Discipline: Biological Sciences
Josiah Moore - Tougaloo College/Brown University
Co-Author(s): Alison DeLong, Brown University, RI
Ethylene gas is an essential regulator of plant growth and development, with direct effects on plant morphology, germination and defense responses. Ethylene gas is synthesized through a two-step enzymatic process in which 1-aminocyclopropane 1-carboxylate synthase (ACS) performs the first step. Ethylene production is limited by proteasome-mediated degradation of ACS enzymes, and stabilization of ACS proteins leads to increased ethylene synthesis. The plant hormone cytokinin increases ethylene production by increasing the stability of two ACS proteins, ACS5 and ACS9. The heterotrimeric protein phosphatase 2A (PP2A) complex also increases ACS5 stability. In the PP2A complex, C is the catalytic subunit and the A subunit performs a structural or scaffolding role, while B is the regulatory subunit and is thought to confer substrate specificity on the complex. The three Arabidopsis B subunit gene families (B55, B56 and B72) are comprised of 17 genes. To identify the PP2A B subunits that positively regulate ethylene synthesis by stabilizing ACS5, we screened a set of pp2a-b subunit mutants for defects in cytokinin-induced ethylene production, using a shoot elongation assay in dark-grown seedlings. Seedlings were grown in the dark for approximately 72 hours. The plates were scanned and hypocotyl lengths were measured using NIH ImageJ. Previous experiments did not identify loss-of-function pp2a-b mutants with obvious defects in regulation of ethylene synthesis. However, a B72 gain-of-function mutant (b16-1) shows phenotypes consistent with increased ethylene synthesis. Additionally, cytokinin-induced ethylene responses are reduced in a pp2a-a mutant with decreased PP2A activity. We believe that members of the B72 gene family are required for PP2A-mediated stabilization of ACS5. The b17-4 loss-of-function mutant shows normal cytokinin response, and the slight decrease in cytokinin response in the b14-1 single mutant is not statistically significant. The slight decreases in rcn1-6 and lcmt1 don’t look significant on the histogram, but the lcmt results are significant by 2-way ANOVA (rcn1 sample size is too small unfortunately). The decreases in the rcn1-6 lcmt1 double mutant and the b16-1 single mutant are clearly significant (p <0.005. B subunit mutants that show decreased response to cytokinin may function as positive regulators of type 2 ACS stability. Results from the kinetin dose response assay suggest that we should repeat the assay with the b72 mutants for further investigation. Also, including a double b16, b17 mutant in the assay will suggest whether these genes are positive or negative regulators of ethylene synthesis. This research is applicable to species within the plant genome. Future Research Questions: Do B-55 Family genes positively regulate ethylene synthesis?
Funder Acknowledgement(s): This work was partially supported by the National Science Foundation (IOS-1145585). This work was also supported by the Jackson Heart Study.
Faculty Advisor: Alison DeLong, email@example.com
Role: I conducted experiments and data analysis under my advisor at Brown University.