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.