A New York University-led research team has used single-cell RNA sequencing technology to compare individual cells across corn, sorghum, and millet, and reveal evolutionary differences among these important cereal crops. The study results, reported in Nature, bring scientists closer to pinpointing which genes control key agricultural traits such as drought tolerance, and could help develop approaches to adapting crops to drier environments brought about by climate change.

Senior study author Kenneth Birnbaum, PhD, a professor in NYU’s Department of Biology and Center for Genomics and Systems Biology, and colleagues reported on their findings in a paper titled “A pan-grass transcriptome reveals patterns of cellular divergence in crops,” in which they concluded, “… we illustrate how single-cell techniques can rapidly generate a pan-transcriptome to yield insights into plant cell-type evolution and provide new methods to explore the connection between genetic modules and cellular traits in important crop species.”

Maize/corn (Zea mays) and sorghum (Sorghum bicolor) are grasses that provide food for humans and animals around the world, and which are close relatives that evolved into two different species millions of years ago, the authors explained. “Z. mays (maize) is a staple crop and S. bicolor (sorghum) is an important dryland crop and biofuel candidate that is closely related to maize, sharing a common ancestor about 12 million years ago.”

Despite their shared ancestry, the two crops exhibit substantial differences in key traits such as drought-resistance and cold tolerance. For example, sorghum is far more tolerant to drought than is corn, and the plants release unique gooey substances from their roots to shape how they interact with their surrounding soil. These differences may be traced back to corn undergoing a whole genome duplication (WGD) after its split from sorghum.

First author Bruno Guillotin, a postdoctoral associate in NYU’s Department of Biology, pointed out, “While these three crops are similar, how they differ from each other is important because they have traits that we may want to transfer from one to the other, such as drought tolerance.”

“The importance of the two crops, their evolutionary proximity, and their functional differences present an opportunity for comparative  analysis of cellular evolution in plants,” the team continued. “Comparing patterns of gene expression at the cell level in maize, sorghum and outgroup S. viridis (Setaria) provides an opportunity to examine cellular evolution and the role of gene duplications.” The third grass, millet (Setaria viridis), is a more distant relative of corn and sorghum.

For their study the researchers conducted single-cell mRNA profiling of the roots of corn, sorghum, and millet, dissecting the roots to look at the cells individually and observing precisely where genes are expressed in a particular cell. They then compared the same specialized cells across the three crops.

“Roots are the first line of defense against drought and heat,” Birbaum said. “You can think of the root as a machine with many working parts—in this case, cell types—so knowing how the machine works to collect water and to deal with drought and heat is really important. Comparing the different species helps us tease apart which genes lead to key agricultural traits.”

In examining how cells have evolved and diverged in the different species, the researchers identified several trends that point to rearrangement of existing elements of cells over time. First, they observed that cells often trade gene expression modules, or groups of 10 or 50 genes with coordinated functions, between cell types over evolution.

“This gene module swapping has been shown in animal systems, but the data we generated is the first time it’s been illustrated on a large level in plants,” noted Birnbaum. Swapping of modules was demonstrated in a discovery about root slime—the gooey substance filled with nutrients that roots emit into the soil. Slime is useful for lubricating the soil so roots can pass through and can attract beneficial bacteria that protect the plant or provide hard-to-get nutrients.

The researchers found that the genes that help produce root slime were located in different parts of the corn, sorghum, and millet root. In sorghum, the slime genes were found in the root’s outer tissue, while in corn these were swapped into a new cell type in the root cap, an evolutionary change that may enable corn to attract bacteria that helps the plant to gain nitrogen. “.., homologous cell types appear to diverge in part by swapping gene expression modules, such as the mucilage genes found to be expressed in the maize columella,” they stated.

They also identified other gene regulators that were switched around in different cell types depending on the crop, providing researchers with prime candidates for testing genes that convey specific traits. “… we identified more than 50 swapped modules across cell types,” the scientists wrote. “The swapped modules are prime candidates for genes that could mediate differences in cellular traits between maize and related species.”

The researchers used a technology called Molecular Cartography, a spatial transcriptomics method, in collaboration with Resolve Biosciences to map the cellular location of about a hundred genes at once. This allowed them to validate the cell-by-cell cross-species map of gene expression. [Bruno Guillotin/Ramin Rahni]
In addition, the researchers found that the whole genome duplication in corn after its split from sorghum 12 million years ago affected specific types of cells, allowing corn cells to rapidly specialize. They also observed that certain kinds of cells acted as the donors of new genes while others seemed to collect new gene duplicates, which may suggest that gene duplication sped up the evolution of certain cells. “The comparative cellular analysis shows that the transcriptomes of some cell types diverged more rapidly than those of others—driven, in part, by recruitment of gene modules from other cell types” the investigators stated “The data also show that a recent whole-genome duplication provides a rich source of new, highly localized gene expression domains that favor fast-evolving cell types.”

Recent advances in single-cell sequencing techniques made this research possible and open up new methods to explore the connection between genes and cellular traits in crops.“A decade ago, we were only able to analyze a dozen or a few dozen cells with the early single-cell sequencing techniques. Now we can profile tens of thousands of cells in a pretty routine experiment,” said Birnbaum.

The authors concluded, “Together, the cell-by-cell comparative analysis shows how fine-scale cellular profiling can extract conserved modules from a pan transcriptome and provide insight on the evolution of cells that mediate key functions in crops.”

Future studies will compare how single cells of these three crops respond to stress, such as drought. “It’s that response that may be the key to finding that set of genes that are really important for drought tolerance,” said Birnbaum.

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