The Arabidopsis thaliana secretome was analyzed by the proteomic approach, which led to the identification of secreted proteins implicated in many aspects of cell biology. We then investigated the change in the Arabidopsis secretome in response to salicylic acid and identified several proteins involved in pathogen response. One of these, a secreted lipase with a GDSL-like motif designated GDSL LIPASE1 (GLIP1), was further characterized for its function in disease resistance. glip1 plants were markedly more susceptible to infection by the necrotrophic fungus Alternaria brassicicola compared with the parental wild-type plants. The recombinant GLIP1 protein possessed lipase and antimicrobial activities that directly disrupt fungal spore integrity. Furthermore, GLIP1 appeared to trigger systemic resistance signaling in plants when challenged with A. brassicicola, because pretreatment of the glip1 mutant with recombinant GLIP1 protein inhibited A. brassicicola-induced cell death in both peripheral and distal leaves. Moreover, glip1 showed altered expression of defense-and ethylene-related genes. GLIP1 transcription was increased by ethephon, the ethylene releaser, but not by salicylic acid or jasmonic acid. These results suggest that GLIP1, in association with ethylene signaling, may be a critical component in plant resistance to A. brassicicola.

Stomatal opening provides access to inner leaf tissues for many plant pathogens, so narrowing stomatal apertures may be advantageous for plant defense. We investigated how guard cells respond to elicitors that can be generated from cell walls of plants or pathogens during pathogen infection. The effect of oligogalacturonic acid (OGA), a degradation product of the plant cell wall, and chitosan (␤-1,4-linked glucosamine), a component of the fungal cell wall, on stomatal movements were examined in leaf epidermis of tomato (Lycopersicon esculentum L.) and Commelina communis L. These elicitors reduced the size of the stomatal aperture. OGA not only inhibited light-induced stomatal opening, but also accelerated stomatal closing in both species; chitosan inhibited light-induced stomatal opening in tomato epidermis. The effects of OGA and chitosan were suppressed when EGTA, catalase, or ascorbic acid was present in the medium, suggesting that Ca 2؉ and H 2 O 2 mediate the elicitor-induced decrease of stomatal apertures. We show that the H 2 O 2 that is involved in this process is produced by guard cells in response to elicitors. Our results suggest that guard cells infected by pathogens may close their stomata via a pathway involving H 2 O 2 production, thus interfering with the continuous invasion of pathogens through the stomatal pores.

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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...

Gibberellins control various aspects of growth and development. Here, we identified a gene, designated paclobutrazol resistance1 (PRE1), by screening Arabidopsis activation-tagged lines. PRE1 encodes a helix-loop-helix protein and belongs to a small gene family. Physiological and genetic analysis indicated that overexpression of PRE1 altered various aspects of gibberellin-dependent responses such as germination, elongation of hypocotyl/petiole, floral induction and fruit development, and suppressed gibberellin-deficient phenotypes of the ga2 mutant. Expression of some gibberellin-responsive genes was also affected by PRE1. Expression of PRE1 was shown to be early gibberellin inducible in the wild-type plants and under control of SPY and GAI, upstream negative regulators of gibberellin signaling. The shortened hypocotyl length phenotype of the gai-1 mutant was suppressed by PRE1 overexpression. Ectopic overexpression of each of the four PRE1-related genes conferred pleiotropic phenotypes similar to PRE1 overexpression, indicative of overlapping functions among the PRE gene family. Our results of gain-of-function studies suggest that PRE genes may have a regulatory role in gibberellin-dependent development in Arabidopsis thaliana.

The N-terminal transit peptides of nuclear-encoded plastid proteins are necessary and sufficient for their import into plastids, but the information encoded by these transit peptides remains elusive, as they have a high sequence diversity and lack consensus sequences or common sequence motifs. Here, we investigated the sequence information contained in transit peptides. Hierarchical clustering on transit peptides of 208 plastid proteins showed that the transit peptide sequences are grouped to multiple sequence subgroups. We selected representative proteins from seven of these multiple subgroups and confirmed that their transit peptide sequences are highly dissimilar. Protein import experiments revealed that each protein contained transit peptide-specific sequence motifs critical for protein import into chloroplasts. Bioinformatics analysis identified sequence motifs that were conserved among members of the identified subgroups. The sequence motifs identified by the two independent approaches were nearly identical or significantly overlapped. Furthermore, the accuracy of predicting a chloroplast protein was greatly increased by grouping the transit peptides into multiple sequence subgroups. Based on these data, we propose that the transit peptides are composed of multiple sequence subgroups that contain distinctive sequence motifs for chloroplast targeting.

A new phosphorescent zinc sensor (ZIrF) was constructed based on an Ir(III) complex bearing two 2-(2,4-difluorophenyl)pyridine (dfppy) cyclometalating ligands and a neutral 1,10-phenanthroline (phen) ligand. A zinc-specific di(2-picolyl)amino (DPA) receptor was introduced at the 4-position of the phen ligand via a methylene linker. The cationic Ir(III) complex exhibited dual phosphorescence bands in CH3CN solutions originating from blue and yellow emission of the dfppy and phen ligands, respectively. Zinc coordination selectively enhanced the latter, affording a phosphorescence ratiometric response. Electrochemical techniques, quantum chemical calculations, and steady-state and femtosecond spectroscopy were employed to establish a photophysical mechanism for this phosphorescence response. The studies revealed that zinc coordination perturbs nonemissive processes of photoinduced electron transfer (PeT) and intraligand charge transfer (ILCT) transition occurring between DPA and phen. ZIrF can detect zinc ions in a reversible and selective manner in buffered solution (pH 7.0, 25 mM PIPES) with Kd = 11 nM and pKa = 4.16. Enhanced signal-to-noise ratios were achieved by time-gated acquisition of long-lived phosphorescence signals. The sensor was applied to image biological free zinc ions in live A549 cells by confocal laser scanning microscopy. A fluorescence lifetime imaging microscope (FLIM) detected an increase in photoluminescence lifetime for zinc-treated A549 cells as compared to controls. ZIrF is the first successful phosphorescent sensor that detects zinc ions in biological samples.

Copper is an essential nutrient for life, but at the same time, hyperaccumulation of this redox-active metal in biological fluids and tissues is a hallmark of pathologies such as Wilson’s and Menkes diseases, various neurodegenerative diseases, and toxic environmental exposure. Diseases characterized by copper hyperaccumulation are currently challenging to identify due to costly diagnostic tools that involve extensive technical workup. Motivated to create simple yet highly selective and sensitive diagnostic tools, we have initiated a program to develop new materials that can enable monitoring of copper levels in biological fluid samples without complex and expensive instrumentation. Herein, we report the design, synthesis, and properties of PAF-1-SMe, a robust three-dimensional porous aromatic framework (PAF) densely functionalized with thioether groups for selective capture and concentration of copper from biofluids as well as aqueous samples. PAF-1-SMe exhibits a high selectivity for copper over other biologically relevant metals, with a saturation capacity reaching over 600 mg/g. Moreover, the combination of PAF-1-SMe as a material for capture and concentration of copper from biological samples with 8-hydroxyquinoline as a colorimetric indicator affords a method for identifying aberrant elevations of copper in urine samples from mice with Wilson’s disease and also tracing exogenously added copper in serum. This divide-and-conquer sensing strategy, where functional and robust porous materials serve as molecular recognition elements that can be used to capture and concentrate analytes in conjunction with molecular indicators for signal readouts, establishes a valuable starting point for the use of porous polymeric materials in noninvasive diagnostic applications.