Every plant cell is a master builder, constructing its own rigid walls from the inside out. But there’s a mystery: how does a cell know exactly where to add new material as it grows?
New research from Dartmouth reveals how plant cells use calcium ions to create a chemical “map” that helps to guide and focus their construction efforts.
Using a combination of microscopy and CRISPR-Cas-9 methods, researchers identified proteins that actively pump calcium ions only in regions that are removed from the growing tips, creating a concentration gradient that marks the construction zone. The steeper this calcium gradient, the faster the cell grows—resulting in bigger plants.
Understanding these fundamental mechanisms of plant growth could eventually help scientists engineer plants with specific traits—such as better candidates for biofuel production, says Magdalena Bezanilla, the Ernest Everett Just 1907 Professor in the Department of Biological Sciences and senior author of the study, which was published in the Journal of Cell Biology.
Magdalena Bezanilla (Photo by Katie Lenhart)
“My lab is really interested in understanding how cells shape themselves and their extracellular environment,” she says. “Plants are a really fantastic system to study that, because every single cell in a plant is surrounded by a wall that is built by individual cells.”
Some plant cells grow by elongating at one end, a pattern known as polar growth. It’s the most common mode of growth in the juvenile phase of the moss plants that Bezanilla works with, Physcomitrium patens. Polar growth is also critical for flowering plants, where it occurs in specialized cell types such as pollen tubes, which are essential for fertilization, and root hairs, which help plants withstand drought.
Previous studies suggested that calcium ions might play a role in orchestrating polar growth, because actively growing cells have a higher concentration of calcium in their elongating tips, and because the concentration of calcium at the cells' tips fluctuates from second to second. However, calcium’s exact role, and how it carries it out, was unclear.
“Calcium is an important signaling molecule in every biological system, but it’s enigmatic, because it's a tiny ion that can diffuse extremely readily, and yet it can do very specific things in cells,” says Bezanilla. “The question is, how does such a tiny molecule manage to convey such complex and rich signals?”
Two signals, two roles
To unravel the dual roles of calcium signaling in polar growing cells, Bezanilla’s team used fluorescently tagged proteins to observe and track changes in calcium ion concentration within growing moss cells. They found that cells with a more pronounced calcium concentration gradient—meaning there was a bigger difference between the concentration at the cell's tip compared to its interior—grew faster than cells with a smaller concentration gradient.
Since calcium ions would naturally diffuse throughout the cell, the fact that polar growing cells have a higher concentration at their tips means that they actively control the calcium there. To understand how the cells do this, the team investigated a group of proteins called autoinhibitory calcium ATPases (ACAs), which use energy to pump calcium out of the cell’s interior.
When the researchers used CRISPR/Cas-9 to create moss cells with non-functional ACAs, they found that the plants were smaller, the cells grew more slowly and had a much less pronounced calcium concentration gradient. However, moss cells without functional ACAs still displayed normal calcium fluctuations, indicating that the two components of calcium signaling—temporal and spatial—act independently of each other.
“This gives us a tool to separate the temporal and spatial elements of calcium signaling for the first time,” says Bezanilla. “The spatial calcium signal seems to define a region in the cell where there is enhanced secretion of cell wall material. The next step will be to identify the molecules that actually respond to this signal by doing that secretion.”
The team also showed that the calcium concentration gradient regulates cell growth independently of a protein called actin that is also known to regulate plant cell growth. When they used gene editing to create moss cells that lacked both ACAs and actin specifically at the cell tip, most of these mutant cells died, and the ones that survived were only 1–2% the size of normal plants.
“We previously showed that the temporal calcium signal is directly anti-correlated with actin, and now we've discovered that the ACAs don't interact with actin at all,” says Bezanilla. “Now that we know this, we can start to define what these two signals are doing to the cell.”
Though polar growth only occurs in certain types of plant cell, the principles uncovered in this study are likely to apply to other modes of plant cell growth. And because calcium signaling occurs in all branches of the tree of life, these insights could even have applications beyond plants.
“Calcium is used in our cells every day to signal many different processes, so being able to separate temporal and spatial calcium signals and knowing that they’re doing two independent things provides a whole new way of looking at how to work with calcium,” says Bezanilla.