Calcium-dependent phosphorylation as a regulator of metabolic processes in chloroplasts
Zusammenfassung der Projektergebnisse
Aim of this project was the analysis of the role of Ca2+ signalling for chloroplast metabolism with a special focus on Ca2+-dependent protein phosphorylation. Numerous studies have indicated that chloroplasts participate in cellular Ca2+ homeostasis and signaling and that this requires a tight, fine-tuned control of the organellar Ca2+ concentration. We have now created compatible sensor lines to measure Ca2+ changes in several chloroplast subcompartments, especially for the first time in the thylakoid lumen. Initial experiments have confirmed the usability of these sensors to measure Ca2+ changes and have provided some interesting data on the involvement of the thylakoid lumen in chloroplast Ca2+ signaling that need to be substantiated in further studies. We have also, after a long and elaborate effort obtained Arabidopsis lines that contain nonphosphorylatable (TKLS428A) and phosphomimetic (TKLS428D) transketolase variants in a tkl/tkl and a wild type genetic background. While viable, both variants show growth deficiency in the tlk/tlk background and the TKLS428A variant even in the wild type background causing much difficulties in obtaining these plant lines. This strongly support the notion that phosphorylation of TKL plays an important role in carbon metabolism and that maintenance of a proper ratio of phosphorylated to non-phosphorylated TKL is an important factor for plant growth. Some initial thin sectioning of leaves from these lines suggest that the TKLS428A plants have less starch and their cells are much more loosely packed with wider spaces in both the palisade and the spongy mesophyll. However, these results are very preliminary and a more detailed analysis is required in the future. Unfortunately, despite several different approaches, no kinase could until now be identified that phosphorylates TKL in a Ca2+-dependent manner. We could further show that the chloroplast calcium sensor protein CaS undergoes differential regulation by Ca2+-dependent and independent phosphorylation involving at least two kinases localized in the stroma and the thylakoid membrane, respectively. qPCR and western blot analysis further showed that CaS expression undergoes a diurnal rhythm indicating a very differential need for the regulation of CaS activity. By crossing a cas knock-out mutant with established sensor lines, we have created Arabidopsis lines that will allow to measure Ca2+ and ROS changes in the cas knock-out background. We have furthermore employed the CRISPR/CAS9 system to produce plants with CaS proteins that lack the C-terminal phosphorylation hot-spot and the N-terminal Ca2+- binding site, respectively. These lines are the basis for ongoing experiments to elucidate the role of CaS in chloroplast and cytosolic Ca2+ signaling.
Projektbezogene Publikationen (Auswahl)
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(2018) Chloroplast Ca2+ Fluxes into and across Thylakoids Revealed by Thylakoid-Targeted Aequorin Probes. Plant physiology 177 (1) 38–51
Sello, Simone; Moscatiello, Roberto; Mehlmer, Norbert; Leonardelli, Manuela; Carraretto, Luca; Cortese, Enrico; Zanella, Filippo G.; Baldan, Barbara; Szabò, Ildikò; Vothknecht, Ute C.; Navazio, Lorella
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(2013) Identification of CP12 as a novel calcium-binding protein in chloroplasts. Plants 2: 530-540
Rocha A.G. and Vothknecht U.C.
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(2014) Phosphorylation of Arabidopsis transketolase at Ser-428 provides a potential paradigm for metabolic control of chloroplast carbon metabolism. Biochemical Journal 458: 313-322
Rocha A.G., Mehlmer N., Stael S., Mair A., Parvin N., Chigri F., Teige M. and Vothknecht U.C.
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(2016) Dissecting stimulus-specific Ca2+ signals in amyloplasts and chloroplasts of Arabidopsis thaliana cell suspension cultures. Journal of Experimental Botany 67: 3965-3974
Sello S., Perotto J., Carraretto L., Szabò I., Vothknecht U.C., Navazio L.
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(2017) Structural basis for the magnesiumdependent activation of transketolase from Chlamydomonas reinhardtii. BBA - General Subjects, 1861: 2132-2145
Pasquini M., Fermani S., Tedesco D., Sciabolini C., Crozet P., Naldi M., Henri J., Vothknecht U.C., Bertucci C., Lemaire S.D., Zaffagnini M. and Francia F.
