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Study solves mystery of how plants use sunlight to tell time via cell protein signaling

Discovery may someday allow farmers to grow crops in climates where they currently won’t grow and allows scientists to make a subtle, targeted mutation to a specific native plant protein

Findings of a new study solve a key mystery about the chemistry of how plants tell time so they can flower and metabolize nutrients.

The process — a subtle chemical event — takes place in the cells of every plant every second of every day.

The new understanding means farmers may someday grow crops under conditions or in climates where they currently can’t grow, said chemist Brian D. Zoltowski, Southern Methodist University, Dallas, who led the study.

“We now understand the chemistry allowing plants to maintain a natural 24-hour rhythm in sync with their environment. This allows us to tune the chemistry, like turning a dimmer switch up or down, to alter the organism’s ability to keep time,” Zoltowski said. “So we can either make the plant’s clock run faster, or make it run slower. By altering these subtle chemical events we might be able to rationally redesign a plant’s photochemistry to allow it to adapt to a new climate.”

Specifically, the researchers figured out the chemical nuts and bolts of how a chemical bond in the protein Zeitlupe forms and breaks in reaction to sunlight, and the rate at which it does so, to understand how proteins in a plant’s cells signal the plant when to bloom, metabolize, store energy and perform other functions.

Zoltowski’s team, with collaborators at the University of Washington and Ohio State University, have made plant strains with specific changes to the way they are able to respond to blue-light.

“With these plants we demonstrate that indeed we can tune how the organisms respond to their environment in an intelligible manner,” Zoltowski said.

Zoltowski and his colleagues made the discovery by mapping the crystal structure of a plant protein whose function is to measure the intensity of sunlight. The protein is able to translate light intensity to a bond formation event that allows the plant to track the time of day and tell the plant when to bloom or metabolize nutrients.

A plant uses visual cues to constantly read every aspect of its environment and retune its physiological functions to adapt accordingly. Some of these cues are monitored by plant proteins that absorb and transmit light signals — called photoreceptors. The research team specifically studied two key photoreceptors, Zeitlupe (Zite-LOO-puh) and FKF-1.

“Plants have a very complex array of photoreceptors absorbing all different wavelengths of light to recognize every aspect of their environment and adapt accordingly,” said Zoltowski, an assistant professor in the SMU Department of Chemistry. “All their cells and tissue types are working in concert with each other.”

The finding was reported in the article “Kinetics of the LOV domain of Zeitlupe determine its circadian function in Arabidopsis” in the journal eLIFE online in advance of print publication.

Co-author and lead author is Ashutosh Pudasaini, a doctoral graduate from the SMU Department of Chemistry who is now a postdoctoral fellow at the University of Texas Southwestern Medical School, Dallas. Other co-authors are Jae Sung Shim, Young Hun Song and Takato Imaizumi, University of Washington, Seattle; Hua Shi and David E. Somers, Ohio State University; and Takatoshi Kiba, RIKEN Center for Sustainable Resource Science, Japan.

The research is funded through a grant from the National Institute of General Medical Sciences of the National Institutes of Health awarded to Zoltowski’s lab.

Nighttime is the right time for plants to grow
“If you live in the Midwest, people say you hear the corn growing at night,” said Zoltowski, who grew up in rural Wisconsin.

“During the day, a plant is storing as much energy as it can by absorbing photons of sunlight, so that during the evening it can do all its metabolism and growth and development. So there’s this separation between day and night.”

Plants measure these day and night oscillations as well as seasonal changes. Knowledge already existed of the initial chemistry, biology and physiology of that process.

In addition, Zoltowski and colleagues published in 2013 the discovery that the amino acids in Zeitlupe — working like a dimmer switch — gradually get more active as daytime turns to evening, thereby managing the 24-hour Circadian rhythm. Additionally, they found that FKF-1 is very different from Zeitlupe. FKF-1 switches on with morning light and measures seasonal changes, otherwise called photoperiodism.

But a knowledge gap remained. It was a mystery how the information is integrated by the organism.

“Ultimately that has to be related to some kind of chemical event occurring, some kind of chemical timekeeper,” Zoltowski said. “So by following that trail we figured out how the chemistry works.”

Dark state and light state snapshots
The problem required a two-pronged approach: Solving the structure of the protein to understand how forming and breaking bonds changes how the organism perceives its environment; and solving the chemistry, specifically the crystal structures of the protein’s dark and light states.

That process yielded a snapshot of the protein in the dark state and a snapshot of the protein in the light state, so the researchers could watch changes in protein structure in response to the bond-forming event.

From there, the researchers made mathematical models 1) that explain how the chemistry of the bond breaking and bond forming event, and the rate at which it occurs, should affect the organism; and 2) that design mutations to the protein that affect how it goes from the dark state to the light state to block that process.

Standard techniques yielded the discovery
The team used a few standard techniques. To get at the chemistry, they deployed ultra-violet visible spectroscopy to measure how efficiently proteins absorb light. They followed differences in the absorption spectrum, seeing what wavelengths are absorbed, to track chemical changes between the dark and the light states.

On the structure side, they crystallized the proteins and collected data at synchrotron sources at Cornell University, then mapped out like a puzzle where all the electrons are located in the crystal. From there they could fit and build — amino acid by amino acid — the protein, yielding a three-dimensional image of where every atom in the protein is located.

“This gives us pictures and snapshots of all those discrete events, where then we can look at how the atoms are moving and changing from one to the other,” Zoltowski said. “That allows us to see the bonds forming, the bonds breaking, and how the rest of the protein changes in response to that.”

Why didn’t we think of that?
The question has been an important one in the field, but challenging technical hurdles thwarted solutions, said Zoltowski. The key for his team was persistence and years of experience.

“This is not an easy protein to work with — it’s difficult to get crystals of these proteins. It requires a protein that is stable enough and will interact in a way that it yields a perfectly ordered crystal. So it’s difficult to do the chemistry and the structures. Researchers have struggled with getting adequate amounts of protein to be able to do these types of characterizations,” he said.

Think of it like a diamond, Zoltowski said, which is a perfectly ordered crystal that is just carbon atoms arranged in a specific way.

“Zeitlupe and FKF-1 have thousands of atoms in each protein, and in order to get a crystal, each molecule of the protein needs to arrange itself with the same type of accuracy and precision as carbon atoms in a diamond. Getting that to occur, where they pack nicely together, is non trivial. And some proteins just are really challenging to work with.”

Zoltowski and his colleagues have been fortunate in having years of experience working with these families of proteins, called the Light-oxygen-voltage-sensing domains, or LOV domains, for short.

“So we’ve developed a lot of skills and techniques over the years that can get over some of the technical hurdles,” he said. “So just from gaining experience over time, we’ve gotten better with working with some very difficult proteins. It makes something that is challenging, much more tractable for our lab.”

Does this apply to all LOV proteins in every plant?
Zeitlupe is a German word that means slow motion. The protein was dubbed Zeitlupe because scientists discovered when they found mutations of this protein previously that it made the Circadian clock run slower. It naturally altered the way the organism perceived time.

“We wanted to understand the proteins well enough that we could selectively alter the chemistry, or selectively alter the structure, to create mutations that would be testable in the organism,” Zoltowski said. “We wanted a predictive model that would tell us that these mutations that affect the kinetics — the rate at which this bond breaks — should do ‘X’ in the organism.”

The team’s new discovery results in hybrid plants — something nature already does and has done for millions of years through the process of evolution so that plants adapt to survive.

“We’re not putting anything into the plant or changing its genetics,” Zoltowski said. “We’re making a very subtle, targeted mutation to a specific protein that already is a native plant protein — and one that we’ve shown in this paper has evolved considerably throughout various different agricultural crops to already do this.”

The discovery gives scientists the ability to rationally interpret environmental information affecting a plant in order to introduce mutations, instead of relying on selective breeding to achieve a targeted mutation to generate phenotypes that potentially allow the plant to grow in a different environment.

What’s next?
The research opens a lot of new doors, including new questions about how these proteins are changing their configuration and how other variables, like oxidative stress, couple with the plant’s global sensory networks to also alter proteins and send multiple signals from the environment.

“What we’ve learned is that you need to pay careful attention to specific parts of the protein because they’re modulating activity selectively in different categories of this family,” Zoltowski said. “If we look at the whole family of these proteins, there are key amino acids that are evolutionarily selected, so they evolve specific modulations of this activity for their own independent niche in the environment. One of the take-homes is there are areas in the protein we need to look at to see how the amino acids are now different.”

Besides the NIH grant, the lab operates with $250,000 from the American Chemical Society’s Herman Frasch Foundation for Chemical Research Grants in Agricultural Chemistry. — Margaret Allen

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CBS DFW 11: Too Much ‘Blue Light’ Hinders Sleep

The negative consequences of blue light are associated with people’s metabolic clock being offset from their brain clock.

CBS DFW Channel 11 reporter Doug Dunbar covered the blue light research of Brian Zoltowski, an assistant professor in the SMU Department of Chemistry.

“As a society, we are using more technology, and there’s increasing evidence that artificial light has had a negative consequence on our health,” says Zoltowski, who was awarded $320,500 from the National Institute of General Medical Sciences of the National Institutes of Health to continue its research on the impact of blue light.

“Our study uses physical techniques and chemical approaches to probe an inherently biological problem,” Zoltowski said. “We want to understand the chemical basis for how organisms use light as an environmental cue to regulate growth and development.”

Dunbar’s piece featuring Zoltowski’s research and lab, “Too Much “Blue Light” Hinders Sleep,” was published online Dec. 12.

Watch the full coverage.

EXCERPT:

By Doug Dunbar
CBS DFW 11

Can’t get a good night’s sleep. You might be getting a little too much blue light.

What’s that? It’s a big issue the Federal government is asking researchers at SMU to study.

There’s a reason why it’s dark in this lab. It’s because they’re studying light.

They have the lights off so they can purify the proteins in the dark.

So that we can study the activation process when we first expose them to light. But not just any light. Blue light. The stuff in fluorescents, and devices like laptops and phones. But also daylight.

One of the negative consequences of blue light is associated with our metabolic clock being offset from our brain clock. That can lead to problems for diabetes, cancer, mood disorders.

[ …] But Zoltowski and his crew could potentially tackle problems much bigger than sleeping.

“If we understand how these proteins that respond to light work we can create new biotechnology.”

Maybe new ways to deliver drugs, or even targeted cancer treatments.

“We can shine light on a very specific spot and that can allow us to activate any biological event we want at that very precise location and time.”

Watch the full coverage.

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KERA: The Bright Side And Dark Side Of Blue Light

“We’re introducing blue light into the environment and into our daily lives at times when we’re not supposed to see it – and that basically causes dysfunction in our biological processes.” — Brian Zoltowski

KERA Public Radio journalist Justin Martin explored the good and bad of blue light in our environment with Brian Zoltowski, an assistant professor in the SMU Department of Chemistry.

“As a society, we are using more technology, and there’s increasing evidence that artificial light has had a negative consequence on our health,” says Zoltowski, who was awarded $320,500 from the National Institute of General Medical Sciences of the National Institutes of Health to continue its research on the impact of blue light.

“Our study uses physical techniques and chemical approaches to probe an inherently biological problem,” Zoltowski said. “We want to understand the chemical basis for how organisms use light as an environmental cue to regulate growth and development.”

Martin’s interview with Zoltowski, “The Bright Side And Dark Side Of Blue Light,” was published online Dec. 8.

Listen to the interview.

EXCERPT:

By Justin Martin
KERA

Light is necessary for life on earth, but scientists believe that too much of a certain wavelength can cause everything from crop diseases to changes in the migratory patterns of animals. SMU professor Brian Zoltowski is working to unravel the mystery of blue light in a study funded by the National Institutes of Health.

Interview Highlights: Brian Zoltowski

… On what defines blue light:

“Blue light refers to a part of the electromagnetic spectrum that has specific energy. Usually we define things by basically their wavelength of light. So typically blue light can be centered around 450 nanometers but can range … from about 425 nanometers to 475 nanometers.”

… On the origin of excess blue light:

“Blue light is very abundant in nature in general. That’s why organisms actually use that wavelength of light to drive their biological processes. But it turns out though that because it’s abundant in nature, we like to have it to be abundant in our products like our lights, our computers, our laptops and everything else. So we introduce a lot more foreign blue light into the environment compared to what should naturally be there.”

… On blue light’s effects in animals vs. plants:

“A lot of that is not known, which is one of the reasons we’re actually doing a lot of this research. What we do know is that blue is extremely important for basically growth and development of any organism you can conceive of. How that’s ultimately regulated and when there can be too much is a big question. The bigger question is when you get the blue light, nature is designed to use blue light as a signal as to what time of day it is. So when we’re introducing blue light into the environment or into our daily lives through computers and laptops, we’re introducing blue light at times of day like evening when we’re not supposed to see it – and that basically causes dysfunction in our biological processes.”

… On how blue light fosters fungal growth:

“There’s growing understanding that a lot of these fungal pathogens of plants – there are several that actually attack wine, which causes billions of dollars of crop loss each year. There are some that we’re interested in that also basically attack a lot of your grain crops — so you’re looking at wheat, alfalfa — that have very detrimental aspects to agriculture. Their ability to infect the plant is regulated by blue light.”

Listen to the interview.

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Study funded by NIH is decoding blue light’s mysterious ability to alter body’s natural clock

Blue light from artificial lighting and electronic devices knocks circadian rhythms off-kilter, resulting in health problems, sleep, cancer development, mood disorders, drug addiction, crop disease and even confused migratory animals

A study funded by the National Institutes of Health is unraveling the mystery of how blue light from residential and commercial lighting, electronic devices and outdoor lights can throw off-kilter the natural body clock of humans, plants and animals, leading to disease.

Exposure to blue light is on the increase, says chemist Brian D. Zoltowski, Southern Methodist University, Dallas, who leads the study, “Protein : Protein interaction networks in the circadian clock.”

At the right time of day, blue light is a good thing. It talks to our 24-hour circadian clock, telling our bodies, for example, when to wake up, eat and carry out specific metabolic functions.

In plants, blue light signals them to leaf out, grow, blossom and bloom. In animals, it aids migratory patterns, sleep and wake cycles, regulation of metabolism, as well as mood and the immune system.

But too much blue light — especially at the wrong time — throws biological signaling out of whack.

“As a society, we are using more technology, and there’s increasing evidence that artificial light has had a negative consequence on our health,” said Zoltowski, an assistant professor in SMU’s Department of Chemistry.

“Our study uses physical techniques and chemical approaches to probe an inherently biological problem,” he said. “We want to understand the chemical basis for how organisms use light as an environmental cue to regulate growth and development.”

Zoltowski’s lab was awarded $320,500 from the National Institute of General Medical Sciences of the National Institutes of Health to continue its research on the impact of blue light.

The lab studies a small flowering plant native to Europe and Asia, Arabidopsis thaliana. The flower is a popular model organism in plant biology and genetics, Zoltowski said.

Although signaling pathways differ in organisms such as Arabidopsis when compared to animals, the flower still serves an important research purpose. How the signaling networks are interconnected is similar in both animals and Arabidopsis. That allows researchers to use simpler genetic models to provide insight into how similar networks are controlled in more complicated species like humans.

Understanding the mechanism can lead to targeted drug treatments
In humans, the protein melanopsin absorbs blue light and sends signals to photoreceptor cells in our eyes. In plants and animals, the protein cryptochrome performs similar signaling.

Much is known already about the way blue light and other light wavelengths, such as red and UV light, trigger biological functions through proteins that interact with our circadian clock. But the exact mechanism in that chemical signaling process remains a mystery.

“Light is energy, and that energy can be absorbed by melanopsin proteins that act as a switch that basically activates everything downstream,” Zoltowski said.

Melanopsin is a little-understood photoreceptor protein with the singular job of measuring time of day.

When light enters the eye, melanopsin proteins within unique cells in the retina absorb the wavelength as a photon and convert it to energy. That activates cells found only in the eye — called intrinsically photosensitive retinal ganglian cells, of which there are only about 160 in our body. The cells signal the suprachiasmatic nucleus region of the brain.

“We keep a master clock in the suprachiasmatic nucleus — it controls our circadian rhythms,” he said. “But we also have other time pieces in our body; think of them as watches, and they keep getting reset by the blue light that strikes the master clock, generating chemical signals.”

The switch activates many biological functions, including metabolism, sleep, cancer development, drug addiction and mood disorders, to name a few.

“There’s a very small molecule that absorbs the light, acting like a spring, pushing out the protein and changing its shape, sending the signal. We want to understand the energy absorption by the small molecule and what that does biologically.”

The answer can lead to new ways to target diabetes, sleep disorders and cancer development, for example.

“If we understand how all these pathways work,” he said, “we can design newer, better, more efficacious drugs to help people.”

Chemical signal from retina’s “atomic clock” synchronizes circadian rhythms
Besides increased reliance on artificial lighting indoors and outdoors, electronic devices also now contribute in a big way to blue light exposure. Endless evening hours on our smartphones and tablets with Candy Crush, Minecraft or Instagram don’t really help us relax and go to sleep. Just the opposite, in fact.

The blue glow those devices emit signals our circadian clock that it’s daytime, Zoltowski said. Red light, on the other hand, tells us to go to sleep.

Awareness of the problem has prompted lighting manufacturers to develop new lighting strategies and products that transition blue light to red light toward evening and at night, Zoltowski said.

Targeted solutions could neutralize destructive blight in staple crops
In plants, the researchers study how the absence of “true dark” in nature due to artificial light can reduce yields of farm crops and promote crop disease.

For example, fungal systems rely on blue light to proliferate, forming pathogens known as blight in crops resulting in leaves that look chewed on and reducing yields.

“We study fusarium and verticillium,” Zoltowski said. “They cause about $3 billion worth of crop damage a year to wheat, corn, soybeans — the staple food crops.”

Understanding their ability to infect crops would allow scientists to potentially design small molecules that target and disrupt the fungal system’s circadian clock and neutralize their proliferation.

Research to understand how light and clock regulation are coupled
In animals, Zoltowski’s lab studies the blue light pathway that signals direction to birds and other animals that migrate. Blue light activates the protein that allows various species to measure the earth’s magnetic field for directionality. For example, Monarch butterflies rely on the cryptochrome photoreceptor for their annual migration to Mexico.

“We’re interested in how these pathways are regulated in a diverse range of organisms to understand how we can manipulate these pathways to our advantage,” he said, “for health consequences and to improve agriculture yields.”

The researchers will map the reaction trajectory beginning from the initial absorption of the photon to the point it alters an organism’s physiology.

Zoltowski notes that light is just one of a handful of external cues from our environment that trigger biological processes regulating the circadian clock. Others include temperature changes, feeding and metabolites.

Besides the NIH grant, the lab operates with $250,000 from the American Chemical Society’s Herman Frasch Foundation for Chemical Research Grants in Agricultural Chemistry. — Margaret Allen

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Scientific American: How your smartphone messes with your brain — and your sleep

Scientific American science blogger Josh Fischman drew on the sleep expertise of SMU Assistant Professor of Chemistry Brian D. Zoltowski to explain how artificial light from our smartphones and other digital devices causes sleep deprivation. His blog article, “How your smartphone messes with your brain — and your sleep,” published May 20 and has been heavily shared through social media.

Zoltowski’s lab at SMU studies one of the many proteins involved in an organism’s circadian clocks. Called a photoreceptor, the protein responds to light to predict time of day and season by measuring day length.

The circadian clock is an internal biological mechanism that responds to light, darkness and temperature in a natural 24-hour biological cycle. The clock synchronizes body systems with the environment to regulate everything from sleep patterns and hunger in humans to growth patterns and flowering in plants.

“Our research focuses on understanding the chemical basis for how organisms perceive their surroundings and use light as an environmental cue to regulate growth and development,” Zoltowski says.

Zoltowski and the American Chemical Society created a video explaining the light-sleep deprivation relationship.

Read the full story.

EXCERPT:

By Josh Fischman
Scientific American

It’s not the Angry Birds, streaming videos, emails from your boss, or your Facebook updates that disturb your sleep when you spend an evening staring at your smartphone or tablet. OK, the apps can keep you glued to your screen until the wee hours, and that doesn’t help. But it is the specific type of light from that screen that is throwing off your natural sleep-wake cycles, even after you power down. In a new video from Reactions: Everyday Chemistry, a sleep researcher explains the eerie power of blue light over your brain.

Cells at the back of your eyes pick up particular light wavelengths and, with a light-sensitive protein called melanopsin, signal the brain’s master clock, which controls the body’s circadian rhythms. Blue light, which in nature is most abundant in the morning, tells you to get up and get moving. Red light is more common at dusk and it slows you down. Now, guess what kind of light is streaming from that little screen in your hand at 11:59 P.M.? “Your iPad, your phone, your computer emit large quantities of blue light,” says sleep researcher and chemist Brian Zoltowski of Southern Methodist University

Read the full story.