Arabidopsis Projects

Studies of novel cytoskeletal regulatory proteins that are involved in abiotic stress signaling
A collaboration with Dr. Einat Sadot
Volcani Institute, Israel
Supported by BARD (IS-4038-07)
Staff: Dr. Mohamad Abu-Abied

Employing a functional genomic screen for new plant cytoskeleton binding proteins, we discovered two novel actin-associated proteins that are induced by abiotic stress. These are the dehydrin family member, ERD10, and the touch-induced calmodulin-like protein 2 (TCH2), which bind actin directly and indirectly respectively (Abu-Abied et al. 2006) . In a co-sedimentation assay, ERD10 demonstrated binding to actin filaments in a dose-dependent manner. At steady state, ERD10 decreased the critical concentration (Cc) for actin filament assembly, consistent with stabilization of actin filaments. ERD10 also reduced the rate and extent of actin filament disassembly following dilution in a dose-dependent manner. Furthermore, ERD10 protected actin from depolymerization in leaves treated with the actin disrupting reagent Latrunculin B. The collective results strongly support the notion that ERD10 is capable of binding to the side or ends of actin filaments in vitro and in vivo and alters actin dynamics. Calmodulin and calmodulin-like proteins can be myosin light chains through binding to the IQ domain(s) on the heavy chains of both conventional and unconventional myosins. Indeed, we found that TCH2 bound to the 4XIQ domain of plant myosin VIII (ATM1) in a GST pull down assay. Another touch-induced calmodulin, TCH1, did not bind to the ATM1 IQ domain, which reinforces the specificity of the TCH2/IQ interaction. In this proposal we aim to extend our research regarding these findings at the biochemical, molecular, biological, and physiological levels. We will test the effect of different signaling messengers such as Ca2+, pH, phospholipids, and post-translational modifications such as phosphorylation on the interaction of ERD10 with actin and TCH2 with myosin VIII in vitro. We will check other dehydrin family members, like COR47 and ERD14, for actin binding activity and TCH family members 1, 2 and 3 for the ability to bind myosin IX and TCH3 for the ability to bind myosin VIII. We will establish a motility assay for recombinant plant myosins and TCH proteins expressed in insect cells. Transgenic plants with fluorescent chimeras of TCH2 and ERD10 under their own promoters, as well as mutant plants lacking ERD10 and TCH2, will be generated and characterized. Detailed study of these plants will allow us to address the question of how these proteins affect the integrity and dynamics of the actin cytoskeleton in live cells, both in normal conditions and in response to various abiotic stresses.

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