The cycle of light: Analyzing how cellular proteins in leaves change through day, night

Because next-generation biofuels will depend on the growth and hardiness of woody feedstocks, scientists have sought to better understand how leaf cells quickly respond to environmental cues such as light, temperature and water. These rapid responses take the form of “post-translational modifications” that occur when a plant cell chemically modifies a protein to alter its function within seconds.

Scientists at the Center for Bioenergy Innovation, or CBI, have studied this phenomenon in leaves from poplar trees during normal daily cycles of daylight and darkness. Until now, the effect of these modifications at the cellular protein level was not well understood, partly because of the technical limitations of the analytical tools available.

“What we’re trying to do is understand what healthy looks like across a 24-hour period,” said lead researcher Paul Abraham, a bioanalytical chemist at the Department of Energy’s Oak Ridge National Laboratory. This information provides a new baseline data set that can be used as a comparison in future studies of how leaves respond to stresses, such as drought. Such research can eventually help scientists develop biofuel feedstocks that can thrive in climate extremes, including harsher cold snaps or saltier water.

For this study, CBI scientists established poplar trees in a greenhouse before placing them in a growth chamber, where the plants experienced 12 hours of daylight followed by 12 hours of darkness. Leaf tissue was sampled every two hours. Researchers used chemical processes and high-resolution mass spectrometry to break down the samples into proteins and then peptides and ions. An advanced software program was used to search and sequence the mass spectrometry results. This revealed specific changes in the abundance of proteins and post-transitional modifications over the course of time.

The results showed not only that different classes of proteins were more abundant during daylight or darkness, but also whether their presence peaked when lighting mimicked dawn, noon or some other specific period in the light cycle.

Perhaps the most revealing results came from analyzing the protein modifications and when they occurred. “We are just now able to show that a single protein can take hundreds of different forms through the post-translational modifications that can be attached to it,” Abraham said. “I think what surprised me was the number of those modifications that can occur on a protein, and how many of those can exist across a 24-hour period.”

These results provide a new perspective and opportunity for scientists seeking to develop a hardier variety of biofuel feedstock. Genome editing often involves removing or modifying genes. “When you knock out a particular gene, that’s going to be permanent,” Abraham said. “But plants respond to stresses with these very sensitive, time-specific changes that are dynamic, and you want them to be able to go back to that original state. You don’t always want to be stuck.”

A new research path could focus on altering these post-translational modifications rather than making permanent changes, Abraham said. This option could prove especially valuable for feedstocks like trees that often grow for many years — both during and after phases of severe drought, for example.

The Center for Bioenergy Innovation at ORNL is one of four DOE Bioenergy Research Centers focused on advancing biofuels and bioproducts for a vibrant domestic bioeconomy. CBI is accelerating the development of bioenergy-relevant plants and microbes to enable production of drop-in sustainable aviation fuel, bioproducts that sequester carbon, and sustainable replacements for plastics and other environmentally harmful products.

CBI research is supported by the Biological and Environmental Research program in DOE’s Office of Science.

UT-Battelle manages Oak Ridge National Laboratory for DOE’s Office of Science, the single largest supporter of basic research in the physical sciences in the United States. DOE’s Office of Science is working to address some of the most pressing challenges of our time. For more information, visit energy.gov/science.  –S. Heather Duncan