The RNA-binding protein RNP29 is an unusual Toc159 transport substrate

The precursors of RNP29 and Ferredoxin (Fd2) were previously identified in the cytosol of ppi2 plant cells with their N-terminal amino acid acetylated. Here, we explore whether precursor accumulation in ppi2 is characteristic for Toc159 client proteins, by characterizing the import properties of the RNP29 precursor in comparison to Fd2 and other Toc159-dependent or independent substrates. We find specific accumulation of the RNP29 precursor in ppi2 but not in wild type or ppi1 protoplasts. With the exception of Lhcb4, precursor accumulation is also detected with all other tested constructs in ppi2. However, RNP29 is clearly different from the other proteins because only precursor but almost no mature protein is detectable in protoplast extracts. Co-transformation of RNP29 with Toc159 complements its plastid import, supporting the hypothesis that RNP29 is a Toc159-dependent substrate. Exchange of the second amino acid in the RNP29 transit peptide to Glu or Asn prevents methionine excision but not N-terminal acetylation, suggesting that different N-acetyltransferases may act on chloroplast precursor proteins in vivo. All different RNP29 constructs are efficiently imported into wild type but not into ppi2 plastids, arguing for a minor impact of the N-terminal amino acid on the import process

Identification of protein N-termini in Cyanophora paradoxa cyanelles: Transit peptide composition and sequence determinants for precursor maturation

Glaucophyta, rhodophyta and chloroplastida represent the three main evolutionary lineages that diverged from a common ancestor after primary endosymbiosis. Comparative analyses between members of these three lineages are a rich source of information on ancestral plastid features. We analyzed the composition and the cleavage site of cyanelle transit peptides from the glaucophyte Cyanophora paradoxa by terminal amine labelling of substrates (TAILS), and compared their characteristics to those of representatives of the chloroplastida. Our data show that transit peptide architecture is similar between members of these two lineages. This entails a comparable modular structure, an overrepresentation of serine or alanine and similarities in the amino acid composition around the processing peptidase cleavage site. The most distinctive difference is the overrepresentation of phenylalanine in the N-terminal 1-10 amino acids of cyanelle transit peptides. A quantitative proteome analysis with periplasm-free cyanelles identified 42 out of 262 proteins without the N-terminal phenylalanine, suggesting that the requirement for phenylalanine in the N-terminal region is not absolute. Proteins in this set are on average of low abundance, suggesting that either alternative import pathways are operating specifically for low abundance proteins or that the gene model annotation is incorrect for proteins with fewer EST sequences. We discuss these two possibilities and provide examples for both interpretations

Regulation of WRKY46 Transcription Factor Function by Mitogen-Activated Protein Kinases in Arabidopsis thaliana

Mitogen-activated protein kinase (MAPK) cascades are central signaling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs), such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defense as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defense