Plastid-targeted proteins pass through the cytosol as unfolded precursors. If proteins accumulate in the cytosol, they can form nonspecific aggregates that cause severe cellular damage. Here, we demonstrate that high levels of plastid precursors are degraded through the ubiquitin-proteasome system (UPS) in Arabidopsis thaliana cells. The cytosolic heat shock protein cognate 70-4 (Hsc70-4) and E3 ligase carboxy terminus of Hsc70-interacting protein (CHIP) were highly induced in plastid protein import2 plants, which had a T-DNA insertion at Toc159 and showed an albino phenotype and a severe defect in protein import into chloroplasts. Hsc70-4 and CHIP together mediated plastid precursor degradation when import-defective chloroplast-targeted reporter proteins were transiently expressed in protoplasts. Hsc70-4 recognized specific sequence motifs in transit peptides and thereby led to precursor degradation through the UPS. CHIP, which interacted with Hsc70-4, functioned as an E3 ligase in the Hsc70-4-mediated protein degradation. The physiological role of Hsc70-4 was confirmed by analyzing Hsc70-4 RNA interfernce plants in an hsc70-1 mutant background. Plants with lower Hsc70 levels exhibited abnormal embryogenesis, resulting in defective seedlings that displayed high levels of reactive oxygen species and monoubiquitinated Lhcb4 precursors. We propose that Hsc70-4 and CHIP mediate plastid-destined precursor degradation to prevent cytosolic precursor accumulation and thereby play a critical role in embryogenesis. INTRODUCTIONProteins destined for the two endosymbiotic organelles (i.e., plastids and mitochondria) are targeted from the cytoplasm as unfolded precursors (Keegstra and Froehlich, 1999;Koumoto et al., 2001;Jarvis and Soll, 2002;Soll and Schleiff, 2004;Jarvis, 2008). In the cytosol, unfolded proteins have a high tendency to form cytotoxic, life-threatening, and nonspecific aggregates if they accumulate to high levels (Wickner et al., 1999;Esser et al., 2004;Meredith, 2005;Kabashi and Durham, 2006). Therefore, posttranslational targeting to endosymbiotic organelles requires that precursor levels be maintained within limits that do not result in nonspecific aggregate formation. At the same time, the cytosolic regulatory mechanism must not jeopardize the supply of sufficient amounts of proteins to the organelles.Eukaryotic cells have a protein quality control (PQC) mechanism to constantly monitor the quality of newly synthesized proteins and preexisting proteins and to actively remove unfolded or misfolded proteins (Hartl and Hayer-Hartl, 2002;Hatakeyama and Nakayama, 2003;Esser et al., 2004). It is reported that as much as 30% of newly synthesized proteins are immediately degraded by the PQC system because of a problem in protein folding (Schubert et al., 2000). The PQC in the cytosol is achieved by two opposing processes: chaperone-assisted folding and ubiquitin/proteasome-mediated degradation. The molecular chaperones heat shock protein 70 (Hsp70) and heat shock protein cognate 70 (Hsc70), whose levels are ele...

N(ε) -lysine acetylation, a reversible and highly regulated PTM, has been shown to occur in the model Gram-negative bacteria Escherichia coli and Salmonella enterica. Here, we extend this acetylproteome analysis to Bacillus subtilis, a model Gram-positive bacterium. Through anti-acetyllysine antibody-based immunoseparation of acetylpeptides followed by nano-HPLC/MS/MS analysis, we identified 332 unique lysine-acetylated sites on 185 proteins. These proteins are mainly involved in cellular housekeeping functions such as central metabolism and protein synthesis. Fifity-nine of the lysine-acetylated proteins showed homology with lysine-acetylated proteins previously identified in E. coli, suggesting that acetylated proteins are more conserved. Notably, acetylation was found at or near the active sites predicted by Prosite signature, including SdhA, RocA, Kbl, YwjH, and YfmT, indicating that lysine acetylation may affect their activities. In 2-amino-3-ketobutyrate CoA ligase Kbl, a class II aminotransferase, a lysine residue involved in pyridoxal phosphate attachment was found to be acetylated. This data set provides evidence for the generality of lysine acetylation in eubacteria and opens opportunities to explore the consequences of acetylation modification on the molecular physiology of B. subtilis.

SummaryRapid tip growth allows for efficient development of highly elongated cells (e.g. neuronal axons, fungal hyphae and pollen tubes) and requires an elaborate spatiotemporal regulation of the growing region. Here, we use the pollen tube as a model to investigate the mechanism regulating the growing region. ROPs (Rho-related GTPases from plants) are essential for pollen tip growth and display oscillatory activity changes in the apical plasma membrane (PM). By manipulating the ROP activity level, we showed that the PM distribution of ROP activity as an apical cap determines the tip growth region and that efficient tip growth requires an optimum level of the apical ROP1 activity. Excessive ROP activation induced the enlargement of the tip growth region, causing growth depolarization and reduced tube elongation. Time-lapse analysis suggests that the apical ROP1 cap is generated by lateral propagation of a localized ROP activity. Subcellular localization and gain-and loss-of-function analyses suggest that RhoGDI-and RhoGAP-mediated global inhibition limits the lateral propagation of apical ROP1 activity. We propose that the balance between the lateral propagation and the global inhibition maintains an optimal apical ROP1 cap and generates the apical ROP1 activity oscillation required for efficient pollentube elongation.

In this study, we investigated the effect of nZVI on plant root elongation in Arabidopsis thaliana and showed, for the first time, that nZVI enhanced root elongation by inducing OH radical-induced cell wall loosening. Exposure of plants to 0.5 g/L nZVI enhanced root elongation by 150-200% over that in the control, and further mechanistic studies showed that this occurred via nZVI-mediated OH radical-induced cell wall loosening. The oxidation capacity of nZVI, leading to release of H2O2, allowed it to cause OH radical-induced cell wall loosening in roots. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometers (MALDI-TOFMS)-based analysis clearly revealed that pectin-polysaccharides in roots were degraded; they are one of the main matrix-polysaccharide-connecting and load-bearing polymers in cell walls. Rapid root elongation led to structural changes in root cell walls: reduction of cell wall thickness and a bias on the orientation of cellulose microfibrils. Additionally, the asymmetrical distribution of tensional strength resulted from the OH radical-induced cell wall loosening enhanced endocytosis. These findings emphasize that OH radical-induced cell wall loosening is important for mechanical regulation of the cell wall and provide new insights into the cellular responses of plants exposed to reactive metal nanoparticles.

The nucleotide sequence around the translational initiation site is an important cis-acting element for post-transcriptional regulation. However, it has not been fully understood how the sequence context at the 5′-untranslated region (5′-UTR) affects the translational efficiency of individual mRNAs. In this study, we provide evidence that the 5′-UTRs of Arabidopsis genes showing a great difference in the nucleotide sequence vary greatly in translational efficiency with more than a 200-fold difference. Of the four types of nucleotides, the A residue was the most favourable nucleotide from positions −1 to −21 of the 5′-UTRs in Arabidopsis genes. In particular, the A residue in the 5′-UTR from positions −1 to −5 was required for a high-level translational efficiency. In contrast, the T residue in the 5′-UTR from positions −1 to −5 was the least favourable nucleotide in translational efficiency. Furthermore, the effect of the sequence context in the −1 to −21 region of the 5′-UTR was conserved in different plant species. Based on these observations, we propose that the sequence context immediately upstream of the AUG initiation codon plays a crucial role in determining the translational efficiency of plant genes.