Nucleoside and RNA metabolism in plant vacuoles
Autophagy or „self eating“ is a process that currently has come into view in plant sciences. It is believed that this process is involved in many breakdown reactions of plants. Ribophagy describes the engulfment of cellular RNAs and the delivery of corresponding vesicles to the central plant vacuole for further catabolism. Furthermore, it has been described that up to 70% of the total cellular RNAse activity resides in the vacuole. We have identified a transport protein able to export the breakdown products that form after complete action of RNAse, phosphodiesterase and phosphatase activity i.e. the equilibrative nucleoside transporter (ENT) 1. In addition mutants of ENT1 and a vacuolar RNAse, RNS2 are in our hands. With help of these mutants we want to analyze the effects of constricted RNA breakdown. Besides the analysis of metabolic intermediates, deep sequencing of vacuolar RNAs by Illumina next generation sequencing is a most important tool in this project. For a proof of concept RNAs from isolated vacuoles of plants grown under standard conditions have been analyzed this way and the presence of high numbers of different small RNAs was confirmed. In this project Arabidopsis and Barley as a crop plant will be subjected to nutrient limitation (phosphate and nitrogen starvation conditions) and the dynamics of RNA catabolism will be studied. Furthermore, mutants will be included in the analysis. In a later period of the project this type of analysis will be extended to mutants in ribophagy. We expect to gain important information about the recycling of valuable nutrients through ribophagy and breakdown which will be useful to optimize phosphate- and nitrogen use efficiency in plants.
Whereas the process of next generation sequencing is highly automated the analysis of the corresponding large scale data is very time consuming and restricted to experts in bioinformatics. Therefore, interaction with the cooperation partners from informatics/mathematics is urgently needed. This implies also the visualization of large scale data. A goal for the long run would be the automatisation of data processing for non expert users or even the development of an easy to use tool.