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 2009-2010, various constructs for objective 1 and 2 have been made and are being introduced into the mutant plants via Agrobacterium-mediate transformation approach. EMS mutation of mutant was generated, and M2 plants from 32 pools were screened for plants with either enhanced or suppressed sensitivity to moderate high temperature treatment. Several putative enhancer/suppressor lines were identified and are in the process to set M3 seeds. In addition, cDNA of FtsH11 genes that are highly homologous to AtFtsH11 were cloned from Pea, Castor bean, and black cottonwood and introduced into the binary vector, respectively. The resulting constructs were introduced into the mutant via Agrobacterium-mediate transformation approach and the resulting T1 seeds harvested.