in plants than animals. Plant cells had started to be grown in culture from early in the 20th century. Plant exudates could induce a proliferation of undifferentiated cells. Plant Cell cultures could be grown up to a point from dissected stems of some plants. As a known plant growth regulator, culture experiments had started to include auxin as well. Auxin was known to induce root formation in cell cultures under many conditions.
The Path to Investigating Cytokinin
In Skoog’s lab, they started using coconut milk that other labs had observed to be a source of a factor that could induce shoot formation in cell cultures. Growing plants in culture was not easy. Contamination, plant growth conditions, and harsh sterilization protocols all caused inconsistencies in cell culture. Skoog’s group sourced coconuts from the local grocery store. It’s a trick that some plant biologists use to this day (it’s a good source of bulk plant tissue/fruits for many experiments). They found that the coconut milk only occasionally induced shoot formation. Skoog sought advice from a an expert in the use of coconut milk in cell culture. Specifically he sought help in the purification of the milk to make it more consistent for his studies in shoot formation. At this time, Skoog did not care about identifying the factor in coconut milk that induced shooting. He just wanted a consistent way of making shoots.
Professor FC Steward at Cornell responded a letter in reply stating that studies of the coconut milk should be left to the Cornell experts. This rebuff sparked Skoog to compete with Steward to figure out just what the factor in coconut milk was that induced cell proliferation. Skoog contacted his colleague Frank Strong in the Department of Biochemistry to collaborate in the effort. Members of both labs developed purification methods and important bio-assays to test extracts at each step of the protocol. One key observation was that the meat of the coconut was an even better source of the factor than the milk. Another was some characterization of what the factor was at the chemical level. It was heat stable, non-volatile, water soluble, and definitely carbon-based amongst other characteristics.
The Discovery process.
This is where Carlos Miller enters the picture as a postdoc in Skoog’s lab. Miller set to work on improving the culture medium for growing plant tissue. He also worked to identify the growth factor that was causing cell proliferation and shoot formation. One thing he tried adding to the growth medium was a bottle of yeast extract. He thought it might enhance the auxin activity in the medium– yeast extract had compounds similar to auxin. He happened to choose an old bottle of yeast extract and it turned out to have some of the growth promoting activity. Miller had thought it would be a source of auxin activity, inducing rooting). New batches of yeast extract ordered didn’t have the same activity. Working with that one special bottle, he narrowed what he thought the factor might be. Classes of chemicals known as purines and pyrimidines: 4 of which are main constituents of DNA: adenine, guanine, cytosine, and thymine. Based on some previous work from the Skoog lab showing that adenine had weak shoot inducing activity, Miller tested a bottle of herring sperm DNA. My sense is that this was a common thing to have around labs. He found that similar to the coconut extract and his special bottle of yeast extract, it also had the shoot inducing activity. Skoog, excited by this finding, ordered a keg of herring sperm DNA for the lab (yes, a keg! Of herring sperm DNA!). However, like new bottles of yeast extract, the new herring sperm DNA showed no shoot inducing activity.
Miller was able to partially purify the factor from his original bottle of herring sperm DNA, achieving activity at a concentration of 1 milligram/1 liter of water
Miller was able to partially purify the factor from his original bottle of herring sperm DNA, achieving activity at a concentration of 1 milligram/1 liter of water. To be clear, that means that this factor was quite potent, like the ethylene and auxin in previous posts. Miller also discovered that preparations of herring sperm DNA left at room temperature for months developed shooting activity. A similar thing result was found with yeast extract. Miller tried autoclaving new herring sperm DNA dissolved in water. And this worked! The factor was present after the heating step. After more work, Miller purified crystals of what he was confident was the factor they were looking for. At this point, the Strong lab helped characterize and determine the structure of the factor. It did appear to be an adenine derivative, an “N6 substituted” derivative, in fact. They even predicted what the N6 substitution structure might be.
With a hypothesized structure in hand, the Strong lab made the compound from scratch. The Skoog lab put it through the bioassays as well as analysis steps to see if it matched what they’d observed in their initial isolation protocols. Everything matched and the labs named the chemical Kinetin. This was the first cytokinin every identified. Others followed from the Skoog lab as well as Steward at Cornell. When Miller was running his own lab at Indiana University, he found the first naturally occurring cytokinin from maize (corn) and named it Zeatin.
The Wake of Curiosity
I want to stress here that cytokinin is an adenine derivative. In DNA, adenine pairs with thymine in the complimentary strand of the DNA double helix. Kinetin came out of what are degradation of sources of DNA (yeast extract, herring sperm DNA). All this was before we knew how DNA worked or even had a physical characterization of a gene. Kinetin was one part of figuring out some of these mechanisms in plants. Cells (all cells) main energy source is another adenine-like compound called ATP (Adenosine tri-phosphate). Cytokinins are part of a chemical family that make up DNA and a cell’s energy source, showing how evolution uses what it has on hand to generate new features.
The discovery of a small molecule that could induce cell division was a big deal. The American Cancer Society funded part of the Kinetin research. Though plants have different inputs into cell division than animals, the core machinery of cell division is much the same. Studying plants has lead to new chemotherapies like taxol from the Pacific Yew tree. And there are mechanisms in plants that do translate into other systems, and vice-versa. In science, the fact that we can learn from other systems can be easy to forget, especially when we’re awash in information of a single discipline.
Fodder for the scientific engine to keep working and shed innovations in the wake of curiosity
An academic slight spurred Skoog to go after the factor that turned out to be cytokinins. This may be where the competitive spirit in academia works best. A person to compete against induces motivation to succeed and spurs efforts to come up with the solution to a biological problem. This is also a story where Skoog initiated a collaboration to help him. Again, the competitive spirit spurring action. The University of Wisconsin, Madison is a giant in research, including the kinetin story. The study of plant cells in culture and shoot formation sounds esoteric on the surface. As does trying to grow human cells in culture. Even the structure of DNA was esoteric in the 1950s. It affected no one’s day-to-day lives. The public may have sort of appreciated the advance, but how, 60 years later, these discoveries have an impact on all our lives. These results yielded new questions. Fodder for the scientific engine to keep working and shed innovations in the wake of curiosity. Think about that next time you read a contemporary story about basic science research. It can take decades for research to yield results.
The story of Miller discovering Kinetin was not straight forward
The current Governor of Wisconsin wants to make professors teach more and only do research that translates into immediate monetary value for the state. And to get away from curiosity driven science. His proposal reads as “We want to train people here who go other places to make great discoveries”. Professors are not lazy. And having them do more may mean missing out on that next great collaboration or discovery. Research enhances teaching and vice versa. Modern research universities do downplay teaching too much. And perhaps the functions of teaching and research ought to be separate. But basic science does enrich a university, a culture where ideas percolate and grow. The story of Miller discovering Kinetin was not straight forward. Discovering something new never is. Scientists’ primary job is to figure out what the good questions are to ask to learn new things about the world. And that is not a straight forward, linear process because we don’t know a priori what will yield great results. The kinetin story illustrates just how fortuitous science often is. There is nothing wrong with curiosity based research. More knowledge is better than less.