Abstract:
Plants are confronted with a very dynamic climate throughout their whole life. These changes challenge the plants each and every day and put them, and especially crops, under high stress. To gain further insides on how plants react upon stress is there for an important task to for plant scientists, but also for agriculture economy, the bases of our modern living. Phosphatidic acid (PA) was shown to be a very important second messenger in plants to response to stress. PA is a glycerophospholipid that acts as a signaling molecule, but is also the precursor for the synthesis of all other glycerophospholipids, underlining its importance for plants. Past studies showed that elevating PA level, especially at the plasma membrane, is a key mechanism to regulate protein function in response to stress. To better understand how PA regulates stress response is there for an important challenge. The development of new and optimized already existing techniques could help to gain further insides on how PA regulates stress response. Phosphatidic Acid Phosphatases (PAPs) are important regulators of PA level in all eukaryotes, that dephosphorylate PA to diacylglycerol. The protein structure of yeast and mammalian PAPs was well described in the past. Today we know that PAPs generally consist of a N-LIP, C-LIP and Tryptophan domain, as well as of a N-terminal helix and a M-LIP domain, both important for proper protein localization. The presence or absence of the N-terminal helix and the M-LIP domain in the Arabidopsis PAP PAH1 remained elusive. Our bioinformatical analysis suggests the presence of both, a N-terminal helix as well as a M-LIP domain for Arabidopsis PAH1. Transient expression in tobacco leaves showed that full length PAH1 localized to the endoplasmic reticulum, plasma membrane and the cytosol, even though these results need to be verified with other approaches. If the N-terminal helix and the M-LIP domain contribute in regulating PAH1 localization still needs to be shown, while our phenotypic studies in transgenic Arabidopsis plants showed that both domains are important for proper PAH1 function. Deleting the N-terminal helix led to more roundish leaves, comparable to the pah1/2 double knock out mutant. PAH1 without the M-LIP domain had no developmental defects, but seedlings treated with salt showed to be less sensitive compared to the controls. Based on recent paper and experiments done in this work, we tried to develop two genetically encoded tools to modify PA level at the plasma membrane in planta. To target both PA modifying tools to the plasma membrane we used the twelve very N-terminal amino acids of CBL1. This amino acid sequence was shown to mediate plasma membrane localization in the past. For protein detection we chose a triple mCherry fusion protein, were the single fluorophores are separated by small linker consisting of eight serine and seven glycine residues. Tei and Baskin introduced in 2020 a tool to upregulate PA level at different membranes in mammalian cells using the Streptomyces sp. strain PMF Phospholipase D (PLD). We tried to use this particular PLD to modify PA level at the plasma membrane in planta. Protein sequence alignments of the PLD against the twelve Arabidopsis thaliana PLDs revealed an amino acid sequence homology of less than 1%, making unwanted protein-protein interactions that regulate the Arabidopsis PLDs unlikely. Transient PLD expression in Nicotiana benthamiana leaves led to a strong cell death response 62 hours after the infiltration. The negative control, the catalytically inactive dPLD, did not lead to this response. Additionally, we tried to develop a tool to downregulate PA level at the plasma membrane called PAP. We used Arabidopsis thaliana PAH1 and modified the protein, based on recent studies and experiments that were done in this work, to minimize the protein in size and avoid unwanted protein-protein interactions. We used different approaches to verify the activity of both, the PLD and the PAP. We co-infiltrated the PLD and the PAP in tobacco leaves with PA specific biosensors and proteins that were shown to be recruited or activated by PA at the plasma membrane. Also, we used thin-layer and gas chromatography to analyze the activity. Unfortunately, all result lead to the conclusion, that both, PLD and PAP, did not modify PA level at the plasma membrane and need further modifications. Taken together this thesis gave novel insides in the structure of Arabidopsis thaliana PAH1 and set the bases for the development of genetically encoded tools to modify PA level at the plasma membrane in planta.
phosphatidic acid