Cell type-specific characterization of nuclear DNA contents within complex tissues and organs

Abstract

Background: Eukaryotic organisms are defined by the presence of a nucleus, which encloses the chromosomal DNA, and is characterized by its DNA content (C-value). Complex eukaryotic organisms contain organs and tissues that comprise interspersions of different cell types, within which polysomaty, endoreduplication, and cell cycle arrest is frequently observed. Little is known about the distribution of C-values across different cell types within these organs and tissues.

Results: We have developed, and describe here, a method to precisely define the C-value status within any specific cell type within complex organs and tissues of plants. We illustrate the application of this method to Arabidopsis thaliana, specifically focusing on the different cell types found within the root.

Conclusion: The method accurately and conveniently charts C-value within specific cell types, and provides novel insight into developmental processes. The method is, in principle, applicable to any transformable organism, including mammals, within which cell type specificity of regulation of endoreduplication, of polysomaty, and of cell cycle arrest is suspected.

Figure 1

Confocal and bright-field images of wild-type plants, and plants transgenically expressing HTA6-GFP and HTA6-YFP under the transcriptional control of the CaMV35S promoter. For the bright-field picture, seeds of the three genotypes were germinated on MS agar plates. 3-day-old, similar-sized seedlings were transferred onto fresh MS agar plates, and were grown in a vertical position for two weeks.

Figure 2

(A) Fresh weights and (B) root growth amounts of plants that are transgenic or non-transgenic for HTA6-GFP expression. Measurements were made on three groups of 20 pooled plants, and the error bars indicate standard deviations. Abbreviations: WT, wild-type; HTA6-GFP, transgenic plant expressing HTA6-GFP.

Figure 3

Flow cytometric analysis of the nuclear DNA contents of plant root homogenates. Homogenates, prepared as described by Galbraith et al. [5], stained with DAPI, were analyzed using a Partec CCAII Flow Cytometer (Partec GmbH, Munster, Germany). The instrument PMT and amplification settings were adjusted between samples to provide distributions conveniently distributed across the abscissae. This means that the numerical positions of the nuclear peaks should not be used for comparative analysis of DNA content between species. (A) Arabidopsis thaliana. (B) Pisum sativum. (C) Cucumis sativus. (D) Daucus carota. (E) Nicotiana tabacum. (F) Lycopersicon esculentum. (G) Petunia hybrida. (H) Vinca rosea. (I) Zea mays. (J) Oryza sativa.

Figure 4

Confocal and biparametric flow cytometric analyses of wild-type and transgenic plants expressing nuclear GFP. Flow cytometry was done using a Cytomation MoFlo flow cytometer with laser excitation at 365/488 nm, and biparametric detection of DAPI fluorescence (418–482 nm; FL4; log units), and GFP fluorescence (505–530 nm; FL1; log units), with triggering based on 90°-light scatter [59]. For confocal microscopy, roots were counterstained by dipping in propidium iodide (1 μg/mL in water) for 2 minutes. Abbreviations: p35S: CaMV 35S promoter; pSCR: SCARECROW promoter; pSHR: SHORTROOT promoter; pRPL16B: ribosomal protein large subunit 16B promoter; pSultr2-1: sulfate transporter 2-1 promoter.

Figure 5

Analysis of the stability and amounts of targeted GFP fluorescence within nuclei following homogenization. Biparametric flow cytometric analyses were done of transgenic plants constitutively expressing nuclear GFP at various times following homogenization. A. The modes of the GFP fluorescence distributions for the four classes of nuclei (2C, 4C, 8C, 16C) are plotted as a function of time after homogenization. B. The modes obtained from the GFP fluorescence distributions were averaged across the time course for the different nuclear classes, and these mean values are plotted against C-value. Error bars indicate standard deviations.

Figure 6

Comparison of the distributions of nuclei within the various C-value classes found in the roots of wild-type plants and plants transgenic for expression of HTA6-GFP. Measurements were made on three replicate samples (wild-type and transgenic) each sample comprising ~100 pooled seedlings. The error bars indicate standard deviations.

Figure 7

Quantification of the proportions of nuclei within the various C-value classes for transgenic plants expressing HTA6-GFP under the control of constitutive and cell type-specific promoters. The proportions were determined by integrating the total number of events within the biparametric plots of Figure 4 falling within a rectangular box containing the specific C-value class and having a GFP-intensity value of 18 or greater. Abbreviations: See Legend to Figure 4.

Figure 8

Production of supernumerary endodermal cell layers is associated with accumulation of 4C and depletion of 2C cells. Supernumerary endodermal cell layers were produced by transgenic expression of SHR under the control of the SCR promoter, and these layers were identified via expression of HTA6-GFP under the control of the SCR promoter. A. Confocal analysis of the transgenic plants. B. C-value distributions of GFP-positive and -negative nuclei as determined through biparametric flow analysis (axis designations as described in the Legend to Figure 4). C. Classification of the proportions of nuclei within the various C-value classes for non-transgenic controls (WT), for all nuclei within transgenic plants producing supernumerary endodermal cells (SN-SCR), and for GFP-positive nuclei (GFP⁺) within these same transgenic plants.