Tag: Alice Barkan

Determining the degree to which chloroplast genome copy number limits the expression of chloroplast genes

Presenter: Dylan Udy, Biology

Poster: D-1

Mentor: Alice Barkan, Institute of Molecular Biology

The relationship that exists between chloroplasts and the plant cells they occupy is very complex. Chloroplasts evolved from a cyanobacterial endosymbiont, and throughout evolution many of the ancestral bacterial genes have been transferred to the plant nuclear genome. The proteins from many such nuclear genes are sent back to the chloroplast where they perform a variety of functions. We identified a non-photosynthetic maize mutant that accumulates reduced levels of several chloroplast mRNAs. We showed that the causal mutation is a transposon insertion in a nuclear gene encoding a protein that is closely-related to bacterial DNA polymerase I. Angiosperm genomes include two closely related paralogs encoding this protein. These have been studied in Arabidopsis (a dicot plant), where they are dual-targeted to both the mitochondria and chloroplast and have redundant functions. I have shown that our maize mutant has a 10-fold reduction in chloroplast DNA but normal levels of mitochondrial DNA, suggesting that the two paralogs have become specialized for either chloroplast or mitochondrial DNA replication in maize (a monocot plant). I am using the maize mutant to investigate the degree to which the abundance of chloroplast DNA limits chloroplast gene expression. I have found that the abundance of some chloroplast mRNAs decreases in parallel with the abundance of chloroplast DNA, whereas the abundance of other mRNAs does not. These results show that different factors limit the expression of different genes in the chloroplast.

Investigating How Light Regulates Protein Synthesis in Chloroplasts

Presenter(s): Carolyn Brewster – Biology, Math and Computer Science

Faculty Mentor(s): Alice Barkan

Poster 51

Research Area: Natural/Physical Science

Funding: O’Day Fellowship

Photosynthesis provides the fuel for earth’s biomes. The protein PsbA is essential for photosynthesis but is also damaged as a consequence of photosynthesis; PsbA must therefore be constantly replaced to maintain photosynthetic activity. Accordingly, PsbA synthesis increases dramatically within minutes after shifting plants from dark to light. The mechanisms underlying this response are not known. We are investigating these mechanisms with a two-pronged approach: we are studying proteins that we suspect may be involved in PsbA light-regulation, and we are designing protein “tags” to isolate potential regulators that are attached to PsbA mRNA. We identified two candidate regulators, HCF244 and TPJ1, based on their patterns of gene expression. Using a combination of techniques, we discovered that HCF244 is required for PsbA synthesis whereas TPJ1 is not. We found that TPJ1 activates production of a different protein involved in photosynthesis. Thus, HCF244 is a good candidate for regulating PsbA synthesis in response to light, but TPJ1 is not. In the second approach, we designed a method to engineer proteins to bind specifically to the PsbA RNA. We expressed these engineered proteins in plants and confirmed that they bind specifically to PsbA RNA in vivo. We are using these proteins as “hooks” to purify PsbA RNA and the proteins bound to it. These will be evaluated for their role in PsbA regulation. In addition to elucidating mechanisms that regulate production of the photosynthetic apparatus, this is the first demonstration that proteins can be designed to purify specific RNA-protein particles from an organism.