Solving the global food shortage is now a race against time, according to Kevin Hines, graduate student in Biochemistry, Molecular, and Cellular Biology (BMCB). Increases in yield rates for staple food crops such as rice and wheat have stalled or ceased in recent years, whereas the world’s population is increasing at an exponential pace. Most scientists today agree that if crop yields are not increased by at least 50 percent by the year 2050, severe food crises could become a common occurrence.

Hines works with the tobacco crop in the Maureen Hanson lab at Cornell University, attempting to increase the plant’s efficiency in turning atmospheric carbon dioxide into energy and biomass through photosynthesis. The ultimate aim, however, is to successfully transfer his implementation of the process to large-scale food crops, especially rice, wheat, and soy.

“Tobacco is a great model for our work; it bears significant similarities to staple food crops. If we find a way to improve tobacco’s photosynthetic efficiency, the hope is to be able to apply these techniques to food crops. Even if our work progresses at its current pace and if we’re successful, we are still likely a decade away from applying potential improvements to food crops on a large scale.”

From an Engaging Internship to an Exciting Research Lab

While working on his undergraduate degree at the University of Massachusetts, Amherst, Hines interned at the Boyce Thompson Institute as a member of the Hanson lab in the summer of 2012. During the internship, Hines worked on a project, expressing foreign cyanobacterial proteins in tobacco plants to observe if they would still form their complex structures in a new cellular environment. He characterizes his initial experience at the lab as engaging and intriguing. Upon acceptance into Cornell’s PhD program in BMCB, Hines joined the Hanson lab.

Increasing the Efficiency of Photosynthesis in Plants for Crop Yield

The Hanson lab’s current project involves engineering carboxysomes, complex multi-protein structures from cyanobacteria, into the chloroplasts of tobacco. RuBisCO, a major chloroplast protein, catalyzes the first step in the Calvin Cycle by converting atmospheric CO₂ into an organic molecule that is then converted to glucose, which can be used as energy or as the building blocks of cell walls. Many important crop plants, however, lack the ability to concentrate CO₂ around RuBisCO. As a result, the opposing process of oxygenation occurs simultaneously and renders the photosynthesizing process highly inefficient.

Carboxysomes, therefore, could increase photosynthetic efficiency in plants by concentrating CO₂ around RuBisCO, and this greater efficiency has the potential to increase crop yields in more than what would be possible by traditional plant breeding. Hines’ specific role in this process is to ensure the carboxysomes can work efficiently by removing enzymes known as carbonic anhydrases from the chloroplast before the carboxysome are inserted. If the anhydrases were to be present in the chloroplast, they would reduce the supply of CO₂ to the carboxysome and RuBisCO, negating any potential benefits.

“Instead of eureka moments or satisfying results, you’re often left with more questions than when you started.”

The Process and How It Works, Using CRISPR-Cas9 and the Gene Gun

The removal of anhydrases from the chloroplast is performed using the CRISPR-Cas9 system, an increasingly popular technology for genome modification. Upon encountering a virus, a bacterial cell eliminates the viral DNA, while simultaneously adding a part of the DNA to its own genome so that it can recognize the virus if it later re-enters the bacteria. The Cas9 protein can be paired with different custom RNA sequences to edit genes in any organism. Using Cas9, Hines creates deletions in tobacco’s carbonic anhydrase genes, thus preventing them from being expressed in its cells. Although Cas9 has recently been utilized for several controversial experiments, Hines asserts that the technology’s effectiveness has facilitated significant progress in genome modification.

The final step in the process involves inserting the carboxysome genes into the chloroplast’s genome. Hines explains that this process is executed with the gene gun, a biolistic particle delivery system. Miniscule gold particles are coated with carboxysome genes along with selectable markers, which help identify transgenic plants after transformation. The particles are then loaded into the gene gun and shot into tobacco seedlings. The gold particles are small enough to enter cell membranes and the chloroplast with minimal damage to the plant tissue. Interestingly, although the gun now utilizes compressed air to shoot the particles, its initial versions used gunpowder to execute the delivery.

The Excitement of Discovering the Unknown

Hines says his favorite aspect of research is frequently discovering what he considers unknowns through experimentation. “Classroom experiments are almost always predictable and are missing the ambiguities, which make research difficult, but also compelling.” For instance, he explains that he recently created a mutated tobacco line lacking two carbon anhydrases. Hines wasn’t expecting any drastic results, but the mutated plants are pale and shriveled at atmospheric CO₂ levels of approximately 400 parts per million (ppm) and only exhibit ordinary growth at highly elevated levels of around 9000 ppm. Why deleting these two genes causes such a strange phenotype is unexplained and now a major part of Hines’ dissertation.

The ambiguities, however, occasionally also irk Hines. “Research can be frustrating at times; science involves constant trouble shooting and repeating the same experiments in order to make them work. Instead of eureka moments or satisfying results, you’re often left with more questions than when you started. It can be a discouraging process.”

For this reason, he enjoys the occasional respite provided by teaching. At Cornell Hines has been a teaching assistant for Intro to Cell and Developmental Biology (BioMG1350) and Lab in Genetics and Genomics (BioMG2801). He says, “Being able to teach has been a great experience. I love talking about science, and it’s an awesome feeling to watch students come to understand the material.” Following the completion of his PhD, Hines plans to teach, alongside pursuing his research, at a research university.