Mechanical forces and cell shape guide how plant stomata form

Sarah Robinson's research team has uncovered how the interplay between cell shape and mechanical stress influences the orientation of stomata (microscopic pores on the leaf surface) during early plant development.

Stomata, from the Greek word for mouth, are essential gateways that regulate the exchange of carbon dioxide and oxygen, as well as water loss.

The stomatal density and how efficiently stomata open and close in response to stress strongly influences how effectively plants capture carbon dioxide for photosynthesis and limit water loss during drought.

In the longer term, a better understanding of stomata could help researchers design crops with improved water-use efficiency and resilience to drought.

Stomata structure: Stomata are microscopic pores on the surface of leaves and stems that regulate gas exchange and water loss by opening and closing through the action of specialised guard cells.

Stomata development

Each pore is formed by a pair of specialised guard cells that open and close in response to environmental cues, playing a central role in plant survival and productivity.

While researchers have long known of the key genetic regulators that control stomatal development, including transcription factors SPEECHLESS and MUTE (which act sequentially, to initiate and terminate the stomatal cell state) it remains unclear what determines the orientation of the stomata on the leaf.

Using the model plant, Arabidopsis thaliana, researchers from Dr Sarah Robinson’s research group at the Sainsbury Laboratory Cambridge University (SLCU) combined live imaging and computational modelling to investigate how stomata are orientated during early leaf development.  The research is published in Cell Reports.

The team tracked more than 10,000 stomata from 72 embryonic leaves (cotyledons) over the first five days after seed germination.

“Our results show that stomatal divisions are strongly guided by the geometry of the cell,” said Dr Leo Serra, who is the first author of the study. “In most cases they align with the long axis of the cell, which is unusual when compared with many other plant cell types.”

However, this did not explain the organ scale alignment of stomata they observed. The team found that cell shape is only part of the story.

Mechanics comes into force

By modelling growth patterns and experimentally altering mechanical forces within the young plants, the researchers demonstrated that when tensile strength was generated as the leaf expanded, it influenced the orientation of guard cell divisions.

“When we altered the mechanical forces applied to the leaf we saw clear changes in stomatal division orientations,” said Dr Euan Smithers, who conducted the modelling. “This suggests that mechanical stress might override geometric cues.

Changes in the tensile stress pattern shift the orientation of stomata divisions: Figure G–H: Computational modelling of differential growth and predicted stress orientation in the cotyledon. (G) Distribution of growth rates applied to the cotyledon simulation, based on the cotyledon shown in panel D and two additional biological replicates. Simulated growth rates differ between the abaxial and adaxial surfaces, with higher growth rates on the adaxial side. Colours indicate relative growth rate across the cellular mesh. (H) Predicted orientation of differential growth-derived tensile stress on the abaxial and adaxial cotyledon surfaces. Solid lines indicate the principal direction of tensile stress and dashed lines indicate the minimal tensile stress direction. Colours represent stress anisotropy, with higher anisotropy shown in yellow and lower anisotropy in purple. Scale bar: 50 μm. Modelling by Euan Smithers.

“We found that stomatal divisions tend to align with the long axis of the cell but can be influenced by mechanical stress,” said Dr Robinson. 

“While stomatal divisions show a strong alignment with the cell’s geometry, mechanical perturbation confirmed the influence of tensile stress on stomata division orientation.” 

The researchers also discovered striking differences between the adaxial (upper) and abaxial (lower) sides of the leaf. 

These two sides grow at different rates (the adaxial side growing faster than the abaxial side), creating distinct patterns and levels of tensile forces across the leaf surface.

As a result, stomatal orientation varies over time and between surfaces.

In the abaxial side (where stomata are more abundant) divisions closely align with the leaf axis, but become more variable as development progresses.

In contrast, on the adaxial side the stomatal orientation becomes disorganised much earlier. 

Stomata orientation: Heat maps showing stomatal orientation relative to the leaf proximodistal axis on the abaxial and adaxial sides of Arabidopsis thaliana cotyledons at 1, 2 and 5 days after germination (DAG). Each line represents a stoma. On the stomata-rich abaxial side, orientations initially align with the leaf axis before becoming more variable over time, whereas stomatal orientation on the adaxial side becomes disorganised earlier in development. Scale bar: 200 μm. Imaging by Leo Serra.

What causes the differences in alignment between adaxial and abaxial sides?

Dr Robinson says these differences most likely come from differential growth between the two sides of the leaf.

“Faster growth on the adaxial side leads to greater tensile stress relaxation, while slower growth on the abaxial side maintains more consistent tensile stress patterns, contributing to more coordinated stomatal alignment,” she said. 

Cotyledon shape: Light-sheet images showing cotyledon opening and the development of distinct mechanical stress patterns between the adaxial and abaxial sides. (A) Seedlings imaged at 1 and 2 days after germination (DAG). Dashed lines indicate the positions of the optical transverse sections shown in panels B and C. Scale bar: 200 μm. (B) Optical transverse section of a cotyledon at 1DAG showing the adaxial and abaxial sides. Scale bar: 100 μm. (C) Optical transverse section of a cotyledon at 2DAG showing increased curvature and differential growth between the two sides of the cotyledon. Scale bar: 100 μm. Imaging by Leo Serra.

Differential growth between the two sides of the cotyledon: (D) Heatmaps showing the growth ratio distribution across the two sides of the same cotyledon over time, imaged between 1 and 2 DAG (scale bar: 100 μm). (E) Growth ratio distributions for both cotyledon sides shown in (D) (replicate 1), alongside two additional biological replicates. (F) Heatmap of abaxial–adaxial cell length distribution in a transverse cotyledon section at 1 DAG, reconstructed from 3D segmentation of an entire cotyledon (scale bar: 50 μm). Imaging by Leo Serra.

Visualising stress patterns using mutants with weakened cells adhesion

To disentangle the relative contributions of geometry, growth, and mechanical stress, the team used time-lapse imaging and advanced image analysis to track cell lineages and quantify division orientations. They used mutant plants with weakened cell adhesion, allowing stress patterns to become visible as cracks on the leaf surface. 

Identifying tensile stress: To experimentally test the model prediction, we imaged cotyledons of the quasimodo2-2 cell adhesion defect mutant, which forms cracks between epidermal cells. The localisation of these cracks has previously been used to infer the main direction of tensile stress in the plant epidermis. Differential localisation of cell adhesion defects on the two sides of cotyledons at 2 days after germination. Arrowheads indicate small cracks on the adaxial epidermis.Imaging by Leo Serra.

Applying mechanical stress by leaf bending

It can be challenging to apply mechanical stress to very small tissues.

"We found a way to bend the leaves to cause a change in the stress patterns at the surface of the leaf. We found that this was sufficient to alter the orientation of the stomata," said Dr Serra.

Dual influences on stomata orientation

Their analyses revealed that, although stomatal divisions are most often aligned with the long axis of the cell, they can also align with predicted patterns of organ-scale tensile stress, particularly in cells with more isotropic shapes.

This finding is consistent with observations in other plant tissues, where cell divisions often follow the direction of maximal mechanical stress.

“Geometry appears to be the dominant factor guiding stomatal orientation, but mechanical stress can take over in certain contexts,” said Dr Robinson. “This dual control may help coordinate cell behaviour across the tissue.”

Although the study did not directly test how stomatal orientation affects function, the researchers suggest aligning stomata with mechanical stress patterns may optimise how efficiently pores open and close.

“Since stomata themselves generate mechanical forces on neighbouring cells, sensitivity to stress may also help position new stomata in ways that improve overall performance,” Dr Robinson said.

“Our work highlights how mechanical context shapes biological outcomes. There is still a lot to learn about how plants generate patterns that can span across whole organs. 

Reference

Leo Serra, Euan T. Smithers, Lucy Bentall, Martin O. Lenz, Sarah Robinson (2026) Geometry and mechanical stress influences stomata division. Cell Reports.

DOI: 10.1016/j.celrep.2026.117366

Naturally occurring and experimentally induced stomatal division orientations are influenced by tissue geometry and are biased toward the organ axis independently of cell growth.
(A) Time-lapse images of PM-YFP cotyledons showing new stomatal divisions occurring between 1 and 2 days after germination (DAG). Newly formed stomata are highlighted in magenta at 2DAG; inset images show representative patterns on the abaxial and adaxial sides. Scale bar: 100 μm.
(B) Time-lapse images of iMUTExPM-YFP cotyledons showing induced stomatal differentiation divisions between 1 and 2DAG. Clones present at 1DAG are outlined in magenta, and newly formed cell walls at 2DAG are highlighted in green. Scale bar: 100 μm. Imaging by Leo Serra.