No abstract

Following a previous microbial inoculation, plants can induce broad-spectrum immunity to pathogen infection, a phenomenon known as systemic acquired resistance (SAR). SAR establishment in Arabidopsis thaliana is regulated by the Lys catabolite pipecolic acid (Pip) and flavin-dependent-monooxygenase1 (FMO1). Here, we show that elevated Pip is sufficient to induce an FMO1-dependent transcriptional reprogramming of leaves that is reminiscent of SAR. In planta and in vitro analyses demonstrate that FMO1 functions as a pipecolate N-hydroxylase, catalyzing the biochemical conversion of Pip to N-hydroxypipecolic acid (NHP). NHP systemically accumulates in plants after microbial attack. When exogenously applied, it overrides the defect of NHP-deficient fmo1 in acquired resistance and acts as a potent inducer of plant immunity to bacterial and oomycete infection. Our work has identified a pathogen-inducible L-Lys catabolic pathway in plants that generates the N-hydroxylated amino acid NHP as a critical regulator of systemic acquired resistance to pathogen infection.

Myeloid-related protein (MRP) 8 and MRP14, two members of the S100 family expressed in myelomonocytic cells, have been ascribed some extracellular functions, e.g. antimicrobial, cytostatic, and chemotactic activities. Since S100 proteins lack structural requirements for secretion via the classical endoplasmic reticulum/Golgi route, the process of secretion is unclear. We now demonstrate the specific, energy-dependent release of MRP8 and MRP14 by human monocytes after activation of protein kinase C. This secretory process is not blocked by inhibitors of vesicular traffic through the endoplasmic reticulum and Golgi, and comparative studies on tumor necrosis factor-alpha and interleukin-1beta indicate that MRP8 and MRP14 follow neither the classical nor the interleukin-1-like alternative route of secretion. Inhibition by microtubule-depolymerizing agents revealed that MRP8/MRP14 secretion requires an intact tubulin network. Accordingly, upon initiation of MRP8/MRP14 secretion, immunofluorescence microscopy showed a co-localization of both proteins with tubulin filaments. Release of MRP8 and MRP14 is associated with down-regulation of their de novo synthesis, suggesting that extracellular signaling via MRP8/MRP14 is restricted to distinct differentiation stages of monocytes. Our data provide evidence that the S100 proteins MRP8 and MRP14 are secreted after activation of protein kinase C via a novel pathway requiring an intact microtubule network.

Changes in cytosolic calcium concentrations regulate a wide variety of cellular processes, and calcium-binding proteins are the key molecules in signal transduction, differentiation, and cell cycle control. S100A12, a recently described member of the S100 protein family, has been shown to be coexpressed in granulocytes and monocytes together with two other S100 proteins, MRP8 (S100A8) and MRP14 (S100A9), and a functional relationship between these three S100 proteins has been suggested. Using Western blotting, calcium overlays, intracellular flow cytometry, and cytospin preparations, we demonstrate that S100A12 expression in leukocytes is specifically restricted to granulocytes and that S100A12 represents one of the major calcium-binding proteins in these cells. S100A12, MRP8, and MRP14 translocate simultaneously from the cytosol to cytoskeletal and membrane structures in a calcium-dependent manner. However, no evidence for direct protein-protein interactions of S100A12 with either MRP8 or MRP14 or the heterodimer was found by chemical cross-linking, density gradient centrifugation, mass spectrometric measurements, or yeast two hybrid detection. Thus, S100A12 acts individually during calcium-dependent signaling, independent of MRP8, MRP14, and the heterodimer MRP8/ MRP14. This granulocyte-specific signal transduction pathway may offer attractive targets for therapeutic intervention with exaggerated granulocyte activity in pathological states.Granulocytes and monocytes are major effector cells during inflammatory processes. Undue activation of these cells is a pathophysiological factor in many diseases, e.g. rheumatoid arthritis and chronic inflammatory bowel disease (1-4). In monocytes and granulocytes, intracellular Ca 2ϩ regulates various acute response activities, such as respiratory burst, phagocytosis, degranulation, and release of degrading enzymes (5-8). One molecular pathway of calcium signal transduction is calcium-binding proteins of the S100 multigene family, which comprises a group of small, acidic proteins with a tissue-and cell cycle-specific expression (9 -11). S100 proteins contain two calcium-binding sites per molecule (12). Two members of this family have been found in human granulocytes and monocytes, called macrophage migration inhibitory factor-related protein 8 (MRP8) 1 (S100A8) and MRP14 (S100A9), which represent up to 40% of the calcium binding capacity in monocytes (13,14). Both proteins form noncovalently associated complexes in a calciumdependent manner (15, 16). These complexes translocate from cytoplasm to membranes, as well as to intermediate filaments, after elevation of intracellular Ca 2ϩ levels, and this correlates with the induction of inflammatory actions of granulocytes and monocytes. Thus, MRP8 and MRP14 seem to be important regulators of cytoskeletal/membrane interactions during phagocyte activation (17-19).The calcium-induced change in the complex pattern of these two proteins has been considered to be important for their biological function (15,19). For example, only the ...

The nonprotein amino acid pipecolic acid (Pip) regulates plant systemic acquired resistance and basal immunity to bacterial pathogen infection. In Arabidopsis (Arabidopsis thaliana), the lysine (Lys) aminotransferase AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) mediates the pathogen-induced accumulation of Pip in inoculated and distal leaf tissue. Here, we show that ALD1 transfers the a-amino group of L-Lys to acceptor oxoacids. Combined mass spectrometric and infrared spectroscopic analyses of in vitro assays and plant extracts indicate that the final product of the ALD1-catalyzed reaction is enaminic 2,3-dehydropipecolic acid (DP), whose formation involves consecutive transamination, cyclization, and isomerization steps. Besides L-Lys, recombinant ALD1 transaminates L-methionine, L-leucine, diaminopimelate, and several other amino acids to generate oxoacids or derived products in vitro. However, detailed in planta analyses suggest that the biosynthesis of 2,3-DP from L-Lys is the major in vivo function of ALD1. Since ald1 mutant plants are able to convert exogenous 2,3-DP into Pip, their Pip deficiency relies on the inability to form the 2,3-DP intermediate. The Arabidopsis reductase ornithine cyclodeaminase/m-crystallin, alias SYSTEMIC ACQUIRED RESISTANCE-DEFICIENT4 (SARD4), converts ALD1-generated 2,3-DP into Pip in vitro. SARD4 significantly contributes to the production of Pip in pathogen-inoculated leaves but is not the exclusive reducing enzyme involved in Pip biosynthesis. Functional SARD4 is required for proper basal immunity to the bacterial pathogen Pseudomonas syringae. Although SARD4 knockout plants show greatly reduced accumulation of Pip in leaves distal to P. syringae inoculation, they display a considerable systemic acquired resistance response. This suggests a triggering function of locally accumulating Pip for systemic resistance induction.