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Design and assessment of the complementation assay in Arabidopsis trol mutants

Background and aims The aim of this research was to functionally and structurally characterise the novel thylakoid protein TROL (thylakoid rhodanese-like) and to explore its possible role in regulation of photosynthesis (Jurić et al., 2009). We are investigating the centrally positioned RHO domain of TROL by complementing the Arabidopsis plants depleted of TROL (attrol) with vectors that contain modified versions of the RHO domain and by identifying the possible novel interacting partners for TROL. Methods The inserts: full-length TROL cDNA (SENSE), TROL cDNA with deletion of 13 amino acids from the RHO domain (ΔRHO), TROL cDNA with substitution of aspartate for glutamic acid (D207E), and TROL cDNA with substitution of aspartate for asparagine ((D207N), were cloned into the pENTRTM/SD/D-TOPO® vector and transferred to the Gateway® destination vector pH7WG2, 0 (Invitrogen, USA). Constructs were introduced into the attrol plants by floral infiltration and the selection procedure was done according to Harrison et al. (2006). Key results The hygromicin-based identification of transformants proved to be reliable and phenotypically distinctive from wild-type. The T1 generation was PCR genotyped for three regions (pH7WG2, 0 promoter region, 35S CaMV ; pH7WG2, 0 selectable marker region, Hyg ; 35S-TROL junction region, TRANS). The T2 generation was assessed for protein expression and the T3 generation for confirmation of non-segregating transgenic lines. The SENSE and D207N lines satisfied all three checkpoints, the D207E line is in the T3 phase, while the ΔRHO line needs to be revaluated. Conclusions The RHO domain belongs to a very interesting protein family whose members are involved in regulation of metabolism, protection against abiotic and biotic stress, regulation of redox homeostasis and other pathways (Papenbrock et al., 2010). Discovery of the possible interacting partner for the RHO domain, which might be a small molecule, proteinaceous molecule, or a phosphorylation event, would surely contribute to the understanding of the transthylakoid signal transmission and the communication between chloroplast(s) and nucleus. References Harrison S.J., Mott E.K., Parsley K., Aspinall S., Gray J.C., and Cottage A. (2006). Plant Methods 2, 19. Jurić S., Hazler-Pilepić K., Tomašić A., Lepeduš H., Jeličić B., Puthiyaveetil S., Bionda T., Vojta L., Allen J.F., Schleiff E., and Fulgosi H. (2009). Plant J 60(5), 783-794. Papenbrock J., Guretzki S., and Henne M. (2010). Amino Acids DOI 10.1007/s00726-010-0478-6

FNR; Plant; Rhodanese

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