The fine structures of plant cell organelles

Transmission electron microscopy (TEM) is usually used for the 2D investigation of the ultrastructure of cells and their organelles (SMITH & WOOD 1996). However, it is not possible to specify the relations in the cells and organelles because usually only a limited number of 80-100 nm ultrathin sections is used for evaluation in the TEM. Therefore, organelles like plastids (chloroplasts) with a size of 4-8 m are only partly cut. Chloroplasts and mitochondria are the two most important organelles of plant cells beside the nucleus because they guarantee the availability of the energy equivalent ATP and the reduction equivalent NADPH+H+ . Cell organelles and their ultrastructure are affected by different factors (stress), but mainly plastids were investigated because alterations/damages were noted mainly in these organelles (PASTOR et al. 1999, REY et al. 2000). Moreover, other organelles like mitochondria or peroxisomes, which are closely associated with chloroplasts in the photorespiratory process, are more difficult to examine due to their small average size (about 1 m). Changes in their structures and areas or volumes can hardly be noticed with conventional methods. Therefore, morphometric measurements and 3D reconstructions are essential for an exact distinction between damaged or daily periodic variable organelles (PERKTOLD et al.2000). With our method, which is successfully used in a current project, both morphometric data (areas and volumes) and 3D images of complete organelles are available by using ultrathin serial sections and computer reconstructions (PERKTOLD et al. 1998, ZELLNIG & PERKTOLD 1999). Distinct structures of interest can be faded out and selected structures can be viewed from various sides thus obtaining more detailed information about the association of organelles or structural continuities. We intend to investigate the 3D fine structure of various cell organelles during diurnal rhythm of selected field grown plants (Spinacia oleracea, Picea abies, Pinus canariensis). Additionally, the influence of stress factors (drought) will be registered. The result of these investigations is a more detailed knowledge about the distribution/ variation of fine structures, including areas and volumes on a very high level of resolution. These fundamental data will be essential for plant physiology by supplying for the first time exact data of complete organelles of field grown plants thus enabling a better interpretation of the functional significance of physiological and biochemical data.

Chloroplasts of three field grown species (spinach, spruce and pine) are the main object of investigation in this project. Chloroplasts are the organelles in green plant cells, which are responsible for photosynthesis, a process where light energy from the sun is converted into chemical energy and used to produce carbohydrates (starch). Plant cell organelles (chloroplasts, mitochondria, peroxisomes) and their ultrastructure are affected by different biotic and abiotic factors, however, usually the evaluation of the condition of these organelles is based on the information of a limited number of thin sections. The fine structures of chloroplasts can only be seen in the transmission electron microscope (TEM), therefore the organelles have to be cut in very thin sections of a ten thousandth part of a millimeter. With conventional electron microscopical methods it is not possible to specify the relations of fine structures in the cell and organelles because ultrathin sections of plant tissue are too thin for evaluation of complete organelles in the TEM. Organelles like chloroplasts have a size of about 4-8 m, therefore usually only a very small part of the organelle can be investigated. With our method 2D data and 3D images of different organelles are obtained by ultrathin serial sections and digital image analyses. These procedures allow 3D reconstructions of complete chloroplasts and the quantification of their fine structures. By means of these investigations daily periodic variations of fine structures can be distinguished from changes caused e.g. by drought stress. Our results clearly showed differences in the chloroplast structures during the day and after drought stress in field grown plants. In a previous work the amplitude of diurnal and stress related structural varieties was already recorded for chloroplasts of plants grown under defined climate chamber conditions. Chloroplasts of field grown spinach plants differed from climate chamber grown plants in the size, starch and content of internal membranes during the daily course. In addition, drought stress caused different changes in field grown chloroplasts when compared to those of the climate chamber. This knowledge has to be regarded as fundamental for further investigations with respect to sampling times and the usage of field grown and climate chamber grown plants. The result of our investigations is a more detailed information about the architecture and variation of fine structures, including areas and volumes, on a very high level of resolution. These data are essential for both further classifications of structural parameters and the interpretation of the functional significance of physiological and biochemical data.