Turnover of RNA and protein in plant vacuoles and related transport processes
Torsten Möhlmann – Frederik Sommer/Michael Schroda – Timo Mühlhaus - Jan Hauth/Andreas Wirsen
The regulated degradation of proteins and RNAs is a process required for cellular homeostasis involving the plant vacuole. Under conditions of carbon or nitrogen starvation this process becomes even more important to counteract limitations of corresponding elements. In our work we focused so far on the characterization of the vacuolar RNA content (RNome) as a readout for such processes. For this we have established the isolation of pure intact vacuoles from young and developed Arabidopsis leaves in cooperation with the group of Ekkehard Neuhaus. Furthermore, the isolation of small RNAs, which represent the major RNA population in vacuoles, and the subsequent cloning and next generation sequencing (NGS) was achieved. In cooperation with the ITWM (Jan Hauth, Christian Salzig, Andreas Wirsen) a platform for the analysis of these NGS data was set up. The whole process was optimized and now allows for a reliable analysis of the vacuolar RNA composition of Arabidopsis leaf samples. First rounds of data analysis revealed a good reproducibility throughout the whole process. We found that fully developed leaves contained a much higher percentage of chloroplast derived ribosomal RNAs compared to undeveloped leaves. In addition, mutants lacking the vacuolar RNase RNS2 or the autophagy related protein ATG5 showed a markedly altered vacuolar RNA type composition compared to wildtype controls.
We propose to continue and expand the project of the first Biocomp funding period and thereby address the following questions: 1. Are the amounts of RNAs and proteins in vacuoles altered dependent on growth conditions (light stress, starvation), in mutants leading to increased ROS generation (NTT1-2) or upon aging (senescence)? 2. How selective is the process of autophagy with respect to protein and RNA degradation especially for chloroplast components? 3. Is vacuolar export of nucleosides and amino acids affecting the upstream processes and cellular homeostasis?
For this, we will expand the experimental approach to the analysis of the vacuolar soluble (sub‑) proteome. By this “cross –omics” approach we will learn whether RNA and protein import are coupled or proceed independently from each other. We will increase the significance of our data by introducing internal RNA and protein controls that will allow a more quantitative readout.
Autophagy facilitates import of macromolecules into vacuoles and most likely this also holds true for RNA. This process is selective for the transported cargo and it can be differentiated between e.g. chlorophagy and the transport of so called rubisco containing bodies both related to chloroplast degradation and ribophagy describing the degradation of ribosomes including ribosomal RNAs. By which ways ribosomal RNAs derived from chloroplasts are transported to the vacuole is so far unclear. Several autophagy related proteins have been identified as essential for the selectivity of autophagy (e.g. ATG4, ATG5, ATG8, Floyd et al., (2012), J. Integr. Plant Biol. 54, 907-920). By using mutants lacking these ATG proteins and investigating the vacuolar contents as described above, we will address the specificity of autophagy. We aim to enrich and investigate autophagosomes in addition to the established analysis of vacuoles and quantify their contents.
Analysis of vacuolar export processes for nucleosides and amino acids will allow to obtain information on an interaction between import and export processes. Moreover, we will learn about the physiological role of metabolite recycling for whole cell metabolism, such as counteracting nutrient starvation or support of DNA repair. For the analysis of vacuolar nucleoside export the responsible ENT1 protein will be knocked out by CRISPR/CAS9 technology. In addition, mutants lacking a newly described vacuolar amino acid exporter will be analysed.