Cacao plants' defense against toxic cadmium unveiled

Researchers from the University Grenoble Alpes (UGA), France, together with the ESRF, the European Synchrotron located in Grenoble, France, used ESRF's bright X-rays to unveil how cacao trees protect themselves from toxic metal cadmium. This knowledge is relevant as new EU regulations restrict cadmium concentration in chocolate. Their results are published in Environmental and Experimental Botany.

Cadmium often accumulates in food, but it is a highly toxic metal, which can be harmful in humans if chronically exposed to it, according to the Food and Agricultural Organization.

The EU has imposed limits to the cadmium maximal concentration in foodstuffs such as rice, wheat, potatoes and more recently chocolate.

Whilst there have been studies on how cadmium is transferred from soil to the edible part of stable crops, there is hardly any research on cadmium in cacao cultivars.

"Understanding how cadmium builds up in cacao trees is paramount to subsequently find strategies to mitigate the accumulation of this metal in the final product," explains Geraldine Sarret, researcher at the University Grenoble Alpes (UGA) and co-corresponding author of the publication.

The UGA scientists travelled to the International Cocoa Genebank in Trinidad and Tobago, which hosts a field cacao collection with approximately 2400 cacao genotypes, to collect their samples in collaboration with the Cocoa Research Centre.

Then they came to the ESRF, the European Synchrotron, located in Grenoble, France, to investigate a particular cacao cultivar/variety that absorbs more cadmium than others do. Using synchrotron techniques -nano X-ray fluorescence on ESRF beamline ID16B and X-ray absorption on ID21-, they delved into the micro and nanoscale composition of the different parts of the plant.

"Thanks to the ESRF, we could map of the presence of cadmium and other elements in an unprecedented resolution, so we could see the big picture but also going to the smallest detail," says Hester Blommaert, PhD student at UGA and co-corresponding author of the publication.

"The concentration of cadmium in the different parts of the plant is very low, so much so that we couldn't have done this research before EBS," says Hiram Castillo-Michel, researcher at the ID21 beamline at the ESRF.

"In the near future, we will see an increasing number of studies on similar food safety topics at ID21, where our recently installed new microscope will offer enhanced resolution and detection limits," he adds.

The results yield a surprise: "We found that part of the cadmium is stored in calcium oxalate crystals in roots and branches of the cacao plant, which was unexpected," explains Blommaert.

In particular, the crystals were most abundant in the branches.

Interestingly, whilst crystals were present in the leaves, they did not seem to help in detoxifying cadmium in this part of the plant.

"We believe that the calcium oxalate crystals are a mechanism of detoxification of the plant against the metal," she adds.

In addition, they also discovered that cadmium combines with sulphur in certain cells in the roots.

This mechanism is well known in roots of cereals, where cadmium is retained in the vacuoles and bound to thiol-containing molecules.

In the case of cacao, this mechanism is less pronounced, and more cadmium is transferred to aerial parts.

Overall, the strategy developed by cacao plants to manage cadmium is different from cereals, in terms of root to shoot transfer, storage compartments and storage forms.

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Among ancient Mayas, cacao was not a food exclusive to the elite

It was the money that grew on trees.

Said to be a gift from the gods, cacao for the ancient Maya was considered sacred, used not only as currency, but in special ceremonies and religious rituals. It's the progenitor plant of chocolate, and notions of luxury are embedded in its lore.

The prevailing belief: Cacao was more available to, even controlled by, the society's very upper echelons, royalty. Past efforts to identify cacao in ceramics focused on highly decorative vessels associated with elite ceremonial contexts -- think ornate drinking vases -- leading to assumptions about how cacao was distributed and who could access it.

What about the farmers who grew cacao and the communities of people who lived amongst these orchards? What of the general populace?

A new study by UC Santa Barbara researchers Anabel Ford and Mattanjah de Vries asks these questions -- and answers them -- by examining cacao residues from ancient ceramics. Their results, published in the Proceedings of the National Academy of Sciences, demonstrate that cacao was, in fact, accessible to the general populace and was used in celebrations at all levels of society.

"It had long been assumed that cacao for the Maya was an elite exclusive," said Ford, an anthropologist and director of the MesoAmerican Research Center at UC Santa Barbara, who for 40 years has been conducting research on the ancient Maya city of El Pilar. "We now know this is not the case. The imbibing of cacao was a luxury accessible to all. The importance is that it was a requirement of the rituals associated with it."

To test the exclusivity of cacao use, the work examines 54 archaeological ceramic sherds. Originating from El Pilar -- located between Belize and Guatemala -- the sherds can be traced to Late Classic period civic and residential contexts, representing a cross section of ancient Maya inhabitants. The study includes a chemical analysis of these sherds -- specifically of the biomarkers for cacao: caffeine, theobromine and theophylline.

"The discovery of chemical signatures of cacao made the investigation possible, but the main active ingredient, theobromine, it turns out is not sufficiently discrete to be certain of the cacao attribution," said Ford. "Mattanjah (de Vries) and his students, in their chemical research, encountered the possibility of detecting theophylline, a specific component of cacao that could not be confused with anything else. His work was not archaeological, but he saw the potential for an interdisciplinary project."

A distinguished professor and department chair of chemistry and biochemistry at UC Santa Barbara, de Vries has long been studying how DNA bases -- the building blocks of life -- and similar molecules respond to UV light and, he said, whether UV light "could have played a role on an early Earth, in the way nature selected those building blocks from a primordial soup of many such compounds.

"At some point I realized that some of the compounds we had been studying in this origin of life chemistry project occur in cacao, and thus can serve as biomarkers for cacao," de Vries said. "Since we had already investigated the spectroscopy of these compounds in great detail, this presented an opportunity to apply that expertise to detection of these biomarkers for archaeology.

"We can find a needle in a haystack, provided we know what the needle looks like; in this case the target molecule was a certain biomarker for cacao," he added. "That ability is what made this analysis possible."

In their selection of ceramics to test, Ford and de Vries prioritized the vases from which cacao was likely drunk. They also tested bowls, jars and plates. All vessel types had evidence of cacao.

"This was a surprise at first," Ford said, "but giving thought to the presence and understanding of their uses, bowls would be good for mixing, jars would be right for warming the drink (a traditional cacao preparation) and plates appropriate for serving food with sauces that can contain cacao (such as mole poblano).

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Locations of ancient Maya sacred groves of cacao trees discovered

For as much as modern society worships chocolate, cacao -- the plant chocolate comes from -- was believed to be even more divine to ancient Mayas. The Maya considered cacao beans to be a gift from the gods and even used them as currency because of their value.

As such, cacao bean production was carefully controlled by the Maya leaders of northern Yucatan, with cacao trees only grown in sacred groves. But no modern researcher has ever been able to pinpoint where these ancient sacred groves were located -- until now.

Researchers at Brigham Young University, including professor emeritus Richard Terry and graduate students Bryce Brown and Christopher Balzotti, worked closely with archaeologists from the U.S. and Mexico to identify locations the Maya used to provide the perfect blend of humidity, calm and shade required by cacao trees. While the drier climate of the Yucatan peninsula is inhospitable to cacao growth, the team realized the vast array of sinkholes common to the peninsula have microclimates with just the right conditions.

As detailed in a study newly published in the Journal of Archaeological Science Reports, the team conducted soil analyses on 11 of those sinkholes and found that the soil of nine of them contained evidence of theobromine and caffeine -- combined biomarkers unique to cacao. Archaeologists also found evidence of ancient ceremonial rituals -- such as staircase ramps for processions, stone carvings, altars and offerings like jade and ceramics (including tiny ceramic cacao pods) -- in several sinkholes.

"We looked for theobromine for several years and found cacao in some places we didn't expect," said Terry, who recently retired from BYU. "We were also amazed to see the ceremonial artifacts. My students rappelled into one of these sinkholes and said, 'Wow! There is a structure in here!' It was a staircase that filled one-third of the sinkhole with stone."

To extract and analyze the sinkhole soil for cacao biomarkers -- specifically theobromine and caffeine -- the team developed a new method of soil extraction. This involved drying the soil samples and passing them through a sieve, covering them with hot water, having them centrifuged and passed through extraction disks, and analyzing the extracts by mass spectrometry. To increase the sensitivity of their testing, the research team compared the results of the soil samples to seven control samples with no history of exposure to the biomarkers.

The findings of the BYU study indicate that cacao groves played an important role in ancient rituals and trade routes of the ancient Maya, impacting the entirety of the Mesoamerican economy. A 70-mile Maya "highway" in the area that was the main artery for trade passes near hundreds of sinkholes, so it is likely that the leaders who commissioned the highway development also controlled cacao production. The evidence of cacao cultivation alongside archaeological findings also supports the idea that cacao was important in the ideological move from a maize god to a sun god.

In one sinkhole near Coba, Mexico, a village 45 minutes from modern day Tulum, the research team found the arm and bracelet of a figurine attached to an incense jar and several ceramic modeled cacao pods. They also found remnant cacao trees growing there, making it quite possible that this sinkhole, named "Dzadz Ion," was the location of a sacred cacao grove during the Late Postclassic period (About A.D. 1000 to 1400).

"Now we have these links between religious structures and the religious crops grown in these sinkholes," Terry said. "Knowing that the cacao beans were used as currency, it means the sinkholes were a place where the money could be grown and controlled. This new understanding creates a rich historical narrative of a highly charged Maya landscape with economic, political and spiritual value."

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