Proper plant growth in tissue culture experiments typically depends on one crucial step: the plant’s ability to form a clump of pluripotent cells that can then regenerate root, stem, and other organs. This cluster of cells, known as a callus, is essential not just to tissue culture experiments but also to plants’ ability to heal from injury.
While researchers knew that the formation of a callus was driven by the two plant hormones auxin and cytokinin, its spatial organization was unclear. In a recent study, researchers used RNA sequencing to identify three distinct layers of cells within the callus. They found that pluripotency originates in the middle layer, driven by the expression of genes in other stem cells. The results “show that not all cells within the callus are the same,” says Kenneth Birnbaum, a plant developmental biologist at New York University who was not involved with the work. “Most importantly, this paper reveals how hormones regulate cellular functions in the callus.”
Textbook definitions had once described the callus as a group of stem cells with no organization or resemblance to other plant tissues. That definition shifted in 2010, when researchers recognized the callus resembled tissues in the root tip. But little else was known about its cellular organization. Plant biologist Lin Xu of the Chinese Academy of Sciences in Shanghai and his colleagues set out to understand how these root-like cells could regenerate various other kinds of plant tissues.
Xu’s team performed single-cell RNA sequencing on Arabidopsis calluses cultured for 6 days in tissue culture media. They found six clusters of cells with distinctly different patterns of expression were present in layers. “There have been a number of efforts to take apart this mystery [of callus organization] over the years,” says Philip Benfey, a plant developmental biologist at Duke University who was not involved with the study. “This paper take us to the next step in our understanding by using single cell RNA sequencing to identify these clusters corresponding to different cell layers.”
The outermost cell layer resembled the root cap, and the innermost layer was comprised of cells resembling those that form plants’ vasculature. Cells in the middle layer expressed genes such as WOX5 and WOX7, which are commonly up-regulated in stem cells. This led the team to suspect this middle layer was the pluripotent niche. Overexpression of these genes increased differentiation and callus growth into roots and shoots. When they knocked out WOX5 and WOX7, a single callus culture produced significantly fewer shoots and roots compared to controls.
In subsequent experiments, they found that WOX5 interacted with a transcription factor to activate a gene responsible for producing auxin in the middle layer—a key step in organ regeneration. WOX5 also regulated the cytokinin signaling pathway by removing a negative feedback system that blocked this hormone’s actions. “It was a big surprise to see a single gene controlling both hormone actions in the middle layer,” Xu says.
In most plant tissues, including the callus, auxin and cytokinin are antagonistic: A callus exposed to high levels of auxin and low amounts of cytokinin forms roots; one exposed to the opposite ratio will spawn shoots. Exceptions occur, and researchers have suspected that the two hormones work together in the early stages of regeneration and plant development. “This paper reveals a very nice potential mechanism to explain how that might happen,” Birnbaum says.
Future studies could extend this single cell RNA sequencing approach to study changes in the callus over time as it differentiates. “This study is a single snapshot in time, but the technique can now be extended to cover the whole process of regeneration,” Benfey says.
Future studies to elucidate WOX5’s function and the significance of a middle layer in the callus could have important implications for crop engineering and biotechnology, he adds. When developing transgenic plants, researchers frequently generate a callus as an intermediate step during experiments. But many species are recalcitrant to the process and fail to regenerate into full-grown plants, Birnbaum says. “Any clues that we can get to improve calluses in a way that would improve regeneration and make cells more potent,” he notes, “could lead to important developments in agriculture and biotechnology.”