The Chloroplast Clp Protease System in Maize leaves

Project Summary

The Clp (caseinolytic protease) is the most abundant stromal protease in chloroplasts and it plays a critical role in plastid development and function, through selective removal of misfolded, aggregated, or otherwise unwanted proteins. ClpPR proteins are found in proplastids in maize leaves and are present through all developmental stages of chloroplasts, but with the highest accumulation levels in early stages of chloroplast development. In our previous studies, we identified two ClpP2-like proteins (ClpP2-like1 and ClpP2-like2) which are the most abundant ClpPR proteins in maize. Importantly, ClpP2 proteins in maize and Arabidopsis are located in mitochondria, whereas these ClpP2-like proteins in maize are likely plastid-localized. We hypothesize that this abundant ClpP2-like protein makes a separate Clp protease core in the plastid, perhaps with or without other ClpPR proteins, and may have a specialized role in maize plastid proteostasis. To clarify the function of ClpP2-like2 protein, I worked on generating an antigen in order to make an antibody. ZmClpP2- like2 gene (partial) was cloned and ligated into the pET21a expression vector as a C-terminal His_–tag fusion and expressed it in E. coli and purified the recombinant protein by affinity chromatography. In addition, 3 independent alleles ofzmclpp2-like2 mutants (Mu insertions) were genotyped and heterozygous plants were identified but no homozygous plant were obtained, suggesting embryo lethality of homozygous mutants. Heterozygous plants showed pale green phenotype at early developmental stage and accumulated less ZmClpP2-like2 transcript compared to wild type. Moreover, 2 independent alleles of zmclpr1 mutants (Mu insertions) were genotyped and the identified null mutants showed seedling lethal albino phenotypes. These results will help to improve our knowledge on the organization of the Clp protease system in maize.

My Experience

This time that I spent doing research has been, more than anything, an amazing learning experience. Research has truly become a part of my daily routine. I had the unique opportunity to belong at the BTI summer research program and be part of the plant genetics and biology laboratory. This was a great experience to learn more about plant genetics and more specific proteins present in maize. I felt great working together with Ph.D. and postdoc students; this was a challenge for me, but I learned the most important thing is the communication with the people in the laboratory. Working with my mentor I learned that we need to work hard to get the most possible results and learn new things every day. I gained further experience working with molecular technique such as PCR, genomic DNA extractions, western blot, genotyping, phenotyping, making antibodies, among others. Working in a Cornell lab allowed me to decide that I want to go graduate school and my plan is to earn a Ph.D. and pursue a career in the genetics and microbiology field.