RNA editing in organelles

 In the process of RNA editing, RNA nucleotides are inserted, deleted, or modified, resulting in a difference between the actual RNA and the RNA predicted from genomic DNA. RNA editing is known to occur in a variety of organisms, including mammals, insects, plants and microorganisms. In humans and other animals, RNA editing creates diversity in certain classes of proteins encoded by the nuclear genome.  In plants, RNA editing occurs in RNAs encoded by the organelle genomes located in chloroplasts and mitochondria. During RNA editing in plants, cytidines encoded by genomic DNAs are modified to uridines in transcripts.  Editing in plants appears to correct defective RNA post-transcriptionally. We are using genetic and biochemical techniques to examine editing in mitochondria and chloroplasts. It is possible to transform chloroplast genomes with chimeric genes to probe the features of transcripts that are important in RNA editing. We wish to understand RNA editing at the molecular level, including how cytidines are selected for editing and what macromolecules are involved in the editing process.  We have identified RNA-binding proteins involved in selection of the correct C for editing.

Probing plant cell organization with organelle-targeted fluorescent proteins

The ability to label different subcellular locations with the green fluorescent protein (GFP) has made it possible to visualize intracellular activities in living cells. We have introduced chimeric genes which express GFP in a variety of organelles within the plant cell in order to study the dynamics of cell organization. Labeling plastids with GFP led to the rediscovery of tubules emanating from plastids. Now termed stromules, for stroma-filled tubules, the function and mechanism of formation of these unexpected structures remains a topic for study.A web essay on stromules, authored by Hanson and Köhler, can be seen at:  http://4e.plantphys.net/article.php?ch=7&id=122&search=Visual

Below: A cultured cell expressing nuclear-encoded, chloroplast-targeted GFP was imaged with an Olympus FluoView 1000 confocal microscope. Stromules sometimes connect two plastid bodies.

Chloroplasts accumulate to improve acquisition of light energy but also move to avoid excess light that might cause photodamage.  The actin cytoskeleton mediates these responses, but how chloroplasts position themselves within the plant cell is not entirely understood.  We are exploring the role of myosins in movement of chloroplasts and other organelles.

Expression of foreign proteins in chloroplasts  

Because there are multiple copies of the chloroplast genome within each organelle and leaf cells each typically contain 100-200 chloroplasts, in one leaf cell, there can be thousands of copies of a transgene inserted into the chloroplast genome.  Such an elevated copy number offers the possibility to create chloroplast transgenic plants that express foreign proteins as high as 30-40% of total soluble leaf protein.  However, some foreign proteins are expressed only at low levels in transgenic chloroplasts.  Factors that can affect foreign protein yield include the gene expression signals incorporated into the transgene, the stability of the foreign protein, and environmental growth conditions.  In collaboration with Prof. Beth Ahner's laboratory (Biological and Environmental Engineering, Cornell), we are determining how to optimize production of cellulolytic enzymes from the bacterium Thermobifida fusca in chloroplast transgenic plants.  Plants expressing cellulases at high levels could be harvested to obtain enzymes for glucose production from biomass.  Chloroplast transgenes can also be engineering to express large quantities of pharmaceutical proteins.

Below: Chloroplast transgenic plant expressing T. fusca cel6A