Hitting the brakes on the cell cycle for the formation of plant stomata

Scientists from Nagoya University and The University of Texas at Austin have unraveled a key mystery in the formation of stomatal cells in plants. 

Stomata are the holes on the surface of plants’ leaves which open and close to allow the exchange of carbon dioxide, oxygen and water, and have a major influence on plants’ growth and survival. They are formed through multiple asymmetric divisions of a stem cell-like precursor cell, followed by a single symmetric division that creates the pair of guard cells that open and close to allow the stomata to function.

What has thus far remained unknown is how the cells’ pattern of division changes from asymmetric to symmetric. Cell division is closely controlled according to the process known as the cell cycle. In this study, the scientists, led by Professor Keiko Torii of The University of Texas at Austin and the Institute of Transformative Bio-Molecules (WPI-ITbM) at Nagoya University, took advantage of a polychromatic marker called PlaCCI (Plant Cell Cycle Indicator), developed by colleagues in Spain, for the live imaging of the cell cycle during the development of stomata. The cell cycle of the precursor cells (the meristemoid cells) during asymmetric division was around 12 hours, whereas the cell cycle of the guard cells undergoing symmetric division was around 20 hours.

But why does the length of the cell cycle increase as the cell differentiates? The change from meristemoid cell to differentiated guard cell is controlled by a transcription factor called MUTE. The researchers hypothesized that MUTE must induce a cell cycle inhibitor, which lengthens the cell cycle, and set about investigating other MUTE-controlled genes, finding that the one which met their criteria was the cell cycle inhibitor SIAMESE RELATED4 (SMR4).

The researchers created an SMR4 deficient variant of the plant Arabidopsis thaliana, and found that its cell cycle during asymmetric division became one hour shorter. On the other hand, a variant with an excess of SMR4 recorded an average cell cycle length of around 18 hours, significantly slower than usual. In both cases there was no change in the length of the cell cycle during symmetric division. At the molecular level, they found that SMR4 binds to and inhibits cyclin D3;1, the molecule which induces asymmetric divisions, but does not bind to cyclin D5;1, which induces symmetric division. 

In summary, the genetic analysis revealed that in order to make the transition from asymmetric division to the final symmetric division, the transcription factor MUTE induces the cell cycle inhibitor SMR4, effectively applying the brakes to the cell cycle by inhibiting cyclin D3;1, thus creating space for the symmetric division to take place. As cyclin D5;1, which is also induced by MUTE, does not bind to SMR4, symmetric division is able to go ahead and the two guard cells are formed.

The decision making of whether a cell proliferates (in this case, the asymmetric division) or differentiates (the symmetric division) takes place in what is known as the G1 phase of the cell cycle. When SMR4 is forcefully expressed in early stage meristemoid cells, the G1 phase is lengthened and the meristemoid cells swell rather than dividing. Even so, the uninhibited cyclin D5;1 still induces symmetric division and the end result is a large and irregularly shaped stoma with an overall appearance similar to an epidermal cell. The researchers determined that it is this deceleration of the cell cycle during the G1 phase, induced by MUTE and facilitated by the specific binding of SMR4, that underpins the transition from asymmetric division to symmetric division leading to differentiation of the guard cells.

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