Brassica Oleracea and an 'Abominable Mystery' By Melody Kroll & Charles E. Reineke. Photos by Nicholas Benner.
For most of the 400-million-year history of life on land, flowering plants were absent from the Earth’s astounding array of flora. That all changed about 120 million years ago. Suddenly, according to the fossil record, a bewildering diversity of flowering plants — “angiosperms” in the parlance of plant-scientists — burst onto the scene. These quickly came to dominate almost every terrestrial environment, seashore to mountaintop, pole to pole.
An exasperated Charles Darwin, whose famous theory postulated that evolutionary change took place almost imperceptibly over vast stretches of time, admitted he couldn’t explain the explosion of flower power. Darwin dubbed the angiosperm outburst an “abominable mystery.”
These days, hundreds of thousands of diverse angiosperms make up 90 percent of all plant life on Earth. Coming to terms with the genetic phenomena that created, and continues to drive, this diversity remains one of the biggest challenges in biology. The reason is obvious: Unraveling the abominable mystery, plant scientists say, would involve uncovering the genomic underpinnings at the very core of plant life. Such knowledge, in turn, might also lead scientists to refine the way plants are bred, thus accelerating the age-old processes by which farmers gradually transformed edible wild plants into the food crops we eat today.
J. Chris Pires, an evolutionary biologist and associate professor at MU, has spent the past decade at MU working with the plant he believes might help solve this “abominable mystery.” The plant he thinks holds the key may come as a bit of a surprise. It’s not one of the giants of plant genetics — Arabidopsis thaliana, maize, or rice. Neither is it orchids, one of the most successful and species-rich family of flowering plants on Earth. Nor is it the shrub Amborella, an endemic of New Caledonia at or near the base of the flowering plant lineage.
The object of Piers' interest is instead the much loved, sometimes loathed, family of cruciferous stalwarts consumed worldwide. Pires knows them members of the genus Brassica. For the rest of us, they go by the appellations of their many forms: among them broccoli, cauliflower, kale, cabbage, Brussels sprouts and kohlrabi.
“When people ask me what I do, I tell them I study the ‘dogs’ of the plant world,” says Pires. “Everyone knows that – whether it’s a Chihuahua or a Great Dane – dogs are one species. Well, these diverse crops are also all one species.”
Most introductory biology textbooks use the different vegetative forms of B. oleracea to illustrate how greatly varieties in one species can vary in appearance. In his The Variation of Animals and Plants Under Domestication, Darwin also points to this plant as an example of artificial selection, noting it as a great example of “Whatever part man values most, that part will be found to present the greatest amount of difference.”
In B. oleracea, those parts include, for example, the terminal buds in cabbage, the lateral buds for Brussels sprouts, and the stem and flowers for broccoli. This selection for different parts of the plant, says Pires, is unique to the brassicas. “Most crops have been domesticated for one part — for tomato you've got the fruit and for corn you've got the ear. In brassica, multiple parts of the plant have been the focus of domestication.”
Pires believes that what drives this morphological variation in B. oleracea lies in the plant’s evolutionary history, a past that includes not one but two genome-doubling events.
Genome duplication, or polyploidy, is the ability of some organisms to accommodate more than two paired sets of chromosomes. Scientists had known that some organisms had multiple sets of chromosomes, but it was Susumu Ohno, a geneticist who worked with the City of Hope Hospital in Los Angeles in the 1970s, who first proposed that polyploidy might be a driver of species variation. The reason why, he said, has to do with an apparent inefficiency in the polyploidy process; polyploidy provided extra copies of genes — “spare parts,” if you will — for evolution to play around with. Ohno put it this way: “Natural selection merely modified while redundancy created.” Or, as Pires asks, “What happens when two genomes come together? Does 1+1=2?”
At its core, the question raises issues related to processes that geneticists call neofunctionalization and subfunctionalization — the idea that duplicated genes are kept around and co-opted for new or more specialized functions over time. Current thinking is that organisms preferentially keep copies of certain classes of duplicate genes, for example, those that are involved in important developmental or signaling functions. In a study published last year in the journal Genome Biology, Pires and colleagues helped to establish that Brassica oleracea has preferentially kept around these genes.
The finding made waves because of what it could portend for the future. If these duplicate genes are indeed those selected during brassica’s domestication, it’s likely a short step to developing a genetic test that could identify which gene is responsible for creating broccoli or cauliflower or cabbage.
“The question is, are those extra copies of genes one reason why we have this spectacular variation?” Pires asks. “If that’s true, then we can test if those genes are more prone to domestication as opposed to, say, some random single copy genes.”
Perhaps even more exciting are the finding’s implications for Darwin’s abominable mystery. Identifying these genes would provide strong evidence supporting the hypothesis that the variation we see in Brassica oleracea is the product of the polyploid event. Evidence, in short, pointing directly to polyploidy as a trigger for diversification.
Evolution of a Plant Person
Pires’ own evolution from nature-loving youth to plant scientist began in the small town of Gridley, Calif., where, as a high school student, he recalls flower-hunting field trips with an inspiring high school teacher high in the Sierra-Nevada mountains.
“Instead of just dissecting pigs and frogs, we spent a lot of time studying plants,” recalls Pires. “So, yeah, I blame Mr. Brent McGhie for my becoming interested in botany.”
He also remembers the influence of Ridley Scott’s 1982 dystopian science-fiction thriller, the Blade Runner. “I thought we were going to be making people in the future, Pires says. “So when I went to do my undergraduate work at the University of California-Berkeley in 1985, I thought I had better major in both genetics and philosophy.” He later switched majors and graduated with a degree in biology with an emphasis in plant evolution.
As an undergraduate, Pires found time to develop education programs and give tours at the university’s botanical garden. It was here that he encountered his first polyploid plants — a group known as Brodiaea. “Like many plants in California, these plants diversify on different substrates — serpentine soil, vernal pools, wetlands—and it was thought that polyploidy is linked to that [ability].” After a four year-hiatus as an environmental consultant and wetland biologist, Pires earned a doctoral degree working with this group of plants at the University of Wisconsin-Madison.
After a brief post-doctoral stint at the Kew Royal Botanic Gardens in London, Pires returned to the University of Wisconsin for a second postdoctoral appointment. Back at UW, Pires not only began his work with brassica, but also learned what it takes to play in the academic big leagues. “Certainly, that postdoc was a big help for me. I learned what it was like to be a professor as well as how to manage a multi-million dollar budget.” An added bonus was meeting Kate Anderson, now his wife.
In 2005, Pires and Anderson moved to Columbia. He joined the biological sciences faculty at MU and became one of the inaugural investigators in the Christopher S. Bond Life Sciences Center. Anderson currently heads MU’s Zalk Veterinary Medical Library.
So how did cabbage, broccoli and all the cruciferous plants come make their way to our dinner tables? The unsatisfying answer is that no one knows for sure. Cabbage and its cousins are not like corn, wheat, or other cereal crops with well-documented archaeological records. Instead, scholars and scientists have had to rely primarily on literary references to recover its domestication story.
The oldest references to cabbage date from ancient Greece and Rome. Scholars have pointed to these and other ancient allusions to argue that the plant was first domesticated somewhere in the ancient Greek-speaking areas of the central Mediterranean. It then, so the story goes, wended its way westward, most likely through Roman expansion. Adding weight to this hypothesis is the presence of several sister species of wild Brassica on the island of Sicily. The assumption is that one, or all of these, is likely the closest relative to B. oleracea.
“This is a classic, center-of-diversity origin idea -- that things originate from where they are most diverse,” says Pires. “The assumption is that high diversity equals high antiquity.”
Hang on a minute, say the English. The wild cabbage that grows along the cold, windy coasts of England — and the European coasts across the channel — must certainly be the progenitor of modern species, they say.
“Yes, the second hypothesis is that they originated in the UK,” acknowledges Pires. “The reason is because there are all these wild species that look a bit like the domesticated ones. Kind of like wild tomatoes or wild corn landraces.”
The plausibility of England’s claim seems strong when walking along the four-mile path atop the iconic White Cliffs of Dover in England’s southeast. There, besides breathtaking views across the English Channel, one is likely to stumble upon plenty of cabbage amongst the flowering knapweed and horseshoe vetch. These brassicas look undeniably wild: “kind of like a half-way cabbage or a half-way kale,” says Pires. This visual distance bolsters the claims of the true believers. “Since we ourselves bear only a slight resemblance to our most primitive ancestors,” they argue, “doesn’t it make sense that wild cabbage should bear only a slight resemblance to its cultivated forms?”
This UK-origin story has been bolstered by some previous genetic studies, though, as one might imagine, advocates for the Mediterranean origins have been less than convinced.
Enter Pires and an international team of plant scientists and archaeologists. In their quest to identify the domestication genes of B. oleracea — those genes that make a broccoli a broccoli and a cabbage a cabbage — they first had to figure out the relationships among Brassica oleracea and its wild relatives; to, in a sense, build its family tree. In so doing, they inadvertently managed to resolve the origins riddle.
The research consortium, including Guy Barker and Graham Teakle at the University of Warwick, obtained seed from 135 wild and cultivated cabbage and closely related species from the Warwick Genetic Resources Unit in the UK. They selected seed from plant populations throughout the greater European region. Their goal was to capture the widest potential gene pool from which wild cabbage and its cultivated forms may have been selected. Seed in hand, they grew all these plants in a greenhouse.
Once grown, RNA was extracted from the leaves and roots and then sequenced to obtain each plant’s “transcriptome,” the subset of genes in a genome that contribute most directly to form and function. They lined up all the transcriptomes and looked for differences. The variations showed the relationships among the different plants. Those with fewer variations were more closely related, and vice versa. Pires and his team then used the data to build a phylogenetic family tree that precisely displayed how all the plants were related.
It’s not the first family tree for cabbage and its wild relatives, but it is the most robust, Pires says. “We not only have the best sampling, but we also looked at tons of different nuclear genes. Previous studies only looked at a couple chloroplast genes.”
Chloroplast genes, Pires explains, are few in number and are inherited differently than nuclear genes. By looking at the genetic information in the nucleus and, specifically, the transcriptome—the RNA that leaves the nucleus—Pires and his colleagues were able to identify genetic changes as the plant moved around and diversified. They were also able to identify its most likely origin.
The result? Neither the UK nor Sicily can claim the cabbage. “Surprise!” Pires says with a laugh. “Just like cattle, wheat, and many other domesticated species, cabbage has a Mesopotamian origin.”
The research revealed that B. oleracea, in all its wild and cultivated forms, descended from a plant in the eastern Mediterranean, specifically a species named Brassica cretica that grows along the coast of Turkey. The modern forms share more genes (i.e., fewer variants) with B. cretica than with any other plant examined.
'We need more of these interdisciplinary interactions if we want to unravel these evolutionary histories and how they’re tied to human history.'
The findings also overturn both previous cabbage-origin stories. The plants in Sicily reputed to have begotten B. oleracea in fact shared fewer genes than expected with it. Though the “wild cabbage” in Britain does share genetic information with B. oleracea, it is clearly of fairly recent origin, says Pires. “We can not rule out that these are just domesticated cabbages that got out of people’s fields and gardens and then became feral and wild looking.”
Dorian Q. Fuller of the Institute of Archaeology at the University College London participated in the research. “I was asked to answer whether the archaeological and historical evidence for past use of cabbage fit with [Pires’] phylogenetic findings,” he explains. “In other words, can we marry what we know about human history through archaeology with that phylogeny, in terms of dispersal processes?”
In fact, he says, it all ends up making quite a bit of sense. “Many crop domesticates come from western Asia, so it fits a broader pattern that there is a lot of domestication and origination in western Asia and a kind of recurrent pattern of dispersal into other parts of Europe,” he says.
While locating cabbage origins in the eastern Mediterranean was a triumph of plant sleuthing and certainly edifying in its own right, it’s not an end-all for Pires.
“We certainly didn’t set out to resolve this riddle,” says Pires. “We only discovered it because we have these two Brassica genomes available, and we were able to leverage our transcriptome data to really make a good phylogeny population genetic study.”
For Pires, disentangling this recent history is a key step toward figuring out the genes that make a broccoli a broccoli or a kale a kale. If those genes turn out to be duplicated genes, then Pires may finally have the proof he needs to solve the riddle of floral diversification.
It won’t be easy, adds Pires.
“Now comes the hard part: we actually have to make crosses and do the genetics of it and do all the development work,” he says. “It’s a totally different ball game.”