According to the USDA, the average American eats some 415 pounds of vegetables a year. To keep up with supply and demand, farmers have to rely on one tool to maximize their plant growth and crop yield: nitrogen.

When a plant takes up the nitrogen from the soil, it creates amino acids to distribute nitrogen throughout the plant to be used in the leaves and stored in the seeds or roots. The plant enlists the help of small proteins, called amino acid transporters, which act as valves that open or close, directing the flow of amino acids to where they are needed most.

But little is known about the mechanisms that are behind these transporters. Amanda Ramirez, a senior biochemistry major, has been studying a specific amino acid transporter called Amino Acid Permease 1 (AAP1) as part of the Fralin Undergraduate Research Fellowship (FURF).

“We studied AAP1 in Arabidopsis thaliana, and expressed it into tobacco and yeast to understand its signaling mechanisms,” said Ramirez. “We wanted to determine how the cells detect whether they need more amino acids, if they already have enough, or if they don’t need to take in any more.”

If scientists are able to fully understand amino acid transporters and how they function, they may be able to help solve a problem in agronomics that is becoming increasingly complex: nitrogen use efficiency - otherwise known as the plant’s efficiency to take up and use nitrogen to produce seeds.

“We know that amino acid transporters play a part in nitrogen use efficiency because they move amino acids across different organs and re-mobilize nitrogen from the leaves to the seeds,” said Guillaume Pilot, an associate professor in the School of Plant and Environmental Sciences, housed in the College of Agriculture and Life Sciences, and Amanda’s principal investigator. “In the future, we could do some targeted breeding or use biotechnology to increase the efficiency of absorption and use of nitrogen.”

This research could also boost farmers’ efforts to reduce the amount of fertilizer that is being applied to crops, which would inherently reduce water pollution. Farmers typically apply a standard amount of nitrogen to the fields, but this overabundance of nitrogen is just too much for most crops to absorb. As a result, excess nitrogen stays in the soil, and rain events can cause run-off, creating algal blooms and other forms of water pollution.

Ramirez first dove into her undergraduate research project in January 2020. Around that time, Pilot introduced her to Fralin’s Summer Undergraduate Research Fellowship. She got into the program, but because of the COVID-19 pandemic, her path transitioned to the year-long Fralin Undergraduate Research Fellowship.

“COVID-19 was difficult for everyone, but especially for her because she really wanted to do this research,” said Pilot, the founding director of the Translational Plant Sciences Center, which is housed in the Fralin Life Sciences Institute. “Everything was put to a hold. In addition to classes, a job, and a sorority, she had to do lab work. It was a lot, especially being a senior. Considering the situation, she learned a lot. She did a presentation, did experiments, and produced results similar to previous undergraduate students who did not have to deal with all those worries.”

But despite the rapid change of plans, Ramirez found comfort in the weekly lunch meetings that were hosted by Dennis Dean, a University Distinguished Professor of biochemistry and a longtime supporter of undergraduate research.

“It was helpful to finally be able to talk about myself professionally,” said Ramirez. “It was interesting to see what other people are doing and to meet people that were going through the same transition as me. It was especially helpful to learn how to simplify complex topics in a way that other people can understand.”

But before she set her eyes on biochemistry, Ramirez was first inclined to study a field of a different kind: mathematics.

Back in high school, Ramirez enrolled in The Mathematics and Science Academy at Ocean Lakes High School to study mathematics. As she began to take more science classes and labs, her passions and interests changed - largely because of spending time with the passionate teachers. In her senior year of high school, she turned away from mathematics and toward biochemistry.

Fast forward to the present day, and Ramirez is taking part in ground-breaking amino acid transporter research in Guillaume Pilot’s lab.

Recently, the lab took a close look at the phosphorylation states of the amino acid transporter AAP1 to learn more about how it functions. Phosphorylation is an important process that makes tiny edits to the proteins to ensure that they are functioning in a way that is best for the organism. For example, phosphorylation can involve activating proteins, deactivating proteins, or modifying their functions.

To test whether or not amino acid transporters are regulated by phosphorylation, Ramirez helped create mutants by altering amino acids within a few AAP1 transporters. By doing so, the lab was able to mimic AAP1 in a phosphorylated state and AAP1 in a de-phosphorylated state. Once the mutants were created, they were expressed in tobacco and yeast.

After performing various assays, the lab was unable to identify any major difference between the mutated and the unmutated proteins in tobacco and yeast. Despite their recent findings, the lab will continue to study the function of the mutants, but now in Arabidopsis roots.

Ramirez completed the FURF program in late April, after her participation in the Dennis Dean Undergraduate Research Symposium. Although she said that it was challenging to summarize a whole semester’s worth of work into five minutes, her presentation at the symposium went well.

Ramirez said that her research project, and the entire FURF experience, solidified her decision to continue research after college.

“We had discovered something that hadn’t been known before,” said Ramirez. “It is a great feeling - being able to contribute to the frontier of scientific research. That experience emphasized that I want to do research as a career.”

As for her future plans, Ramriez has decided to take a gap year or two before jumping into graduate school. During her gap year, she will be applying to jobs that involve laboratory work, so that she can develop more of her skills.

Inspired by her medicinal plants and herbs class that contributed towards her Integrative Health and Wellness minor, Ramirez is realigning her passions once again, as she hopes to get a master’s or doctorate in pharmacology.

“For me, it is so cool being able to understand how drugs affect our body. In my medicinal plants and herbs class, I was fascinated to learn about how these compounds in plants affect our body and how we can look to them for inspiration to develop new drugs.”

— Written by Kendall Daniels

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