ULTRA-STRUCTURAL ANATOMY AND 3D RECONSTRUCTION OF PLANT TISSUES AND ORGANELLES USING TRANSMISSON ELECTRON MICROSCOPY AND FOCUSED ION BEAM SCANNING ELECTRON MICROSCOPY

Abstract

ABSTRACT

Plastids are a group of organelles present in the cells of higher and lower plants and algae. Along with photosynthesis, they provide a variety of biochemical capabilities and are one of the defining features of plant cells. These organelles are developmentally flexible and can convert from one type to another to accommodate for the physiological needs of a plant tissue. The presence and/or transitions from one plastid type to another are not well documented for all cases and the genetic and physiological basis of these transitions are not well understood. The goal for this study was to explore the plastids and their transitions in a single plant to better understand the underlying mechanisms of these changes and their overall effects on plant cells. As a consequence of this goal, I adapted a type of electron microscopy that had not been widely used on plant tissues, catalogued the organelles in each organ of Arabidopsis thaliana, and linked the chloroplasts leucoplast transition in petal cells to an amorphous aggregate seen in vacuoles.

Electron microscopy (EM) was the primary approach used in this dissertation. Transmission Electron Microscopy (TEM) was used to gain better knowledge of plastids. In addition, Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) tomography was used, which offers the ability to produce serial slices of the materials (tissues) along with simultaneous SEM micrographs. The micrographs were used to create three dimensional (3D) renderings of the tissues with micron level resolution.

The successful application of FIB-SEM enabled the production of three dimensional renderings of five different tissues from A. thaliana: seed endosperm, leaf mesophyll, stem cortex, root cortex and petal lamina. The first part of this study demonstrates the efficacy of this technique in plant tissue/cellular studies and its usefulness in studying organelle architecture and distribution.

The second part of this dissertation focuses on petal cells, specifically a structure present inside the petal vacuoles which has been overlooked or ignored in previous EM studies. This structure was found and named Petal Amorphous Aggregate (PAA) in the first part of this study. By utilizing TEM and FIB-SEM to explore the petal cell vacuoles of A. thaliana, Brassica junceae and Cardamine bulbosa, a link to the transition of chloroplasts to leucoplasts is made to PAA development. The micrographs obtained from TEM demonstrate the development of the PAA in a cell vacuole which I have defined in six stages. Micrographs also indicate the interaction between PAA and plastids during their transition from chloroplast to leucoplast and that these transitions are only present in white petal where these transitions occur.