To check the dynamics of this system, Zhang et al.
] performed a careful quantitative analysis of cotyledon pavement cell shape over time, and showed that lobes are initiated early on, within the first 3 days after germination. Most of pavement cell growth occurs after this time and is in fact close to isotropic (uniform in all directions); to reach this conclusion, the authors measured both the top wall area and the length of each cell side, and the measurements were subjected to a simple linear regression analysis. In other words, the puzzle shape of the pavement cells becomes more obvious after day 3 not because of continuous polarized growth, but because of a homothetic magnification of a shape already existing at day 3 (Figure ).
Interestingly, and consistent with these data, there was not always a clear correlation between the pattern of cortical microtubules, as viewed from the top, and cell shape after day 3 (Figure ). Venturing beyond this classical viewpoint of the microtubules, Zhang and collaborators used fixed tissues (to mildly separate each cell and access the cell sides) as well as single cell transformation (through bombardment) to image the microtubules on the anticlinal wall of the cells. From these data, microtubules were shown to populate the lobe domain, in contrast to the predominant model. This questions the supposed specific role of the microtubules in limiting growth in the necks and, conversely, it also suggests a contribution of the microtubules in driving lobe growth, in addition to actin.
One conclusion from this study is that growth of the young leaf epidermis is truly diffuse after day 3, and this can be correlated with reducing the risk of generating shearing stress between neighboring cells. While this study also leaves a lot of questions to address, it pinpoints the need for quantitative approaches in order to generate reliable hypotheses and drive experimental design. In this case, two very distinct processes, involving different growth modes and cytoskeleton dynamics, seem to control cell morphogenesis in the epidermis.
The quantitative analysis also revealed new unexpected behaviors: in contrast to growth in the XY plane, growth in height, a variable that had not been quantified and correlated to pavement cell shape before, followed a non-linear behavior after day 3. Although it is unclear how this could be achieved, these data suggest that growth in the Z direction might have a more important role in pavement cell morphogenesis than previously anticipated.