By Ashley Stender | July 30, 2025

When plants face drought, heat, and other stressors, they activate a complex network of genes to survive. These genetic responses include the production of specialized metabolites, which are compounds that help defend, signal, or coordinate the plant’s biochemical response to stress. But which genes switch on these pathways, and how does the plant decide when and where to make them?

C-SPIRIT’s Aim 3: Gene and Pathway Discovery explores how plants control the production of stress-responsive compounds at the genetic level. By identifying which genes turn on, where, and under what conditions, Aim 3 helps explain how plants manage chemical responses to their environment. This research serves as a bridge between discovering promising compounds and harnessing them for real-world agricultural use.

Where Gene Control Meets Chemical Complexity

Dr. Olivia Wilkins, a systems biologist at the University of Manitoba, is the lead of Aim 3 and coordinator of the Canadian branch of C-SPIRIT. Her lab brings over 15 years of experience in stress assays and regulatory modeling, with a focus on how plants coordinate gene activity under environmental stress.

“That fits with what this Aim is trying to do: figure out who’s turning the knobs when a plant starts producing these compounds,” Wilkins says.

Her group’s deep expertise in transcriptional regulation made them a natural fit for Aim 3’s goals. However, linking gene activity to metabolite accumulation introduced a new layer of complexity. Addressing this challenge required learning how to integrate metabolomic data with gene expression models, which involves mapping when and where key compounds appear in response to stress.

“We’ve generally been really focused on the transcriptional regulation of these responses,” Wilkins says. “Connecting that to metabolite accumulation is new, but a really exciting direction for us.”

She also credits her involvement in the Plant Cell Atlas with shaping the systems-level perspective she brings to the project. The initiative fostered both collaborations and a systems-thinking mindset that now support Aim 3’s multidisciplinary scope.

Mapping the Signals That Trigger Metabolite Production

Aim 3 investigates how plants switch on specialized metabolite production under stress, including the genes and regulatory pathways that control these processes.

“Our aim is trying to understand the regulatory mechanisms contributing to secondary or specialized metabolite production,” Wilkins explains. “We’re trying to piece together what genes are responsible, under what environmental cues, and in which tissues or cell types.”

Because many of these compounds appear only in specific cell types or developmental stages, Aim 3 also investigates where and when gene activity occurs. These insights can help explain, for example, how stress triggers protective chemistry in the roots of cassava or the leaves of tomato.

Modeling this regulation presents significant challenges, especially when using sparse single-cell data. “There’s still a lot of ground to cover,” Wilkins says. “When you’re working with single-cell data, you’re only capturing a small portion of the transcriptome. Trying to predict a global network from such sparse data is a huge challenge.”

To meet this challenge, Aim 3 is generating both single-cell and bulk transcriptomic datasets and linking them to metabolomic profiles from Aims 1 (Metabolite Discovery) and 2 (Metabolite Annotation and Database Development). “Already having purpose-built datasets that include metabolite information is something that’s still fairly rare in the plant world,” Wilkins says. “We hope it becomes a valuable resource.”

She also emphasizes the importance of making the data accessible. “We’re not just generating data. We’re thinking about how to structure it so that it’s reusable,” she adds. “That’s something the plant field really needs more of.”

Modeling Gene Regulation with Multilayered Data

To explore gene regulation in crops like maize, soybean, and pennycress, Aim 3 is building computational pipelines that combine public datasets with new C-SPIRIT-specific data. Initial models use bulk RNA-seq and chromatin accessibility measurements, with plans to add metabolite profiles and stress-response time series from multiple species as they become available

They are also integrating large language models into their workflow to extract enzyme-substrate and gene-regulatory relationships from the literature, a method that accelerates data curation and helps identify new candidates for downstream testing. 

Aim 3 is also collaborating with Gaurav Moghe, who leads Aim 2, and his lab. Moghe’s group originally developed a tool (FuncFetch) to link enzymes with their targets. Now, it is being adapted to map connections between transcription factors and the genes they regulate.

Researchers in the United Kingdom, Republic of Korea, and Japan are contributing expertise in biosynthetic gene clusters. Although microbial and plant systems rely on different strategies, comparing methods across these domains is leading to new insights.

Visualization is another major focus. Nick Provart’s lab at the University of Toronto, part of the C-SPIRIT Canadian team, is also part of developing tools that allow researchers to explore gene networks, expression data, and metabolite accumulation through interactive displays.

“A big part of Aim 3 is making these data accessible to people who didn’t generate them,” Wilkins says. “That’s how we build community and accelerate discovery.”

Together, these collaborations are laying the groundwork for targeted engineering strategies, especially in crops where stress resilience has direct implications for food security.

Translating Gene Insights into Synthetic Biology

Aim 3 outputs play a key role in informing the work of Aim 4: Biological Synthesis of Bioactive Compounds. By modeling when and where certain compounds are produced in crops like potato, maize, and tomato, Aim 3 helps determine how to reconstruct those pathways in plant or microbial systems.

“Plants produce all sorts of things, and not all of them are what we’re looking for,” Wilkins explains. “Understanding which cells produce which compounds and under what conditions is critical to redirecting production in a synthetic context.”

This kind of targeted application depends on coordination across the full C-SPIRIT research pipeline. Accurate timelines and shared outputs ensure that results from Aim 3 can be directly used in downstream engineering efforts.

Wilkins also points to the value of C-SPIRIT’s integrated design. The project integrates diverse data types to better understand how crops respond to real-world stress conditions. “In Canada, we rarely have the resources to generate this scale of data. Time series, stress treatments, genetic diversity—it’s all being brought together in one project. That’s special.”

These systems-level insights have important agricultural implications. If researchers can predict how specific genes respond to environmental stress, they can help develop crops that are more resilient, productive, and tailored to the challenges of a changing climate.

Turning Genetic Models into Agricultural Impact

Looking to the future, Wilkins is excited about the growing momentum across the Aims. From transcriptomics and metabolomics to network modeling and visualization, C-SPIRIT is building a foundation that researchers can use to understand and improve crop resilience.

As new collaborators join and new data becomes available, Aim 3 continues to evolve. “We’re still learning,” Wilkins says. “But the pieces are coming together. And we’re doing it in a way that feels very intentional.”

Ultimately, the goal is to build a model that can predict and manipulate how plants respond to stress, and apply that model to economically important crops such as maize, soybean, and rice. “The dream is a toolbox you can use to predict and tweak metabolite production in response to real-world stress,” Wilkins says. “And to do it in a way that is informed by evolution and grounded in real biology.”

Reflecting on the collaborative nature of the project, Wilkins expresses her appreciation for the depth and diversity of expertise within the C-SPIRIT network: “I’m excited to be part of something this broad. It feels like a real privilege.”

With its emphasis on collaboration, regulatory insight, and translational potential, Aim 3 helps bridge the molecular basis of stress responses with the future of resilient agriculture.