David W Galbraith
Work Summary
I examine the molecular functions of the different cells found in the tissues and organs of plants and animals and how they combine these functions to optimize the health and vigor of the organism.
I examine the molecular functions of the different cells found in the tissues and organs of plants and animals and how they combine these functions to optimize the health and vigor of the organism.
PMID: 21116995;Abstract:
Flow cytometry has been employed for the analysis of higher plants for approximately the last 30 years. For the angiosperms, ∼500,000 species, itself a daunting number, parametric measurements enabled through the use of flow cytometers started with basic descriptors of the individual cells and their contents, and have both inspired the development of novel cytometric methods that subsequently have been applied to organisms within other kingdoms of life, and adopted cytometric methods devised for other species, particularly mammals. Higher plants offer unique challenges in terms of flow cytometric analysis, notably the facts that their organs and tissues are complex three-dimensional assemblies of different cell types, and that their individual cells are, in general, larger than those of mammals.This chapter provides an overview of the general types of parametric measurement that have been applied to plants, and provides detailed methods for selected examples based on the plant model Arabidopsis thaliana. These illustrate the use of flow cytometry for the analysis of protoplasts and nuclear DNA contents (genome size and the cell cycle). These are further integrated with measurements focusing on specific cell types, based on transgenic expression of Fluorescent Proteins (FPs), and on analysis of the spectrum of transcripts found within protoplasts and nuclei. These measurements were chosen in particular to illustrate, respectively, the issues encountered in the flow analysis and sorting of large biological cells, typified by protoplasts; how to handle flow analyses under conditions that require processing of large numbers of samples in which the individual samples contain only a very small minority of objects of interest; and how to deal with exceptionally small amounts of RNA within the sorted samples.
Abstract:
Nuclear movement and targeting of a green fluorescent protein (GFP)-nuclear localization sequence (NLS)-β-glucuronidase (GUS) chimeric protein to the roots of transformed Arabidopsis has been studied using time lapse analysis. To characterize the mechanism governing this movement, metabolic inhibitors, and chemicals that alter the concentrations of divalent cations in the cell were applied. Flow cytometry was used to examine the condition of nuclear membranes. The results showed that nuclear movement in Arabidopsis roots was energy-dependent. The concentration of magnesium ions seemed to play a crucial role in the movement of the nuclei and targeting of the chimeric GFP protein into the nucleoplasm. In contrast, alterations in concentration of calcium ions did not affect either nuclear dynamics or nucleoplasmic GFP accumulation. The results are discussed in terms of some of the physiological processes occurring within the cell. © 2002 Elsevier Science Ireland Ltd. All rights reserved.
PMID: 16668285;PMCID: PMC1080875;Abstract:
Microfluorometric analysis of the nuclear DNA contents of the somatic tissues of Arabidopsis thaliana has revealed extensive endoreduplication, resulting in tissues that comprise mixtures of polyploid cells. Endoreduplication was found in all tissues except those of the inflorescences and was developmentally regulated according to the age of the tissues and their position within the plant.