How a moss keeps its leaf-like organs flat

A thin blade, a complicated task

The moss structures are called phyllids. They evolved their flattened shapes independently of the leaves of vascular plants. Their single-layered blades let researchers investigate coordination within a sheet of cells without the added complication of mechanically different layers growing against one another.

Flatness does not require identical growth everywhere. Developing phyllids have organized gradients: cells progressively stop dividing and expanding from the tip toward the base. The question is how cell division and expansion remain coordinated well enough to produce a reproducible shape.

The team focused on callose, a sugar-based polymer that temporarily helps build the partition between dividing plant cells. During cytokinesis, the final stage of cell division, a structure called the cell plate expands outward until it joins the existing wall. Callose supports the deposition of other wall components during this process.

Disrupting the construction process

The researchers stained developing moss tissue to locate callose. Early on, the fluorescent signal appeared along walls throughout emerging phyllids; later, it was concentrated near the base, where division remained active.

They then used CRISPR-Cas9 to disrupt CALS6, a gene encoding a callose-producing enzyme. Among five callose synthase genes detected in the tissues examined, CALS6 had the highest expression. Independently generated mutant lines showed similar developmental defects, supporting the link between loss of this gene and the altered organs.

The mutants produced shorter phyllids with irregular outlines and crinkled margins. Cell-by-cell reconstructions showed that the normally flat blade regions had become uneven. Instead of orderly patterns of cell size and curvature, the mutant tissue contained patches of small and large cells and more variable curvature.

Microscopy also revealed gaps in the walls separating mutant cells. Most measured gaps were smaller than 5 micrometers, although some exceeded 30 micrometers. Walls were thicker on average and more variable in thickness. Additional fluorescent labeling suggested that some groups of mutant cells had lost membrane integrity.

Watching growth become patchy

To follow the defects as they developed, the team imaged the same phyllids every 12 hours and reconstructed cell lineages. This allowed them to measure division and expansion rather than infer growth solely from mature shapes.

At two days after initiation, mutant organs were comparable in size and shape to controls. Their overall developmental duration also remained approximately 5.5 days. What changed was the activity within that period: the growing region was smaller and closer to the base, fewer cells divided or expanded, and growth rates were lower.

Growth also became more uneven within the active region. The mutants therefore did not simply finish development sooner; they grew less, and in a more disrupted spatial pattern, during a similar developmental window.

Why location and timing matter

Different mutant phyllids developed different shapes, even on the same shoot. The researchers used a computational model to ask whether local growth arrests could explain this variability.

In simulations, dispersed arrested cells reduced organ size but largely preserved the overall outline. Arrests imposed earlier had greater effects than equivalent later arrests. Stopping a central group of cells instead produced pronounced constrictions that worsened as simulated development continued. These outcomes qualitatively resembled patterns observed in mutant tissue.

The model was not a literal reconstruction of every damaged cell. Its arrested cells stopped growing and dividing and were assigned 100 times the stiffness of other cells. That stiffness was a modeling assumption, not a reported measurement of mutant cells. The model was also two-dimensional: its shape changes should not be confused with a direct simulation of the observed three-dimensional crinkling.

One mismatch was informative. Simulated arrests at the margin produced strong indentations, but real mutant margins did not consistently do so. Neighboring cells appeared to expand toward the arrested region, suggesting partial compensation. Mechanical feedback is a possible explanation, not an established mechanism.

The study, available as a bioRxiv preprint, has not been peer-reviewed. Its live-growth comparisons involved three control and four mutant phyllids. Within that evidence, it connects disrupted cell-wall completion with disorganized growth and loss of flatness—and shows why the arrangement of growth defects can matter as much as their presence.