We thank Amanda Rychel for assistance in developing the time lapse protocol and Lynn Pillitteri for constructing POLAR::POLAR-eGFP. We are also grateful to ABRC for providing the pm-rb construct. This protocol was developed through a support from the PRESTO award from Japan Science Technology and Agency. Research on POLAR was also supported by the University of Washington Royalty Research Fund (RRF-4098) and the National Science Foundation (MCB-0855659). KMP is an NSF Graduate Research Fellow (DGE-0718124), and KUT is an HHMI-GBMF investigator.

1. Seed Sterilization
<ol>
	<li>Prepare seed sterilization solution: 33% household bleach, 0.1% Triton X-100.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Incubate at 4 &deg;C for two days or more.</li>
</ol>
<p class="jo...

<ol>
	<li>Sulston, J. E., Schierenberg, E., White, J. G., Thomson, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Embryonic+Cell+Lineage+of+the+Nematode+Caenorhabditis+elegans.">The Embryonic Cell Lineage of the Nematode Caenorhabditis elegans.</a> Developmental Biology. 100, 64 (1983).</li><li>Foe, V., Odell, G., Edgar, B. A., Bate, M., Martinez Arias, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3+Mitosis+and+Morphogenesis:+Point+and+Counterpoint.">Chapter 3 Mitosis and Morphogenesis: Point and Counterpoint.</a> Development of Drosophila melanogaster. , (1993).</li><li>Vincent, C. A., Carpenter, R., Coen, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Lineage+Patterns+and+Homeotic+Gene+Activity+During+Antirrhinum+Flower+Development.">Cell Lineage Patterns and Homeotic Gene Activity During Antirrhinum Flower Development.</a> Current Biology. 5, 1449 (1995).</li><li>Beemster, G. T. S., De Vusser, K., De Tavernier, E., De Bock, K., Inz&#233;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Variation in Growth Rate between Arabidopsis Ecotypes Is Correlated with Cell Division and A-Type Cyclin-Dependent Kinase Activity. Plant Physiology. 129 (2), 854 (2002).</li><li>Grandjean, O., Vernoux, T., Laufs, P., Belcram, K., Mizukami, Y., Traas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Analysis+of+Cell+Division,+Cell+Growth,+and+Differentiation+at+the+Shoot+Apical+Meristem+in+Arabidopsis.">In Vivo Analysis of Cell Division, Cell Growth, and Differentiation at the Shoot Apical Meristem in Arabidopsis.</a> Plant Cell. 16 (1), 74 (2004).</li><li>Pillitteri, L. J., Peterson, K. M., Horst, R. J., Torii, K. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Profiling+of+Stomatal+Meristemoids+Reveals+New+Component+of+Asymmetric+Cell+Division+and+Commonalities+among+Stem+Cell+Populations+in+Arabidopsis.">Molecular Profiling of Stomatal Meristemoids Reveals New Component of Asymmetric Cell Division and Commonalities among Stem Cell Populations in Arabidopsis.</a> Plant Cell. 23 (9), 3260 (2011).</li><li>Abramoff, M. D., Magalhaes, P. J., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+Processing+with+ImageJ.">Image Processing with ImageJ.</a> Biophotonics International. 11 (7), 36 (2004).</li><li>Arganda-Carreras, I., Sorzano, S. &. #. 2. 2. 5. ;. n. c. h. e. z., Marabini, C. O., Carazo, R., de Solorzano, J. M. O. r. t. i. z. -., C, J., Kybic, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consistent+and+Elastic+Registration+of+Histological+Sections+Using+Vector-Spline+Regularization.">Consistent and Elastic Registration of Histological Sections Using Vector-Spline Regularization.</a> Computer Vision Approaches to Medical Image Analysis, ser. Lecture Notes in Computer Science. 4241, 85-95 (2006).</li><li>Peterson, K. M., Rychel, A. L., Torii, K. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Out+of+the+Mouths+of+Plants:+The+Molecular+Basis+of+the+Evolution+and+Diversity+of+Stomatal+Development.">Out of the Mouths of Plants: The Molecular Basis of the Evolution and Diversity of Stomatal Development.</a> Plant Cell. 22 (2), 296 (2010).</li><li>Czymmek, K. J., Fogg, M., Powell, D. H., Sweigard, J., Park, S. -. 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No conflicts of interest declared.

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 growth5,12, but adding the cotyledon epidermis expands the vers...

<table border="1">
 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Bacto Agar</td>
 <td>BD</td>
 <td>214010</td>
 <td></td>
 </tr>
 <tr>
 <td>One-chamber slide</td>
 <td>Nunc (Thermo Scientific)</td>
 <td>155360</td>
 <td>Or two-chamber (155379)</td>
 </tr>
 <tr>
 <td>Laser scanning confocal microscope</td>
 <td>Zeiss</td>
 <td>LSM700</td>
 <td>Zen 2009 software</td>
 </tr>
 <tr>
 <td>20x objective lens</td>
 <td>Zeiss</td>
 <td>420650-9901</td>
 <td>NA 0.8, Plan-APOCHROMAT</td>
 </tr>
 <tr>
 <td>Dissecting microscope</td>
 <td>Benz (National)</td>
 <td>431TBL</td>
 <td>Illuminates from below</td>
 </tr>
 <tr>
 <td>#5 forceps, biology tip</td>
 <td>Roboz Surgical Instrument</td>
 <td>RS-4978</td>
 <td>Very fine tips are critical</td>
 </tr>
 </tbody>
</table>

long-term, high-resolution confocal time lapse imaging of arabidopsis cotyledon epidermis during germination

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.

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...

Watch this Scientific Journal Video about Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination at JoVE.com

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

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.

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

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

long-term-high-resolution-confocal-time-lapse-imaging-arabidopsis

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Biology

13.6K Görüntüleme.  University of Washington. Biz stoma farklılaşma belgeleyen, gelişme birkaç gün içinde epidermis konfokal görüntüleme için bitki kotiledon hareketsiz odası slaytlar ve medya kullanarak bir protokol açıklar. Fluorofor etiketli proteinlerin hücre bölünmesi ve hücre tipi farklılaşması esnasında olası rollerini anlamayı arttırıyor, ifade ve hücre içi lokalizasyonu dinamik izlenebilir.