DREB2C interacts with ABF2, a bZIP protein regulating abscisic acid-responsive gene expression, and its overexpression affects abscisic acid sensitivity.

ABF2 is a basic leucine zipper protein that regulates abscisic acid (ABA)-dependent stress-responsive gene expression. We carried out yeast two-hybrid screens to isolate genes encoding ABF2-interacting proteins in Arabidopsis (Arabidopsis thaliana). Analysis of the resulting positive clones revealed that two of them encode an AP2 domain protein, which is the same as AtERF48/DREB2C. This protein, which will be referred to as DREB2C, could bind C-repeat/dehydration response element in vitro and possesses transcriptional activity. To determine its function, we generated DREB2C overexpression lines and investigated their phenotypes. The transgenic plants were ABA hypersensitive during germination and seedling establishment stages, whereas primary root elongation of seedlings was ABA insensitive, suggesting developmental stage dependence of DREB2C function. The DREB2C overexpression lines also displayed altered stress response; whereas the plants were dehydration sensitive, they were freezing and heat tolerant. We further show that other AP2 domain proteins, DREB1A and DREB2A, interact with ABF2 and that other ABF family members, ABF3 and ABF4, interact with DREB2C. Previously, others demonstrated that ABF and DREB family members cooperate to activate the transcription of an ABA-responsive gene. Our result implies that the cooperation of the two classes of transcription factors may involve physical interaction.

Role of the methionine sulfoxide reductase MsrB3 in cold acclimation in Arabidopsis.

Methionine residues of proteins are a major target for oxidation by reactive oxygen species (ROS), which are generated in response to a variety of stress conditions. Methionine sulfoxide (MetO) reductases are present in most organisms and play protective roles in the cellular response to oxidative stress, reducing oxidized MetO back to Met. Previously, an Arabidopsis MetO reductase, MsrB3, was identified as a cold-responsive protein. Here we report that MsrB3 functions in the process of cold acclimation, thus contributing to cold tolerance. In contrast to normal, wild-type plants, msrb3 mutant plants lost the ability to become tolerant to freezing temperatures following cold pre-treatment. Furthermore, when exposed to low temperature, msrb3 plants exhibited a larger increase in MetO and H(2)O(2) content and electrolyte leakage compared with wild-type and MsrB3 transgenic plants. It is also shown that MsrB3 is localized at the endoplasmic reticulum (ER). We propose that MsrB3 plays an important role in cold tolerance by eliminating MetO and ROS that accumulate at the ER during cold acclimation.

Secretome analysis reveals an Arabidopsis lipase involved in defense against Alternaria brassicicola.

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.

Analysis of the Arabidopsis nuclear proteome and its response to cold stress.

The nucleus is the subcellular organelle that contains nearly all the genetic information required for the regulated expression of cellular proteins. In this study, we comprehensively characterized the Arabidopsis nuclear proteome. Nuclear proteins were isolated and analyzed using two-dimensional (2D) gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Approximately 500-700 spots were detected in reference 2D gels of nuclear proteins. Proteomic analyses led to the identification of 184 spots corresponding to 158 different proteins implicated in a variety of cellular functions. We additionally analyzed the changes in the nuclear proteome in response to cold stress. Of the 184 identified proteins, 54 were up- or downregulated with a greater than twofold change in response to cold treatment. Among these, six proteins were selected for further characterization. Northern analysis data revealed that gene expression of these proteins was also altered by cold stress. Following transient expression in BY-2 protoplasts, two proteins were detected in both the cytoplasm and the nucleus and four others were detected exclusively in the nucleus, which correlates well with the nuclear localization patterns of the proteomic data. Our study provides an initial insight into the Arabidopsis nuclear proteome and its response to cold stress.