Browsing by Subject "Sulfatierung"

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    The formation of an apoplastic diffusion barrier in Arabidopsis seeds is regulated by peptide hormone signaling
    (2022) Royek, Stefanie; Schaller, Andreas
    Diffusion barrier formation is a critical factor in plant development. The most well described diffusion barriers in Arabidopsis are the Casparian strip and the cuticle. They function in the formation of organ boundaries, prevent water and molecule loss, and protect the plant against environmental stresses. The Casparian strip surrounds the root vascular tissue, whereas the cuticle covers aerial plant organs and is formed de novo during seed development. Embryonic cuticle formation is regulated by a peptide hormone signaling pathway, involving the leucine rich repeat receptor like kinases GASSHO1 (GSO1), GASSHO2 (GSO2) (Tsuwamoto et al. 2008) and the subtilisin-like serine protease ABNORMAL LEAF SHAPE 1 (ALE1). Whereas the latter pathway components have been identified in 2001 and 2008, the peptide hormone mediating the signaling has remained elusive. One aim of this work was to identify the missing pathway element. It was hypothesized that the peptide hormone is released from a larger precursor by ALE1 protease activity to trigger cuticle formation via interaction with the GSO receptors. To uncover the unknown element, the signaling pathway for Casparian strip formation, prooved to be a useful lead. Remarkably, Casparian strip and embryonic cuticle formation employ the same receptor (GSO1), and for Casparian strip formation the GSO1 ligands are known to be members of the CASPARIAN STRIP INTEGRITY FACTOR (CIF) protein family (Doblas et al. 2017, Nakayama et al. 2017). Based on its similarity to the mature CIF peptides and on its phenotypic appearance, it was speculated that a seed expressed protein, called TWISTED SEED1 (TWS1), could serve as the sought ALE1 substrate. As it can be challenging to link proteases to their physiological substrates, this work describes methods how to identify protease specific cleavage sites. One of them was applied to test if TWS1 serves as ALE1 substrate. GFP-tagged TWS1 was transiently coexpressed with ALE1 in Nicotiana benthamiana via agroinfiltration. An ALE1-specific TWS1 cleavage product was detected in the protein extract of coinfiltrated leaves. It was identified by pull down via GFP immunoprecipitation, subsequent separation by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS) analysis. Another method, described in this work, is the identification of protease cleavage sites by in-gel reductive dimethylation: cleavage product-containing gel bands are treated with formaldehyde and cyanoborohydride, prior to in-gel tryptic digest, to achieve a dimethylation of N-terminal free amino groups. The chemically modified N-termini can rapidly be identified and assigned to previous cleavage by the protease of interest. With the method described above, it was found that TWS1 is c-terminally cleaved by ALE1. The two amino acids directly flanking the cleavage site were found to be important for ALE1 cleavage site selection, as their substitution caused a loss of ALE1- dependent cleavage. Our cooperation partners demonstrated an interaction of mature TWS1 with the GSO receptors. The binding affinity of mature TWS1 was reduced by a 3 amino acid C-terminal extension, demonstrating the biological relevance of ALE1-mediated TWS1 processing. Like the CIFs, TWS1 contains a DY tyrosine sulfation motive at its N-terminal processing site. The role of tyrosine sulfation in precursor processing is largely unexplored and was addressed in this work by comparing in-vitro cleavage of different sulfated versus nonsulfated TWS1 precursors. SBT1.8 was found to cleave TWS1 at the N-terminal processing site, and cleavage site selection was influenced by the sulfation state of TWS1 P2´ tyrosine. A homology based 3D model of SBT1.8 was created, which suggested that SBT1.8 interacts with the negatively charged sulfate via a positively charged arginine residue (R302). The role of R302 in substrate binding and recognition was confirmed by in-vitro cleavage assays with mutated SBT1.8 versions, in which R302 was replaced. N-terminal TWS1 cleavage was no longer observed when R302 was substituted. Likewise, no N-terminal cleavage was observed for two other seed expressed Arabidopsis subtilases (SBT1.1 and SBT5.4) that feature an arginine at the corresponding position, indicating that the sole presence of R302 is not sufficient for N terminal cleavage site recognition.