Maintenance of the leaf cell proliferation boundary by a cell size at division threshold mechanism.

The sizes and shapes of organs are established by the combined actions of cell proliferation and cell growth. In plants, development of the determinate planar leaf is initiated by primordia formation and establishment of abaxial/adaxial polarity [1,2,3]. Lamina outgrowth is driven by cell division and growth along proximo-distal (PD) and medio-lateral (ML) axes [4], established by mutually repressive PD gradients of miRNA and target transcription factors [5,6,7,8]. These gradients generate proximal regions of competence for cell division and increased growth, with distal regions of reduced growth, endoreduplication and differentiation. The transition from proliferation to growth and differentiation is marked by a cell cycle arrest front, which moves basipetally during leaf growth, progressively restricting proximal proliferative zones as the leaf grows [9,10,11]. Intersection of proximal proliferation-promoting gradients with distal differentiation-promoting gradients may delineate the arrest front, but its dynamics remain poorly understood. We reasoned that mutants affecting cell proliferation patterns may provide insights into the formation, maintenance and dissolution of the arrest front. Spatio-temporal modelling of live imaging data of loss of function mutants of the regulatory peptidase DA1 and its E3 ligase activator Big Brother (BB), which increase cell proliferation [12,13], showed that these proteins effectively establish a threshold cell size at division as a function of distance from the base of the growing leaf and the duration of growth. Loss of BB and DA1 activities increased the persistence of cell divisions and dissolved the arrest front. This suggested that the arrest front emerges from the interactions of threshold areas of cell division with the cessation of division over time, and not from an independently-specified boundary.