Protoplasting and Fluorescence-Activated Cell Sorting of the Shoot Apical Meristem Cell Types.

The shoot apical meristems (SAMs) are located at the tip of the shoot apex. The SAM harbors stem cells that divide continually to provide cells for developing above-ground organs. Several important developmental events occur in SAMs, such as stem cell maintenance, organ differentiation, and flowering commitment which are under genetic control. The SAM is a collection of specialized cells organized in specific spatial domains. Deciphering the gene regulatory networks, guided by the developmental and environmental signals, in these discrete cell types is essential to decoding the SAM function. Here, I provide updates to the previously published protocols for the protoplasting and subsequent purification through fluorescence-activated cell sorting (FACS) of SAM cell types (Reddy, Fluorescence activated cell sorting of shoot apical meristem cell types. In: Riechmann JL, Wellmer F (eds) Flower development. Methods in molecular biology, vol 1110. Humana, New York, pp 315-321, 2014), which has provided genome-wide gene expression patterns at a single cell-type resolution.

Role of turgor-pressure induced boundary tension in the maintenance of the shoot apical meristem of Arabidopsis thaliana.

In plants, the robust maintenance of tissue structure is crucial to supporting its functionality. The multi-layered shoot apical meristem (SAM) of Arabidopsis, containing stem cells, is an approximately radially symmetric tissue whose shape and structure is maintained throughout the life of the plant. In this paper, a new biologically calibrated pseudo-three-dimensional (P3D) computational model of a longitudinal section of the SAM is developed. It includes anisotropic expansion and division of cells out of the cross-section plane, as well as representation of tension experienced by the SAM epidermis. Results from the experimentally calibrated P3D model provide new insights into maintenance of the structure of the SAM epidermal cell monolayer under tension and quantify dependence of epidermal and subepidermal cell anisotropy on the amount of tension. Moreover, the model simulations revealed that out-of-plane cell growth is important in offsetting cell crowding and regulating mechanical stresses experienced by tunica cells. Predictive model simulations show that tension-determined cell division plane orientation in the apical corpus may be regulating cell and tissue shape distributions needed for maintaining structure of the wild-type SAM. This suggests that cells' responses to local mechanical cues may serve as a mechanism to regulate cell- and tissue-scale patterning.

Towards a functional understanding of cell growth dynamics in shoot meristem stem-cell niche.

Shoot apical meristems (SAMs) harbor a set of stem-cells which supply cells for the development of all above-ground structures. A precise spatio-temporal control of growth patterns in stem-cells and the differentiating progeny is critical to maintain a stable set of stem-cells. In recent years, an array of approaches including molecular genetics, transient perturbations, live-imaging, image processing and mathematical modeling have been employed to study the cellular dynamics. In this article, we highlight recent studies that link cell-cell communication mechanisms to cell mechanics and overall growth control that govern stem-cell homeostasis and morphogenesis in SAMs.

ABERRANT TESTA SHAPE encodes a KANADI family member, linking polarity determination to separation and growth of Arabidopsis ovule integuments.

The Arabidopsis aberrant testa shape (ats) mutant produces a single integument instead of the two integuments seen in wild-type ovules. Cellular anatomy and patterns of marker gene expression indicate that the single integument results from congenital fusion of the two integuments of the wild type. Isolation of the ATS locus showed it to encode a member of the KANADI (KAN) family of putative transcription factors, previously referred to as KAN4. ATS was expressed at the border between the two integuments at the time of their initiation, with expression later confined to the abaxial layer of the inner integument. In an inner no outer (ino) mutant background, where an outer integument does not form, the ats mutation led to amorphous inner integument growth. The kan1kan2 double mutant exhibits a similar amorphous growth of the outer integument without affecting inner integument growth. We hypothesize that ATS and KAN1/KAN2 play similar roles in the specification of polarity in the inner and outer integuments, respectively, that parallel the known roles of KAN proteins in promoting abaxial identity during leaf development. INO and other members of the YABBY gene family have been hypothesized to have similar parallel roles in outer integument and leaf development. Together, these two hypotheses lead us to propose a model for normal integument growth that also explains the described mutant phenotypes.

Irregular chiasm-C-roughest, a member of the immunoglobulin superfamily, affects sense organ spacing on the Drosophila antenna by influencing the positioning of founder cells on the disc ectoderm.

We describe a role for Irregular chiasmC-roughest (IrreC-rst), an immunoglobulin (Ig) superfamily member, in patterning sense organs on the Drosophila antenna. IrreC-rst protein is initially expressed homogeneously on apical profiles of ectodermal cells in regions of the antennal disc. During specification of founder cells (FCs), the intracellular protein distribution changes and becomes concentrated in regions where specific intercellular contacts presumably occur. Loss of function mutations as well as misexpression of irreC-rst results in an altered arrangement of FCs within the disc compared to wildtype. Sense organ development occurs normally, although spacing is affected. Unlike its role in interommatidial spacing, irreC-rst does not affect apoptosis during antennal development. We propose that IrreC-rst affects the spatial relationship between sensory and ectodermal cells during FC delamination.