An innovative approach to measuring microscopic changes in plant cells

Every time the temperature drops, a cloud passes overhead, or the sun sets, a plant makes a choice: keep its microscopic pores, called stomata, open to absorb carbon dioxide and continue photosynthesizing or close them to protect its precious stores of water. That capacity to open and close pores requires the plant to respond to subtle environmental changes by adjusting the pressure within the cells of the stomata — a complex ability that plants evolved over hundreds of millions of years.

An interdisciplinary team of biologists, physicists, and engineers, led by researchers at the Yale School of the Environment, developed a pioneering method to observe those pressure changes. The new approach, detailed in a study published in PNAS, vastly expands the rate at which, and the number of species from which, scientists can take measurements,  opening up new possibilities for research on plant evolution and physiology with valuable applications for improving water efficiency, the researchers said.

“Almost every single land plant is using this principle of internal pressure in order to grow, reproduce, and do everything a plant does, but we previously had basically no access to this measurement,” said Craig Brodersen, the Howard and Maryam Newman Professor of Plant Physiological Ecology and the lead author of the study. “So, a lot of the fundamental theory about how plants work is based on an extremely limited set of measurements on just a couple of species.”

The study is the first published application of the method in stomata in Bryophytes (a lineage that includes mosses), which will help better the understanding of the evolutionary trajectory of Earth’s earliest plants, the team noted.

To measure the pressure changes that mechanically force the stomata open and close, scientists traditionally have pierced cells with a fragile, glass tube that measures a fraction of the width of a human hair. The tubes easily break, and the labor-intensive method only works on species with larger cells. In contrast, the new approach uses a laser system creatively adapted from research being conducted at the Yale School of Medicine to understand nerve regeneration in worms

A high pulse of light energy vaporizes liquid within the cell, creating miniscule bubbles. Though the bubbles dissolve in a fraction of a second, the team measured the bubble’s maximum size, which is proportional to the pressure around it, using high speed cameras. The researchers then observed how the pressure changes, based on how big the bubble gets in response to changing light levels. The team successfully tested the method in more than 40 plant species, including several with cells too small to previously study.

Quantifying those changes will help scientists understand how quickly stomata can open and close, which ultimately determines the balance between how much carbon a plant absorbs and how much water it loses while its pores are open. Water use efficiency, as that measure is called, is a central concern in agriculture. These tools are an important first step in developing crop varieties that are more water efficient and improving irrigation management in water-scarce environments, Brodersen said.

The team is continuing to refine the method and was recently awarded funding from Yale Planetary Solutions and the National Science Foundation to engineer a system to get absolute pressure.