Using Physcomitrella patens to reveal key players in salt resistance and elucidate the complex regulatory mechanisms involved in cellular salt metabolism
Final Report Abstract
The moss Physcomitrella patens shows a high resilience towards salinity. We aimed to exploit this capacity to identify key players in salt resistance and to gain deeper insights into the regulation and coordination of different Na+ exclusion mechanisms. One of the main objectives was to identify novel genes that are involved in the adaptation towards salinity stress. For this we used a yeast complementation assay screening Physcomitrella cDNA for genes that are able to confer salt resistance. However, this strategy proved to be not suitable. Hence, we employed a RNAseq approach and were able to identify numerous genes that are up-regulated under salt stress. Identified genes comprise known players within the response to salt stress such as LEA-proteins or dehydrins. Additionally we identified several unknown proteins with unknown function. In subsequent experiments (in an ongoing cooperation with the University of Copenhagen) we aim to verify the expression induction of genes of interest and test their ability to confer salt resistance upon expression in salt sensitive yeast strains. The Na+-ATPase PpENA1 which is encoded in P. patens and has been shown to play an essential role in salinity tolerance and is an attractive candidate gene to generate more salt tolerant crops via a single gene transfer. However, up to now little information about posttranscriptional regulation is available. Therefore we employed split-ubiquitin yeast two hybrid assay to identify potential proteins that interact with PpENA1. Proteins involved in protein trafficking, ATPase subunits as well as unknown proteins. In a third sub-project we elucidated the structure and composition of Photosystem I in P. patens. Work in green algae has shown that PSI can also be a target of salt stress and undergoes structural changes. Furthermore, in P. patens salt stress influences the photosynthtetic apparatus which represent important adaptation mechanisms in order to maintain a functional energy supply for the cells. To investigate the potential influence of salt stress on PSI in Physcomitrella we decided to study its subunit composition and structure since so far only bioinformatical and very little solid biochemical data are published for PSI of Physcomtrella. We established methods to isolate intact and functional PSI from Physcomitrella. Subsequently we characterized the structure, polypeptide composition and light-harvesting function using electron microscopy, mass spectrometry, biochemical and physiological methods.
