Subtle alterations in RNA splicing fine-tune how genes regulate drought tolerance in plants according to new research from Hasna Khan in the Provart lab. This exposes a new way to understand drought response that complements the usual focus on gene expression levels. These findings are published in The Plant Journal as “Differential splicing fine-tunes guard cell gene expression and is required for drought tolerance in Arabidopsis thaliana”
Revealing another layer of regulation
Professor Nicholas Provart studies how plants respond to environmental stress. As climate change increases the frequency and severity of drought, understanding how plants conserve water has become more important.
While many studies focus on which genes become active during drought, Provart and PhD student Hasna Khan suspected another layer of regulation might be hidden within guard cells, the specialized cells that control tiny pores on leaf surfaces.
“We started asking whether we could look at whether expressed gene transcripts were spliced into distinct mRNAs within the same cell”, Khan recalls. “I don’t think either of us expected it to become as complex a project as it did.”
Guard cell-specific changes in drought response
Guard cells play a critical role in drought response. By opening and closing stomata, they regulate both carbon dioxide uptake and water loss. “Drought happens slowly as rains falter and soil dries,” explains Provart. “We anticipated fine-tuning in gene activity in guard cells as drought progresses, in addition to the more widely studied fast responses.”
A previous student in the Provart lab, Dr. Anna van Weringh, spent years developing methods to isolate RNA specifically from guard cells to test this hypothesis. The work presented substantial technical challenges because guard cells represent only a small fraction of the cells within a leaf.
“Getting enough guard cells that you could do RNA work, while also working quickly enough to avoid RNA degradation, was a really fine balance,” Khan says. “At times, it felt like I was elbows deep in guard cell RNA,” she adds with a smile.
Whereas van Weringh focused on changes in gene expression in guard cells from drought-affected plants, Khan analyzed the RNA-seq data to identify different splicing patterns using multiple bioinformatic pipelines. She then validated these results experimentally.
Khan discovered widespread changes in alternative splicing as drought progressed. Many of these changes were specific to guard cells and could not be detected when examining whole-leaf tissue.
“That is really one of our coolest findings,” says Khan. “When we see these guard-cell specific changes, it highlights how import it is to focus on this cell type.”
Drought-responsive alternative splicing required for optimal drought response
This insight suggest that alternative splicing acts as a second layer of drought-responsive gene regulation. While immediate drought responses rely on rapid signaling pathways, alternative splicing may allow plants to gradually adjust their physiology as drought conditions worsen.
Among dozens of candidate genes, SAFE1 emerged as particularly intriguing. This gene reproducibly showed drought-responsive alternative splicing in guard cells but maintained constant gene expression. This allowed the researchers to isolate the effect of splicing from changes in gene activity.
In an impressive confirmation of its importance, when SAFE1 was locked into one splice configuration the plants were more drought sensitive. “That really drove home the idea that this drought-responsive alternative splicing is required for an optimal drought response,” Khan says.
Avenues for new research
This discovery raises new questions about why plants use alternative splicing to regulate drought responses. One possibility is that alternative splicing provides a way to fine-tune the activity of genes with multiple roles throughout the plant, allowing guard cells to respond to drought without affecting other tissues.
Building on these findings, Khan’s next steps will focus on investigating drought-responsive alternative splicing in tomato guard cells. By exploring whether similar mechanisms exist in crop species, this research could help scientists better understand how plants adapt to water stress and potentially inform future strategies for improving drought resilience in agriculture.