QB3-Berkeley scientists peek in on algae at night to discover how these organisms prepare for another stressful day on the job.
After a long day at work, we humans might recharge by sipping tea, soaking in a candle-lit bubble bath, and finally getting some shut eye.
What do algae do to unwind?
These tiny photosynthesizers have a big job: while they make up only about 1% of plant biomass on the planet, they are responsible for roughly half of the world’s CO2 fixation. Day in and day out, they are bombarded with photons and must fine-tune how they harvest them in order to use them for good and avoid photodamage. Then, the sun dips below the horizon, and algae settle into their version of a bedtime routine.
How does the stress of the workday influence the algae’s biological “clock”? In a study recently published in The Plant Cell, researchers led by UC Berkeley Professor Sabeeha Merchant and Research Scientist Masakazu Iwai at Lawrence Berkeley National Laboratory reported that light-induced stress not only impacts what algae do during the day, it also shapes their physiology and gene expression at night.
The team showed that the same tools that algae used to cope with stress during the day were cocked and ready in the dark, too. This likely improves their productivity when they encounter another stressful morning on the job. The study provides time-resolved, systems biology datasets that paint a more complete picture of resiliency in these microbes that have a massive impact on the global carbon cycle.
A day in the life of a model alga
Humans have evolved to eat during the day and then rest and digest in the night. Hunting and gathering are easier when the sun is lighting the way. Similarly, algae have evolved to coordinate their metabolic activities with the rising and setting of the sun.
Merchant’s lab has studied the daily rhythms of one such alga in detail: the model unicellular alga Chlamydomonas reinhardtii, or “Chlamy” for short. The lab grows Chlamy in photobioreactors where researchers can impose environmental conditions that mimic nature in the laboratory: diurnal cycles of warm, bright days and dark, cool nights.
During the daytime, the sun powers photosynthesis: algae harvest sunlight and convert this light energy into chemical energy by fixing CO2 from the atmosphere into sugar. During this time, the algae must grow and synthesize all of the machinery necessary to do their job: proteins, lipids, pigments, and a variety of other molecules. They also store some of the sugar away as starch, saving it for the nighttime when they’ll no longer have access to light energy.
By the afternoon, if Chlamy cells have gotten big enough, they replicate their genomes and divide into daughter cells. Then, Chlamy persists in a quiescent “sleepy” state, living off of their starch reserves and fermenting in the dark. At dawn, they wake up and do it all over again.
In 2019, the Merchant lab discovered that Chlamy’s metabolic routine was driven by diurnal rhythms in the expression of 85% of the alga’s genome: transcripts for their cilia, ribosomes, histones, and photosynthetic complexes each flood the cell at a specific time of day.
As for other organisms, the temporal orchestration of gene expression is key to optimizing how algae allocate their resources over day-night cycles as the environment changes predictably.
“Looking at Chlamy’s gene expression over the diurnal cycle is like seeing the symphony,” Merchant explains. “The conductor cues the violins, then the drums, then the flutes, so all these pieces can come together into a beautiful piece of music. Studying the way the piece comes together across the diurnal cycle in the Chlamy cell has revealed fascinating genetic regulatory mechanisms that we wouldn’t have known existed otherwise.”
Not only can studying Chlamy’s daily routine reveal the secrets behind its genetic programs, it’s also critically important for understanding global carbon cycling. Algae remove billions of tons of CO2 from our atmosphere each year. At the base of aquatic food chains, their metabolism sustains a host of other microbes, shaping Earth’s ecosystems.
So, a wholistic picture of the whole 24 hours—what’s going on in both daytime and nighttime, how that routine is orchestrated, and what happens when it is perturbed— is necessary to understand our planet.
Photosynthesis is no walk in the park
Photosynthesis may sound sweet: vibrant green cells baking sugar and releasing oxygen for their friends? Lovely. But harvesting sunlight is serious business. Light is absorbed by chlorophyll in light-harvesting proteins that are nestled in a network of green membranes called thylakoids. The light energy must then get passed to the reaction center of the photosystem complexes, where it splits a water molecule apart and gets converted to chemical energy.
When too much light is absorbed, the path to the reaction center gets backed up, and the light energy takes a turn for the worse. It forms reactive oxygen species that damage the photosynthetic apparatus and other parts of the cell.
Algae have many strategies to manage the stress of photosynthesis and avoid oxidative “photodamage.” They fine-tune their chlorophyll content and organize their thylakoid membranes to carefully modulate the amount of light they are absorbing. They can also deploy stress-related light-harvesting chlorophyll proteins, LHCSRs, to the sites of absorption. These special proteins take excess light off of the photosystems’ hands and safely dissipate it as heat. Yet, deploy LHCSRs at the wrong time, and they could dissipate light energy unnecessarily, reducing photosynthetic productivity.
Iwai and UC Berkeley Professor Kris Niyogi, another author of the study, have been investigating LHCSRs and other photoprotective tools for decades. They and others have found that algae need some time to adjust to excess light before they can use these tools for managing light stress effectively. Yet, most studies on photodamage and photoprotection have been performed in continuous light, without a nighttime.
If algae need time to be able to protect themselves, are they more susceptible to photodamage in the morning than in the afternoon? More broadly, how do rhythms in Chlamy’s gene expression and metabolism impact photoprotection? And on the flip side, how are those rhythms themselves shaped by light stress?
“Systems biology” study reveals a physiological memory of stressful days passed
To answer these questions, former postdoctoral researcher Valle Ojeda grew Chlamy in the photobioreactors for several weeks of repetitive day-night cycles where the alga would experience the same amount of light each day: either low, moderate, or excess.
“We knew a lot about how the cells looked during light stress from Dr. Iwai’s work,” Ojeda explains. “He had used Airyscan microscopy to look inside live Chlamy cells that had been acclimated to excess light, and what he found was that the lobes of Chlamy’s cup-shaped chloroplast had become very thin and short in these cells.” This kind of behavior helps Chlamy reduce the amount of light it is absorbing to avoid photodamage.
But Ojeda and her colleagues had no idea what to expect during the nighttime. “Would the cells recover and forget about the light stress after some ‘rest’ time?”
Ojeda’s microscopy images of cells during the daytime recapitulated Iwai’s previous work: cells acclimated to low-lit days had chloroplasts thick with fluorescent thylakoid membranes reaching from one end of the cell to the other. The glowing membranes were much slimmer and shorter in cells during excess light days.
“What was shocking was that the cells maintained these differences in their chloroplasts even after ten hours of darkness,” says Ojeda. “It was as if they remembered the previous day and were preparing for what was to come”.
She and her colleagues took a “systems approach” to the investigation, integrating microscopy of thylakoid membranes with measurements of gene expression, metabolism, lipids, and pigments to paint a picture of how light stress influences the alga’s daily rituals. They found that Chlamy’s thylakoid membranes were not the only structures that differed in the aftermath of a stressful day’s work.
The alga’s gene expression program was still rhythmic, and its bedtime routine was still the same: the cells divided at the end of the day and then entered into their “rest and digest” phase of the night regardless of daylight intensity. However, the amount of light stress they had experienced during the day impacted the abundance of hundreds of proteins, lipids, and pigments during the night. Many of these molecules were part of the strategies that help Chlamy withstand environmental stresses during the day.
Slumber party
In order to study the algae as they wind down at the end of the day, Ojeda had to sacrifice her own bedtime routine. “The experiments are long and stressful not just for Chlamy, but also for me. After weeks of preparing the cultures, I would spend over 24 h in the lab pulling samples from the photobioreactors to capture Chlamy at the right times of the diurnal cycle.”
The experiments required careful planning and focus. One wrong move and the photobioreactors could crash, setting researchers back weeks as they acclimate new algal cultures to the right environmental conditions. Ojeda and her colleagues worked hard to make sure that each diurnal cycle experiment went perfectly so that no sleepless night was wasted—for themselves, nor for Chlamy.
Ojeda says, “what kept me going through those times was my curiosity to see what happens in the night, my amazing colleagues, and the pho that I would treat myself to for lunch when it was over. In the end, it is all worth it, because we discovered something new together.”
So worth it, in fact, that more late nights are already on the books as the Merchant lab continues to uncover the mysteries of Chlamy’s bedtime.
Sunnyjoy Dupuis is a postdoctoral scholar in the Merchant group. She received her PhD in Microbiology from UC Berkeley and was awarded the UC Dissertation Year Fellowship. She investigates the ecophysiology of algae: how they are shaped by the environment, and how they contribute to that environment in return. She is co-author of the featured article in The Plant Cell.