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 2011-2012, transgenic plants generated for objective #1 and #2 were evaluated for their ability to complement thermosensitivity of mutant plants. Results have shown that mutations in either ATPase or proteolytic domains affect the normal function of FTSH11. The inner chloroplast envelop membrane (IM) location of FtsH11 is being validated. Biochemical and proteomic analyses aiming at identifying its interactive partners and substrates were performed. Proteomic data collected are being analyzed currently. Objective #3 &4, F2 mapping populations were generated for several enhancer or suppressor lines. Initial mapping analysis identified 4 chromosome regions that associated with either suppression or enhancement of FtsH11 gene activity. Complementation study using FtsH11 homolog genes from other plant species has signified the conserved roles of FtsH11 genes in thermotolerance in higher plants.