Genetic basis of phenotypic evolution

Group leader: Angela Hay

Research project:

Microtubule dynamics in exploding seed pods

Exploding seed pods of the common weed Cardamine hirsuta have the remarkable ability to launch their seeds far from the plant. The energy for this explosion comes from tension that builds up in the fruit valves. Structural elements of the valve fail above a critical threshold, causing the valves to rapidly coil and accelerate the seeds at speeds greater than ten meters per second.
We found that the gradual build-up of tension in these fruits is achieved by active contraction of the outer exocarp layer of the valve. Since the inner layer of the valve is stiffened by lignin, continued contraction of the exocarp cell layer produces more and more tension in the fruit. We recently showed that microtubule dynamics are essential for the patterns of cellular growth and cell wall anisotropy that determine this contraction.

Microtubules are highly dynamic filaments that form ordered arrays beneath the plasma membrane. During growth, these cortical microtubule arrays guide the movement of cellulose synthase to orient the extrusion of cellulose microfibrils into the cell wall. The orientation of these load-bearing microfibrils largely determines the direction of cellular growth. Exactly how microtubule arrays reorient during growth is unknown. Rather than moving individual microtubules, arrays reorganize based on fundamental properties of how microtubules are assembled or disassembled. As such, cortical microtubule arrays have self-organizing properties arising from rules governing the outcomes of encounters between microtubules and with the geometry of the cell. These properties of a self-organizing system are amenable to computational modelling, and the Coen lab (JIC) has been developing modelling approaches to study microtubule dynamics in relation to growth.

In C. hirsuta fruit, we found that a developmental switch in microtubule orientation, coupled to microfibril reorientation and a consequent change in growth pattern, was critical for exocarp cells to actively contract and generate tension in the fruit valve. Therefore, we aim to understand how exocarp microtubule arrays reorient. To do this, we will follow an interdisciplinary approach, using quantitative imaging, genetics and computational modelling in collaboration with the Coen lab (JIC).

Objective 1: Reconstruct switch in microtubule orientation.
Time-lapse confocal microscopy of fruit exocarp cells expressing a fluorescent microtubule reporter will be used to acquire detailed images of cortical microtubules. Microtubule arrays switch orientation from transverse to longitudinal in these cells at a well-defined stage of fruit development, which is accessible to imaging in growing fruit. Image analysis tools will be used to quantify microtubule dynamics in relation to cell geometry. This data will inform computational modelling efforts in the Coen lab, JIC, to reconstruct a switch in microtubule orientation based on the dynamics of microtubule catastrophe and nucleation. Iterations between modelling and imaging will be used to converge on a model that best explains how microtubule arrays reorient during exocarp cell growth.

Objective 2: Test model predictions using genetic perturbations.
To test predictions from this model, we will use mutants and transgenic lines to perturb microtubule dynamics and assess the effect on microtubule reorientation in fruit exocarp cells. We will analyse C. hirsuta mutants of the microtubule-associated proteins KATANIN1, SPIRAL2 and CLASP, which show reduction or failure to reorient microtubule arrays in Arabidopsis. We will also make use of an inducible system (pML1::LhGR>>PHS1ΔP:mCherry) to depolymerise microtubules with precise spatial and temporal resolution. Results from these perturbation studies will help validate and refine the model.

Key publication

Perez-Anton et al. Explosive seed dispersal depends on SPL7 to ensure sufficient copper for localized lignin deposition via laccases. PNAS, 119:e2202287119 (2022).

Potential collaborations with other research groups

John Innes Centre (JIC)

Coen group/Enrico Coen

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