Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Summary

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

Abstract

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles¹; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr². In contrast, cell divisions during plant development are slow, typically on the order of a day ^3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).

This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR⁶, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Protocol

1. Seed Sterilization Prepare seed sterilization solution: 33% household bleach, 0.1% Triton X-100. Place seeds carrying desired fluorescent reporter construct(s) and genotype(s) in 1.7 ml tube and apply 1 ml of sterilization solution. Incubate on nutator for 15 min. In a sterile hood, use a pipettor to remove sterilization solution from tube, leaving seeds behind. Rinse with 1 ml sterile water. Repeat four times. Incubate at 4 °C for two days or more. <p class="jo…

Representative Results

A set of informative time points collected with this method is shown in Figure 3. Cell membranes are labeled with RFP (pm-rb) and GFP is fused to POLAR protein under its native promoter (POLAR::POLAR-GFP)6 At the 30-min time scale, we see cell divisions along with the changes in protein localization preceding them. Asymmetric cell divisions in the stomatal lineage form stem-cell-like stomatal precursors called meristemoids, which retain the ability to undergo further asymmetric divisi…

Discussion

This time-lapse confocal technique allows longitudinal studies of fluorescently tagged protein expression and localization in individual cells of the Arabidopsis cotyledon epidermis, which in the case of POLAR and other dynamically changing proteins is critical to a proper understanding of their function. Previously, sustained time lapse imaging has been used to examine Arabidopsis root fungal infection¹¹ and meristem growth^5,12, but adding the cotyledon epidermis expands the vers…

Materials

Name of reagent	Company	Catalog Number	Comments
Bacto Agar	BD	214010
One-chamber slide	Nunc (Thermo Scientific)	155360	Or two-chamber (155379)
Laser scanning confocal microscope	Zeiss	LSM700	Zen 2009 software
20x objective lens	Zeiss	420650-9901	NA 0.8, Plan-APOCHROMAT
Dissecting microscope	Benz (National)	431TBL	Illuminates from below
#5 forceps, biology tip	Roboz Surgical Instrument	RS-4978	Very fine tips are critical