Probing thein-situvolumes of Arabidopsis leaf plastids using 3D confocal and scanning electron microscopy

Abstract

AbstractLeaf plastids harbor a plethora of biochemical reactions including photosynthesis, the most important pathway on earth. Scientists are eager to unveil the physiological processes within the organelle but also their interconnection with the rest of the plant cell. An increasingly important feature of this venture is to use experimental data in the design of metabolic models. A remaining obstacle has been the limited in situ volume information of plastids and other cell organelles. To fill this gap for chloroplasts, we established three microscopy protocols deliveringin situvolumes based on: 1) chlorophyll florescence emerging from the thylakoid membrane, 2) a CFP marker embedded in the envelope, and 3) calculations from serial block-face scanning electron microscopy (SBFSEM). The obtained data were corroborated by comparing wild-type data with two mutant lines affected in the plastid division machinery known to produce small and large mesophyll chloroplasts, respectively. Furthermore, we also determined the volume of the much smaller guard cell plastids. Interestingly, their volume is not governed by the same components of the division machinery which defines mesophyll plastid size. Based on our three approaches the average volume of a mature Col-0 wild-type mesophyll chloroplasts is 93 µm3. Wild-type guard cell plastids are approximately 18 µm3. Lastly, our comparative analysis shows that the chlorophyll florescence analysis can accurately determine chloroplast volumes, providing an important tool to research groups without access to transgenic marker lines expressing genetically encoded fluorescence proteins or high-end costly SBFSEM equipment.KeywordsOne-sentence summaryThis work describes and compares three different strategies to obtain accurate volumes of leaf plastids from Arabidopsis, the most widely used model plant.