Salicylic acid (SA), the known mediator of systemic acquired resistance, induced stomatal closure of Vicia faba L. Application of SA to the epidermal peels evoked an elevation of chemiluminescence of Cripridina lucigenin-derived chemiluminescent reagent (CLA) which is sensitive to superoxide anion (O₂^·–). The SA-induced generation of chemiluminescence was suppressed by O₂^·–-specific scavengers superoxide dismutase (SOD) and 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron). These results suggest that O₂^·– was generated in epidermal peels by SA-treatment. A peroxidase inhibitor salicylhydroxamic acid (SHAM) inhibited guaiacol peroxidase activity and suppressed the SA-induced CLA chemiluminescence in the epidermal peels, suggesting that O₂^·– generation occurred by the peroxidase-catalyzed reaction as proposed for SA-treated tobacco cell suspension culture [Kawano et al. (1998)Plant Cell Physiol. 39: 721]. SOD, Tiron or SHAM suppressed the SA-induced stomatal closure. Moreover, application of superoxide-generating system also induced stomatal closure. These results support the concept of involvement of reactive oxygen species in signal transduction in SA-induced stomatal closure.

Addition of salicylic acid (SA) to tobacco (Nicotiana tabacum) suspension culture immediately induced a rapid and transient generation of superoxide anion (O⁻₂), followed by a transient increase in cytosolic free calcium ion concentration ([Ca²⁺]_c). The level of SA-induced O⁻₂ was lowered by treatment with several scavengers of active oxygen species and a peroxidase inhibitor, but not with an NADPH oxidase inhibitor. The SA-induced [Ca²⁺]_c elevation was also lowered by inhibitors which effectively lowered the O⁻₂ level. Inhibition of [Ca²⁺]_c elevation by Ca²⁺ channel blockers and a Ca²⁺ chelator indicated that extracellular Ca²⁺ was responsible for the increased [Ca²⁺]_c. Among the several SA analogs, only compounds that actively induced the O⁻₂ generation also elevated [Ca²⁺]_cIn addition, the inhibitory effects of SA analogs on catalase activity correlated well with their effects on the O⁻₂ generation and the [Ca²⁺]_c elevation. SA-dependent O⁻₂ generation was shown to occur extracellularly, requiring both H₂O₂ and at least one proteinaceous factor excreted from the cells. This factor was determined to be a salicylhydroxamic acid-sensitive extracellular guaiacol-utilizing peroxidase.

Arabidopsis roots are routinely exposed to light both during their cultivation within transparent Petri dishes and during their confocal microscopy analysis. Here we report that illumination of roo...

Monocyte chemotactic protein-1 (MCP-1) binding to its receptor, CCR2B, plays an important role in a variety of diseases involving infection, inflammation, and/or injury. In our effort to understand the molecular basis of this interaction and its biological consequences, we recognized a conserved hexad of amino acids at the N-terminal extracellular domain of several chemokine receptors, including CCR2B. Human embryonic kidney 293 cells expressing Flag-tagged CCR2B containing site-directed mutations in this region, 21–26, including a consensus tyrosine sulfation site were used to determine MCP-1 binding and its biological consequences. The results showed that several of these amino acids are important for MCP-1 binding and consequent lamellipodium formation, chemotaxis, and signal transduction involving adenylate cyclase inhibition and Ca²⁺ influx into cytoplasm. Mutations that prevented adenylate cyclase inhibition and Ca²⁺ influx did not significantly inhibit lamellipodium formation and chemotaxis, suggesting that these signaling events are not involved in chemotaxis. CCR2B was found to be sulfated at Tyr²⁶; this sulfation was abolished by the substitution of Tyr with Ala and severely reduced by substitution of Asp²⁵, a part of the consensus sulfation site. The expressed CCR2B was found to be N-glycosylated, as N-glycosidase F treatment of the receptor or growth of the cells in tunicamycin reduced the receptor size to the same level, from 50 to 45 kDa. Thus, CCR2B is the first member of the CC chemokine receptor family shown to be a glycoprotein that is sulfated at the N-terminal Tyr. These post-translational modifications probably have significant biological functions.