1a.Objectives (from AD-416)
1. To determine the localization, topology, oligomerization status, size and identity of components of the FtsH11 complex.
2. To identify substrates involved in the FtsH11 and downstream components in maintaining thermostability.
3. Identify elements involved in the FtsH11 protease regulatory networks related to high temperature adaptation processes in chloroplast biogenesis an din chloroplast development.
4. Study the role of FtsH11 homologs from crop species in HT tolerance of photosynthesis systems.
1b.Approach (from AD-416)
1. Generate transgenic plants expressing epitope-tagged FtsH11 and their analysis, and purification of the tagged complex and its analysis.
2. Identify trapped substrates and comparative proteomics, respectively.
3. Generate EMS mutation of mutant; isolate and characterize the suppressors and possible enhancers of ftsh11 knockout mutant.
4. Generate overexpression and knockdown transgenic lines and comduct complementation analysis of these lines.
In 2010-2011, constructs made for objective #1 and #2 were introduced into the ftsh11 mutant plants via Agrobacterium-mediated transformation approach. Transgenic plants generated are currently being evaluated for their ability to complement thermosensitivity of mutant plants. Preliminary results indicate that FtsH11 protease is located on chloroplast envelope. For objective #3, several putative enhancer/suppressor lines identified were advanced to M5-M6 generation. M6 plants of selected suppressor/enhancer lines are going to be crossed to appropriate Arabidopsis lines to generate mapping population. For objective #4, transgenic plants generated for cDNA constructs of Pea, Castor bean, and black cottonwood FtsH11 homolog gene were evaluated in various thermotolerance assays, and results indicate that these FtsH11 homolog genes play important roles in thermotolerance similar to that of Arabidopsis FtsH11.