In preprints: shrinking boundary cells reveal fluid flux in organogenesis

Water is the most abundant molecule in living organisms and makes up the largest fraction of fresh weight and volume in almost all cell types. It provides the aqueous environment, and sometimes acts as a substrate, for many biochemical reactions in and outside the cells. Water uptake also contributes to volume changes during cell expansion and lumenogenesis, often compared to filling up a balloon, and can subsequently generate forces in the forms of fluid pressure gradient, propellant, and tissue tension (reviewed by Li et al., 2020; Chan and Hiiragi, 2020). Although water flux is generally recognized in fast fluid flows, such as blood circulation and plant sap movement, the kinetics of water distribution are often overlooked in slower processes, such as growth and morphogenesis. To challenge this oversight, Alonso-Serra and colleagues studied in detail cell expansion and, surprisingly, shrinkage in the shoot apical meristem (SAM) of the model plant Arabidopsis thaliana, and proposed that water fluxes can explain the intricate growth patterns during organogenesis (Alonso-Serra et al., 2023 preprint).The biomechanical basis of plant cell expansion is heavily centred around osmosis, whereby absorbed water fills up the cell and generates an intracellular pressure called ‘turgor pressure’ higher than the environment. Turgor pressure stretches the cell wall to generate mechanical stress that elongates the cell wall both reversibly, through elastic deformation, and irreversibly, through cell wall yielding and synthesis. The combined reversible and irreversible cell wall deformations underlie plant cell expansion (reviewed by Ali et al., 2023). Although one hallmark of plant growth is its irreversibility, a handful of shrinking cells were repeatedly noticed in the Arabidopsis SAM (Willis et al., 2016; Long et al., 2020). To confirm the existence and the distribution of the shrinking cells, Alonso-Serra and colleagues conducted a comprehensive investigation by first examining the precise cell volumetric changes throughout all cell layers in the growing Arabidopsis inflorescence SAM over time. The analysis revealed that, between the meristematic cells and the rapidly expanding organ primordial cells, the boundary cells progressively shrunk and appeared ‘compressed’ with reduced sphericity as the boundary deepened. To eliminate potential artefacts from fluid leakage by dissection, the authors further confirmed the presence of shrinking cells in the intact SAM recovered from N-1-naphthylphthalamic acid treatment, which blocks organogenesis, and found that recovered primordial outgrowth triggered cell shrinkage both in the deepening organ boundary and the outer flank of the primordia. This implies that shrinking cells are associated with tissue folding and the rapidly expanding cell neighbours.To understand better how organ outgrowth affects the neighbouring cells, particularly those in the boundary, the authors simulated the biomechanical dynamics of cell wall expansion and fluid redistribution by utilising a multicellular extension of the classic Lockhart–Ortega model (Cheddadi et al., 2019). Simply, local cell wall softening triggers cell wall expansion and tissue outgrowth, which creates more intracellular space that must be filled by water influx. This primordial-like outgrowth effectively turns the tissue into a water ‘sink’, rapidly drawing water from adjacent cells. The water ‘source’, the xylem, is, however, far from the rapid outgrowth, which puts the cells adjacent to the primordium in a challenging situation between high water demand of the ‘sink’ and limited water access from the ‘source’. With less water supply and heavier depletion, the primordium-flanking cells thus experience a net water loss, shrink and fold inward to become organ boundaries.This model prediction elegantly highlighted that the dynamics of fluid fluxes, or ‘tissue hydraulics’, may not simply be a passive response to growth but an active, regulated process that influences tissue patterning and development. One interesting observation is that the simulations expanded by both reversible and irreversible cell wall deformations, but shrinkage only occurred through reversible, elastic deformation. This is supported by the observation that the simulated shrinkage is always smaller than the imposed elastic strain threshold and implies that the significant shrinkage measured in experiments, precisely 10.1±4.6% over a 12-h period, is a result of elastic deformation. This suggests that Arabidopsis SAM cells are very elastic and may have a higher yielding threshold than previously expected.In an attempt to visualise the predicted hydraulic pattern, the authors tracked the intensity of 8-hydroxypyrene 1,3,6 trisulphonic acid (HPTS), a water-soluble dye impermeable to membranes as a proxy for water, to provide indirect evidence of water movement in the SAM. The pattern of HPTS distribution revealed preferential staining of fast-growing cells in younger primordia, and consistent exclusion from deep boundaries. This demonstrated that HPTS level is tissue specific; however, the interpretation could be complex. Since HPTS concentrates in the primordia, it suggests that water must carry HPTS to the cells and then leave to ‘condense’ the dye, thus it may not capture the complete water fluxes in SAM. If HPTS intensity is enriched in water ‘sinks’, then it is surprising to find a lower HPTS signal in, for example, the fast-growing primordium 7 (P7), and a higher signal in the meristematic cells in front of the P7 boundary that was not a predicted ‘sink’. In short, it is difficult to distinguish influx from retention or outflux from dilution. Additionally, HPTS has been used as an intensity-based pH indicator that preferentially stained the acidic vacuoles in SAM; it remains unclear how accurately vacuolar HPTS staining represents water movement within plant tissues.Finally, the authors demonstrated that genes responsive to osmotic stress and salt stress were preferentially enriched in SAM boundaries, indicating that boundary cells may interpret shrinkage as a highly localised hyperosmotic stress. This aligns with the group's previous finding of nuclear compression and stress-induced chromatin remodelling in boundary cells (Fal et al., 2021), and suggests that single-cell abiotic stress response may be an intrinsic component of differentiation. Further validation of stress-responsive pathways in boundary formation is required to unravel the full implications of cell shrinkage during plant development.This study highlights the complex interplay between growth-induced deformation, biomechanical stress patterns, and hydraulic dynamics in plant morphogenesis. Deformation caused by growth not only affects local stress patterns but also intricately influences hydraulic patterns, which are crucial in determining tissue boundaries. Together with the group's recent discovery of cell shrinkage in a transcription machinery mutant (Trinh et al., 2023), this challenges the paradigm that plant cell expansion is irreversible. The findings highlight the role of water as a pivotal patterning factor, which works in synergy with mechanical and biochemical cues to sculpt tissue identity. To consolidate this proposition, it will be exciting to see further explorations of: (1) Poisson ratio of the boundary cells based on their aspect ratio changes to estimate their ‘compressibility’; (2) plasmolysis or other methods to estimate the yield threshold in the boundary; (3) correlation between cell shrinkage, nuclear compression and transcriptional changes to interpret boundary hydraulic status; and (4) differential intensity analysis of HPTS or other pulse-chase tests to determine the hydraulic flow in the SAM.Looking ahead, including tissue hydraulics as an emergent property should improve model predictions for plants, animals or other multicellular structures that are ‘fluid-filled’, especially where folding and alternate fast-slow growth patterns are found.