Chloroplast protease/chaperone AtDeg2 influences cotyledons opening and reproductive development in Arabidopsis

Abstract

AtDeg2 is a chloroplast protein with dual protease/chaperone activity. Since data on how the individual activities of AtDeg2 affect growth and development of Arabidopsis thaliana plants is missing, two transgenic lines were prepared that express mutated AtDeg2 versions that have either only protease or chaperone activity and a comprehensive ontogenesis stage-based study was performed comprising wild type (WT) plants and insertional mutants that do not express AtDeg2, as well as the two transgenic lines. The repression of both AtDeg2 activities in deg2-3 mutants altered just a few phenotypic traits including the time when cotyledons were fully opened, the time when 10% flowers were open as well as the number of inflorescence branches and seed length in plants which have completed their generative development. It was demonstrated that complete opening of cotyledons as well as the number of inflorescence branches and seed length in plants which have completed their generative development required involvement of both AtDeg2 activities, whereas the time when 10% of flowers were open was controlled by AtDeg2 protease activity. These results show for the first time that the chaperone activity of AtDeg2 is needed for some elements of generative development of A. thaliana plants to proceed normally. So far, the chaperone activity of AtDeg2 was confirmed based on in vitro assays only.

Chloroplast protease/chaperone AtDeg2 holds γ₁ subunit of ATP synthase in an unaggregated state under high irradiance conditions in Arabidopsis thaliana

“…Transgenic lines: To search for chloroplast proteins protected by AtDeg2 acting as a holdase from high irradiance-dependent interprotein aggregation via disulphide bridges, we applied 35S:AtDEG2 ΔPDZ1 -GFP transgenic lines (represented by T2 progeny) generated in the deg2-3 background (Luciński et al 2011, Adamiec et al 2018. The 35S:AtDEG2 ΔPDZ1 -GFP transformants express -in fusion with GFP -the mutant AtDeg2 ΔPDZ1 protein which is proteolytically active but lacks holdase and disaggregase activities (Adamiec et al 2018). We attempted to choose the line(s) for which the abundance of AtDeg2 ΔPDZ1-GFP fusion was under high irradiance stress the most similar to that of native AtDeg2 in WT plants.…”

“…Since both these stresses were demonstrated to induce the accumulation of reactive oxygen species in plant cells as well (Mittler et al 2004, Grabsztunowicz et al 2015, we expected that under conditions of high irradiance stress, some oxidatively modified chloroplast proteins might participate in the formation of aggregates held by disulphide bridges and hypothesized that a certain pool of these proteins might be protected from the aggregation by AtDeg2. Our analyses included WT plants and transgenic plants 35S:AtDEG2 ΔPDZ1 -GFP that expressed AtDeg2 lacking both chaperone activities but retaining its protease activity (Adamiec et al 2018). Our results indicate that AtDeg2 operates in vivo as holdase by inhibiting the formation of homodimers of γ1 subunit of ATP synthase in chloroplasts of plants exposed to long-term high irradiance stress.…”

“…The vector construct for stable transformation (35S:AtDEG2 ΔPDZ1 -GFP) was prepared and the transformation performed exactly as described previously (Adamiec et al 2018). Stable transformants were selected on MS media supplemented with gluphosinate ammonium (10 mg L -1 ).…”

“…However, data concerning the role played by AtDeg2 in vivo as chaperone are rather limited. Namely, we have shown recently that under non-stressing conditions, AtDeg2 influenced a few phenotypic features of Arabidopsis thaliana through its chaperone activity in conjunction with the protease activity, i.e., the time when cotyledons were fully opened, the number of inflorescence branches, and seed length in plants which have terminated their generative development (Adamiec et al 2018). To date, no chloroplast proteins, whose potential aggregation were inhibited by AtDeg2 or proteins which form aggregates subsequently disaggregated by AtDeg2, have been identified (neither under non-stressing conditions nor any abiotic stress).…”