Ancient Ferns Uncover A Hidden ‘Stress Memory’ That Let Plants Survive Drought

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Plants have thrived on land for hundreds of millions of years, and their success owes much to tiny pores called stomata (singular: stoma). Stomata are microscopic openings on the surfaces of leaves and stems, flanked by specialized cells called guard cells.

ABA-Driven Stomatal Closure in Fern Evolution

These pores act like adjustable valves, allowing plants to absorb carbon dioxide (CO₂) for photosynthesis while managing water loss through transpiration. In flowering plants, such as the well-studied Arabidopsis thaliana, stomata actively close during droughts through a hormone-driven process.

When water is scarce, the hormone abscisic acid (ABA) triggers a cascade of signals that cause guard cells to lose water and shrink, reducing turgor pressure (the internal water pressure that keeps cells rigid). This loss of turgor collapses the guard cells, closing the pore and preventing excessive water loss.

For decades, scientists debated whether older plant lineages, like ferns, share this sophisticated regulatory system. A groundbreaking study on the fern Ceratopteris richardii now reveals that active stomatal closure is not unique to flowering plants. Instead, it is an ancient trait rooted in shared genetic pathways, reshaping our understanding of plant evolution.

The research, published in bioRxiv in March 2021, tackles a long-standing mystery. While stomata exist in nearly all land plants (except liverworts), their responsiveness to environmental cues like ABA and humidity has been inconsistent in non-flowering species.

Some studies suggested that fern stomata close passively, relying on physical changes in leaf hydration rather than active signaling. Others hinted at ABA sensitivity but failed to explain why results varied across experiments.

The new study resolves these contradictions by demonstrating that fern stomata can actively close—but only after being “primed” by prior exposure to stress. This process, called sensitization, involves genetic and physiological changes that prepare stomata to respond rapidly to threats like drought.

By combining detailed experiments with advanced genetic analysis, the researchers uncovered conserved molecular pathways linking ferns to flowering plants, offering a glimpse into the deep evolutionary history of stomatal regulation.

The Role of Sensitization in Fern Stomatal Responses

To understand how fern stomata behave under stress, the researchers focused on Ceratopteris richardii, a fast-growing fern often used in laboratories due to its short life cycle and genetic tractability. Previous studies concluded that its stomata lack ABA sensitivity, but these experiments were conducted on plants grown in high humidity (around 95% relative humidity).

The new study hypothesized that such conditions might mask the fern’s true capabilities. To test this, the team grew ferns under two distinct environments: one group was kept in constant high humidity (“wet-grown”), while another was periodically exposed to drier air (“dry-pretreated”).

After three weeks, both groups were subjected to low humidity, and their stomatal responses were measured using microscopy and image analysis. The results were striking.

Stomata from dry-pretreated plants closed progressively over two hours, reducing pore area by 46% (from an initial average of 79.7% to 54.0%).

Adding ABA to the dry-pretreated plants accelerated this response, shrinking pores to 40% of their original size. In contrast, wet-grown plants showed minimal closure (only 11% reduction) and no response to ABA.

This demonstrated that prior exposure to stress—whether through low humidity or ABA treatment—is essential for active stomatal closure in ferns. Without sensitization, the stomata remain largely unresponsive.

Further experiments confirmed that ABA alone could prime the stomata. When wet-grown plants were pretreated with ABA (without prior humidity stress), their stomata closed rapidly upon exposure to low humidity, achieving a 51% reduction in pore area within 60 minutes.

Mock-pretreated controls, which received no ABA, showed only a transient 12% closure. These findings overturned earlier assumptions by showing that fern stomata do possess active closure mechanisms—but only when “trained” by prior stress.

Conservation of Ancient Genetic Pathways in Ferns

To uncover the molecular basis of these responses, the researchers turned to RNA sequencing (RNA-seq), a technique that measures gene expression by identifying and quantifying RNA molecules in a biological sample.

They compared the transcriptomes—the complete set of active genes—of sensitized (dry-pretreated or ABA-pretreated) and non-sensitized (wet-grown) fern fronds. The analysis revealed two key genetic signatures: one linked to sensitization and another to active closure.

First, the team identified a “sensitization signature” of 47 genes whose expression levels consistently changed during priming. Among these were homologs (genes shared across species due to common ancestry) of Arabidopsis genes involved in ABA transport and stress signaling.

For example, a homolog of AtPDR12, an ABCG transporter that facilitates ABA uptake in guard cells, was downregulated by 2.3-fold in sensitized plants. ABCG transporters are proteins that move molecules across cell membranes using energy from ATP.

Conversely, a homolog of AtMRP4, an ABCC transporter that regulates stomatal aperture independently of ABA, was upregulated by 1.8-fold. These changes suggest that sensitization alters the guard cells’ capacity to transport hormones and ions, lowering the threshold for stomatal responses. Next, the researchers analyzed genes associated with active closure.

By comparing transcriptomes of closing versus non-closing stomata, they identified 877 genes linked to the closure process.

Notably, many were homologs of Arabidopsis genes critical for stomatal regulation, including potassium channels (SHAKER), which control ion flux during turgor loss; calcium-dependent protein kinases (CPK), which mediate ABA and ROS signaling; and respiratory burst oxidase homologs (RBOH), enzymes that produce reactive oxygen species (ROS) to amplify closure signals.

These genes form part of the well-characterized ABA signaling network in flowering plants, implying that the core genetic toolkit for stomatal closure evolved over 300 million years ago, before ferns and flowering plants diverged.

Surprisingly, ABA application under high humidity did not alter the expression of closure-associated genes, even though it accelerated closure under low humidity.

This suggests that ABA’s role in ferns operates largely at the protein level—through post-translational modifications like phosphorylation (the addition of phosphate groups to proteins to activate or deactivate them)—rather than by turning genes on or off. Such mechanisms are also seen in flowering plants, where ABA activates pre-existing proteins to trigger rapid responses.

Ferns and Flowering Plant Evolution

The study’s most profound conclusion is that the genetic machinery for active stomatal regulation predates the split between ferns and flowering plants. This challenges the notion that sophisticated stress responses are recent innovations.

Instead, early land plants likely possessed rudimentary versions of these pathways, which were later refined in different lineages. For instance, homologs of SLAC1 and OST1—genes encoding an anion channel and a kinase critical for ABA signaling in Arabidopsis—were present in the fern transcriptome.

SLAC1 facilitates the release of negatively charged ions (like chloride) from guard cells, driving water loss and closure, while OST1 activates SLAC1 via phosphorylation. While their exact roles in ferns remain to be validated, their conservation hints at deep evolutionary roots for ABA-driven closure.

The findings also reconcile conflicting reports about stomatal behavior in non-flowering plants. For example, earlier studies on the lycophyte Selaginella found passive closure in some experiments but active responses in others.

The new research suggests these discrepancies arose because sensitization was overlooked. Without prior stress exposure, stomata in basal plants may appear unresponsive, masking their true capabilities.

Universal Plant Stress Adaptation Mechanisms

The conditional nature of fern stomatal responses mirrors a phenomenon called acclimation in flowering plants. Acclimation refers to the ability of plants to adjust their physiology to cope with recurring environmental stress.

For instance, Arabidopsis stomata become more sensitive to ABA as leaves mature, enabling better drought tolerance. Conversely, prolonged exposure to high humidity can reduce closure capacity, even in the presence of ABA. Both processes involve adjusting the guard cells’ sensitivity to stimuli, likely by modulating receptor abundance or signaling thresholds.

In ferns, sensitization appears to work by “rewiring” guard cell physiology. Downregulating inhibitors like GRDP1 (a negative regulator of ABA responses) may heighten sensitivity to stress signals, while upregulating transporters like MRP4 could alter ion gradients to prime the cells for rapid action.

These changes collectively lower the threshold for stomatal closure, allowing ferns to respond swiftly to fluctuating environments—a trait that may have been crucial for early land plants colonizing harsh habitats.

Pioneering Genetic Analysis in Ferns Stomata

A key strength of the study lies in its technical rigor. Ferns lack the genomic resources available for model plants like Arabidopsis, making large-scale genetic analysis challenging.

The researchers overcame this by assembling a high-quality de novo transcriptome—a comprehensive catalog of genes expressed in C. richardii, constructed without relying on a reference genome.

This transcriptome achieved 64.5% completeness based on Benchmarking Universal Single-Copy Orthologs (BUSCO), a tool that assesses the quality of genome or transcriptome assemblies by comparing them to conserved genes expected in a species. This score surpassed the fern’s published genome, demonstrating the robustness of the data.

Orthology mapping, which identifies evolutionarily related genes across species, revealed that 49% of fern genes had direct counterparts in Arabidopsis. This allowed the team to link fern stomatal responses to well-characterized pathways in flowering plants, bridging a 300-million-year evolutionary gap.

For example, homologs of Arabidopsis calcium sensors and ROS-producing enzymes were found in the fern’s closure-associated gene set, suggesting conserved roles in stress signaling.

While the study answers many questions, it also opens new avenues for research. For example, fern genomes may contain duplicated copies of key genes (e.g., SnRK2 kinases), creating functional redundancy (where multiple genes perform similar roles, masking the impact of single-gene mutations).

Additionally, recent work shows that fern stomata open in response to blue light via specialized photoreceptors called cryptochromes. How these light-sensing pathways interact with ABA signaling remains unknown.

Future experiments could use CRISPR-Cas9, a gene-editing tool, to disrupt specific genes in C. richardii, testing their roles in stomatal regulation. Comparative studies across fern species—including ancient lineages like eusporangiate ferns (which have larger, more complex spore structures)—could reveal how these mechanisms diversified over time.

Furthermore, understanding stomatal evolution has practical implications. As climate change intensifies droughts and heatwaves, engineering crops with enhanced stomatal regulation could improve water-use efficiency and yield stability. Ferns, with their ancient stress-response pathways, might offer novel genes for this purpose.

For instance, overexpressing AtPDR12 homologs in crops could enhance ABA uptake, improving drought tolerance. Beyond agriculture, the study underscores the importance of conserving plant biodiversity.

Ferns and other non-flowering plants are often overlooked in conservation efforts, yet they hold genetic secrets critical for understanding plant adaptation. By studying these “living fossils,” scientists can unravel how life coped with past environmental shifts—and apply those lessons to modern challenges.

Conclusion

The discovery of active, condition-dependent stomatal closure in Ceratopteris richardii reshapes our understanding of plant evolution. It reveals that the genetic toolkit for stomatal regulation predates the split between ferns and flowering plants, highlighting the ingenuity of early land plants. These findings bridge gaps between physiology, genetics, and evolutionary biology, offering a unified narrative of how plants conquered land.

As we face a climate-altered future, such insights are more than academic curiosities. They provide a roadmap for engineering resilient crops, conserving biodiversity, and unraveling the mysteries of life’s adaptation to Earth’s ever-changing environments. In the humble stomata of ferns, we find echoes of ancient survival strategies—and hope for modern solutions.