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The cells of higher plants are generally nonmotile and thus the coordination of cell division and cell expansion is crucial in allowing normal growth and development. Little is known, however, about the mechanisms by which particular groups of cells coordinate their division and expansion relative to neighbooring cells during normal development. We are investigating the importance of localised coordination of cell division in determining the architecture of the root meristem of Arabidopsis thaliana. Using a GAL4-GFP transactivation system, the rate of cell division is altered in specific cell layers via targeted misexpression of cell cycle regulatory genes. The consequences of these perturbations are then visualized using high-resolution confocal imaging, followed by computerized three-dimensional reconstruction of cellular architecture. We have shown that altering the rate of cell division in particular layers of the root during embryonic development can have a marked effect on cell division rates and/or expansion in neighboring cells. For example, accelerating cell division rates within the columella region of the root cap significantly increases the number of quiescent center cells and vascular initials. This suggests that perturbation of cell division in the columella is perceived by neighboring cells which adjust their own division rates to compensate. This approach allows us to determine the range and direction of cell-to-cell communication between cell layers during root development and may provide insights into intercellular signaling within the root meristem.

Background and Aims: The root meristem of the Arabidopsis thaliana mature embryo is a highly organized structure in which individual cell shape and size must be regulated in co-ordination with the surrounding cells. The objective of this study was to determine the role of the AUX1 LAX family of auxin import carriers during the establishment of the embryonic root cell pattern., Methods: The radicle apex of single and multiple aux1 lax mutant mature embryos was used to evaluate the effect of this gene family upon embryonic root organization and root cap size, cell number and cell size., Key Results: It was demonstrated here that mutations within the AUX1 LAX family are associated with changes in cell pattern establishment in the embryonic quiescent centre and columella. aux1 lax mutants have a larger radicle root cap than the wild type and this is associated with a significant increase in the root-cap cell number, average cell size, or both. Extreme disorganization of the radicle apex was observed among quadruple aux1 lax1 lax2 lax3 mutant embryos, but not in single aux1 null or in lax1, lax2 and lax3 single mutants, indicating redundancy within the AUX1 LAX family., Conclusions: It was determined that the AUX1 LAX family of auxin influx facilitators participates in the establishment of cell pattern within the apex of the embryonic root in a gene-redundant fashion. It was demonstrated that aux1 lax mutants are affected in cell proliferation and cell growth within the radicle tip. Thus AUX1 LAX auxin importers emerge as new players in morphogenetic processes involved in patterning during embryonic root formation.

A new method is described for fluorescent imaging of mature Arabidopsis embryos that enables their cellular architecture to be visualized without the need for histological sectioning. Mature embryos are stained with aniline blue and cleared with chloral hydrate to allow high-resolution confocal imaging of individual cells within the embryo prior to germination. The technique allows the collection of longitudinal optical sections throughout the cotyledon, hypocotyl and root of wild-type Arabidopsis C24 embryos. Every cell within the mature embryo can be visualized with sufficient clarity and resolution to allow three-dimensional analysis of cellular architecture. Optical sectioning of mutant gnom, short-root and scarecrow embryos, and through root meristems disrupted as a consequence of targeted misexpression of diphtheria toxin, demonstrate the potential of this technique for visualizing the cellular organization of mutant and perturbed embryonic phenotypes.