Analysis of Thionin Proprotein Processing in Arabidopsis

Antimicrobial peptides are widespread in animals and plants as part of the innate immune system. Thionins are a group of plant-specific antimicrobial peptides which are often stabilized by 3 or 4 disulfide bridges. They are produced as precursor preproproteins containing a signal peptide for secretion, the thionin domain and a so-called acidic-domain. The mature peptides are found in cell wall, vacuoles, and protein bodies. Plants must therefore contain a Thionin Proprotein Processing Enzyme (TPPE) to remove the acidic domain. In a previous FWF project we have isolated such a TPPE from barley. We produced the TPPE in E. coli and showed that it was able to process a barley leaf thionin proprotein (which was also produced in E. coli) in vitro. The acidic domain was cleaved at several sites, which explains why a peptide corresponding to an acidic domain has never been isolated from plants. The thionin was protected from processing by the TPPE by its threedimensional structure which is stabilised by disulfide bridges. In this project it is proposed to further study thionin proprotein processing in planta in order to confirm the in vitro results. This would be done in Arabidopsis which is a much better experimental system than barley for this purpose. Arabidopsis contains 4 thionin genes which are expressed in different tissues. Our unpublished results indicate that Arabidopsis contains a single TPPE (AtSbt1.4) which is homologous to the barley TPPE. The AtSbt1.4 and 2 related subtilases (AtSbt1.2 and AtSbt1.7) have already been cloned as have the proproteins for all 4 Arabidopsis thionins. Processing of these proproteins by the TPPE and related enzymes (including the barley TPPE) would be first studied in vitro using especially mass spectrometry as has been done before for the barley TPPE. These studies would then be extended to study processing in planta using available T-DNA mutants and the AGROBEST system for transient expression in Arabidopsis seedlings. On top of this, transgenic lines which express FLAG-tagged proproteins will be produced which will be useful to study processing in tissues other than seedlings. These experiments should confirm AtSbt1.4 as Arabidopsis TPPE. Furthermore, sequence requirements for the cleavage site of the proprotein will be analyzed using a range of different peptides which will be incubated with the TPPE and cleavage analysed by mass spectroscopy. Thionins have been found in vacuoles, cell walls and protein bodies but it is not clear where in the cell processing occurs. Therefore, the localization of the Arabidopsis thionins will be analyzed using antibodies and the localization of the TPPE will be studied using also antibodies as well as tagging with GFP and FLAG. Finally, it is proposed to study the function of the PA domain and the C-terminal domain of AtSbt1.4 using a mutant approach and analysis of the function of these mutants in vitro and in planta.

Thionins are antimicrobial peptides found only in plants. They are first produced as preproproteins and then processed to yield the usually 5 kd, basic thionin peptide with 3 or 4 disulfide bridges. We had previously purified a barley TPPE (Thionin Proprotein Processing Enzyme). Here we used the sequence of the barley TPPE to identify Arabidopsis TPPE as subtilase Sbt1.4. We expressed all 4 Arabidopsis proproteins, viscotoxin proprotein and barley leaf-thionin proprotein in E. coli and purified them to high purity. The Arabidopsis TPPE was also expressed in E. coli. However, compared to the barley TPPE, the Arabidopsis TPPE could only be induced to a low level and we were only able to obtain a low amount of rather impure Arabidopsis TPPE. The enzyme was tested with all proproteins in vitro and was able to process all of them as shown by gel electrophoresis. Closer inspection with mass spectrometry has still to be done. Expression of the Arabidopsis TPPE was studied with a promoter::GUS fusion. Staining was seen in seedlings immediately after germination and was very strong in young seedlings and there especially in the hypocotyl region. Staining in roots was generally very low. Staining was found in young and old leaves and also in stem leaves. In leaves the base of trichomes was stained and also stomata. Staining was also found in flowers and siliques. This expression is in accordance with what is known about the expression of the four thionin genes. One important question was if the subtilase Sbt1.4 encoded by the gene At3g14067 is the only Arabidopsis TPPE. To address that question we produced homozygous transgenic lines in wildtype and in the At3g14067 mutant with a FLAG tagged proprotein construct for Thi2.4. We used Western blots probed with an Anti-FLAG antibody to detect the tagged thionin proprotein which showed that in the mutant there was the unprocessed proprotein containing the FLAG tag. Western blots with the Thi2.4 antibody showed the Thi2.4 peptide in the wildtype extracts. From our results we conclude that Sbt1.4 is the only TPPE in Arabidopsis for Thi2.4 while we cannot exclude that there are additional TPPEs for the other Arabidopsis thionins. While this project was running, we gained access to the data from the One Thousand Plant Transcriptomes Initiative (1KP project) which has sequenced the transcriptomes of more than 1000 plant species. The new data from the 1KP project led us to revise the thionin classification. We now recognize only class one thionins with 8 cysteine residues and class 2 thionins with 6 cysteine residues. The different variants that we found (and also previously known variants) can all be traced back to one of these 2 classes.