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Four years after plant sciences professor Kentaro Inoue was struck and killed while riding his bike, the last three graduate students from his lab are ensuring his scientific legacy lives on through their published research, careers in industry and academia, and mentoring of future science students.

Philip Day, Laura Klasek and Lucas McKinnon successfully completed their doctoral degrees in the past year, having continued their studies with the support of plant biology professor Steven Theg, one of Inoue’s colleagues, and the Department of Plant Sciences.

Inoue, a member of the faculty for 15 years, died while cycling through West Sacramento, en route to campus, Aug. 31, 2016.

Day, Klasek and McKinnon “had to make a decision to stay in science, stay with projects Kentaro gave them, not knowing how they would turn out, so they really persevered,” Theg said. “I thought it took a lot of fortitude.”

Theg would become their mentor — fortunate, he said, to have inherited such talented students. The trio has flourished, he said, citing publication of their work in one of the leading journals in plant sciences, The Plant Cell — research that focuses on chloroplasts, which are responsible for photosynthesis in plants. Theg’s own work involves protein translocation across chloroplast membranes.

“They should be very proud of their research, and I’m proud of them, too,” he said. “Having their papers all in the same top plant sciences journal is a really nice way to celebrate Kentaro’s final scientific contributions.”

‘A beautiful collaboration’

Georgia Drakakaki, associate professor in plant sciences and a colleague of Inoue, described the work of Theg and the students as “a beautiful collaboration — something really good that came out of a tragic moment that Kentaro would be proud of.”

Inoue, whose appointment was in the College of Agricultural and Environmental Sciences, and Theg, of the College of Biological Sciences, were close colleagues, had overlapping research and served on committees for each other’s students. To work with Day, Klasek and McKinnon just made sense, Theg said, because he was so familiar with Inoue’s research.

“It’s a tribute to Kentaro that all three students decided to keep their existing projects rather than start new ones,” Theg said. “They were good projects — Kentaro had really thought about what was important in the field.”

It takes a village

The grad students acknowledged that what Theg did was no easy task, accepting established students and meeting with them weekly.

“All three of us are grateful for what Professor Theg did, taking us on,” Day said. “It must have been very difficult to start thinking about new projects that weren’t originally his, but he did a great job and pushed us through to publish and graduate.”

Klasek added: “Professor Theg was an important part of being able to continue research. He was a solid sounding board, a great source of support and a great mentor for the last two-thirds of my Ph.D.”

The Department of Plant Sciences also invested in the students to help them finish theirs and Inoue’s research. Chairs Joe DiTomaso and later, Gail Taylor, along with the chief administrative officer, Dee Madderra, arranged to continue the students’ support until they graduated, including travel funding.

“We wanted to do what was right — whatever we could to make sure Philip, Lucas and Laura were well taken care of,” Madderra said. “We hoped that with the department’s support, it would help them move through the transition.”

Good scientists

The students said Inoue prepared them well and taught them to be good scientists — to think like scientists and to be good scientific citizens. Klasek recalled how Inoue emphasized that scientists should always acknowledge anyone who helped with their work, whether it was someone who prepared a reagent or someone who, in discussion, spurred new ideas or direction.

“He was really conscientious about recognizing those contributions and how we would never get everything done without everyone in the lab and other people doing research in our field and beyond,” Klasek said.

She added that Inoue’s students always knew they had managed to do something really well if the professor simply said, “good,” and moved on with the conversation.

“That was cause for celebration,” she said.

As understated as he was, though, Inoue’s influence went a long way — inspiring these three graduate students to their Ph.D.s. even in his absence.

“Kentaro was demanding and pushed us very hard to defend our conclusions and support our data,” Klasek said. “But he also had our backs 100 percent. If we started to get discouraged or had other stuff going on, he wanted to see how he could help.”

LAST OF THE INOUE LAB

‘Think like a scientist’

Philip Day completed his Ph.D. in 2019. helped determine how beta-barrel proteins are sorted to the correct location in the outer envelope membrane of plant chloroplasts. Protein sorting to this membrane is one of the least understood aspects of chloroplast protein targeting.

Day continued his chloroplast research with Professor Henning Kunz at Washington State University for a year, until Kunz moved his laboratory to Germany. Due to travel difficulties during the pandemic, Day decided not to follow. He is considering his next career steps.

Working with Inoue for more than 3½ years, Day said he credits him with teaching him the importance of rigor, how to design a good experiment and carry it out, and, above all, making sure he knew how to think like a scientist.

Said Day: “I find myself always thinking, ‘Are these the results that Kentaro would have agreed with, would they have convinced him they were true?’ We’re carrying on his scientific legacy, and we’re doing things the way that he taught us.”

Day joined Inoue’s lab because they were both interested in evolution and the origin of chloroplasts. Chloroplasts evolved about a billion years ago from an ancient endosymbiotic relationship between a cyanobacterial species and a eukaryotic cell.

“I think Professor Inoue would have been pretty proud that we kept putting in the work, and that we did justice to those projects,” Day said.

‘A scientific citizen’

Laura Klasek completed her Ph.D. in June and joined Elemental Enzymes in St. Louis, Mo., as a research scientist, providing support to research and development efforts and regulatory efforts.

In July, at the annual summit of the American Society of Plant Biologists, or ASPB, she gave a presentation that comprised the bulk of her dissertation, on how molecular chaperones help proteins reach their final destination in the chloroplast. She discovered a previously unappreciated new role for these chaperones, one that had escaped detection in 30 years of study. Her paper is under review.

She spent two years in Inoue’s lab, studying the targeting of chloroplast proteins. She recalled she was 13 days short of taking her qualifying exams at the time of Inoue’s death, and faced a very tough decision about whether to proceed. Ultimately, she took her exams, then started making choices about continuing her project. It was its early stages, she said, “but I was invested in it.”

“And there was an element of wanting to honor the trust Kentaro had placed in me as a student and pushing the research forward,” Klasek said.

Inoue had an indelible impact on who she is as a scientist, she said. “He was also an enormous influence in how I consider myself in the broader scientific community, what makes a good scientific citizen.”

She also cited Inoue’s influence in her interest in mentoring. She serves on an ASPB early career committee in support of programming and research promotion for graduate students and postdoctoral students.

‘He prepared me well’

Lucas McKinnon, who worked under Inoue for more than three years, completed his studies in March, earning his doctorate in plant biology with a designated emphasis in biotechnology. was one of the first to show how the membrane environment can help to chaperone proteins to their final folded state that confers their activity.

In July, McKinnon joined Bayer Crop Science in Chesterfield, Mo., as a protein scientist in the regulatory science group; he is involved in safety assessment studies to support Bayer’s submissions of its genetically modified organism, or GMO, crop products to regulatory agencies around the globe.

“My work is similar to the types of things I did in both Kentaro Inoue’s lab and Steven Theg’s lab,” McKinnon said. “I did lots of protein work, purification experiments and biochemical analyses to study the biology of proteins and their properties, and I’ll be doing that at Bayer at a much larger scale.”

On working with Inoue: “Even though I was in grad school for another three years after he passed, I still feel like I was prepared enough to finish — he prepared me to graduate ... and I’m very thankful for that.”

In a tribute he posted online shortly after Inoue’s death, McKinnon wrote: “I have never known or worked with someone so dedicated to ensuring his/her students would be successful in the future. He relentlessly pushed us to be the best scientists we could be, and I am grateful to have worked with him.”