Localization of Hydrogen Peroxide Accumulation during the Hypersensitive Reaction of Lettuce Cells to Pseudomonas syringae pv phaseolicola

366

286

Cell death plays important roles in the development and defense of plants as in other multicellular organisms. Rapid production of reactive oxygen species often is associated with plant defense against pathogens, but their molecular mechanisms are not known. We introduced the constitutively active and the dominant negative forms of the small GTP-binding protein OsRac1, a rice homolog of human Rac, into the wild type and a lesion mimic mutant of rice and analyzed H 2 O 2 production and cell death in transformed cell cultures and plants. The results indicate that Rac is a regulator of reactive oxygen species production as well as cell death in rice.Cell death is important in the development and defense of multicellular organisms (1). Cell death occurs in normal plant development and during infection by avirulent pathogens (2-5). It plays a key role in plant defense and shares several features with apoptosis in mammalian cells (3,5,6). Rapid production of reactive oxygen species (ROS) often is associated with cell death during resistance reactions to pathogens, and a plasma membrane NADPH oxidase similar to the neutrophil enzyme is suggested to be responsible for ROS production (5-7). However, the molecular mechanisms for ROS production and cell death in plants are largely unknown.In phagocytic cells, activation of NADPH oxidase leads to production of superoxide, which effectively kills invading microorganisms (8, 9). The neutrophil NADPH oxidase is a multicomplex enzyme consisting of two membrane proteins, gp91 phox and p22 phox , and three cytosolic factors, p47 phox , p67 phox , and Rac (8, 9), and the plant enzyme is thought to be similar to the neutrophil enzyme (10-13). Genes whose deduced amino acid sequences are similar to those of the Rho͞Rac family of the small GTP-binding proteins have been isolated in plants (14-18), and some were shown to play a role in the control of pollen tube growth (19). However, functions of most of these genes are not known. Genes whose amino acid sequences are similar to that of the animal gp91 phox also have been isolated from plants (20, 21), but their functions have not been investigated. In this study we analyzed functions of Rac in rice by expressing constitutively active and dominant negative Rac genes in transgenic cell cultures and plants and found that Rac plays an important role in ROS production as well as cell death in rice. MATERIALS AND METHODSBiochemical Analysis of Recombinant OsRac1. OsRac1 cDNA was cloned in an expression vector, pGEX-4T-1 (Pharmacia), and transformed into Escherichia coli. The glutathione S-transferase (GST)-fusion protein was purified by glutathioneSepharose beads (Pharmacia) and used for assays of GTPbinding and GTPase activities according to published protocol (22). For the assay of the GTP-binding activity, the binding of Rice Transformation. OsRac1-G19V was made by substitution of the glycine corresponding to G12 of human Rac1 by valine by the use of an LA PCR in vitro Mutagenesis kit (Takara Shuzo, Kyoto). OsRac1-T24N similarly ...

Section: Resultssupporting

confidence: 89%

The small GTP-binding protein Rac is a regulator of cell death in plants

Kawasaki

Henmi

Ono

et al. 1999

366

286

“…1B, H 2 O 2 is shown to be present in the upper unwounded leaves of young tomato plants that were wounded on the lower leaves, indicating that the generation of H 2 O 2 production was systemic and localized in the vascular tissues. In lettuce tissue, H 2 O 2 was reported to be localized within the cell walls of xylem vessels with secondary thickening and within walls of surrounding cells (18), and DAB detected H 2 O 2 in barley epidermal cells (19) and in microbursts in Arabidopsis leaf periveinal cells in response to pathogen attacks (4). DAB can detect H 2 O 2 in leaves at levels as low as 0.1 M concentrations, but strong color develops only at higher concentrations of about 1-10 M (18).…”

Section: Resultsmentioning

confidence: 99%

Hydrogen peroxide is generated systemically in plant leaves by wounding and systemin via the octadecanoid pathway

Orozco-Cárdenas

Ryan

1999

739

448

“…Transmission electron microscopy (TEM) and H 2 O 2 localization were performed as described (18,31). Electron probe x-rays were microanalyzed with a CM12 Scanning Transmission Electron Microscope equipped with a TRACOR TN 54 EDX spectrometer probe (Philips, Eindhoven, The Netherlands).…”

Section: Methodsmentioning

confidence: 99%

“…To localize H 2 O 2 , TEM was used to visualize electron-dense precipitates of cerium perhydroxides caused by the reaction of H 2 O 2 with CeCl 3 (31). Adventitious rootlets were harvested 50 and 70 h post inoculation (hpi) with ORS571.…”

Section: H2o2 Colocalizes With Plant Cell Death During Bacterial Invamentioning

confidence: 99%

Reactive oxygen species and ethylene play a positive role in lateral root base nodulation of a semiaquatic legume

D’Haeze

Rycke

Mathis

et al. 2003

173

128

Lateral root base nodulation on the tropical, semiaquatic legume Sesbania rostrata results from two coordinated, Nod factor-dependent processes: formation of intercellular infection pockets and induction of cell division. Infection pocket formation is associated with cell death and production of hydrogen peroxide. Pharmacological experiments showed that ethylene and reactive oxygen species mediate Nod factor responses and are required for nodule initiation, whereby induction of division and infection could not be uncoupled. Application of purified Nod factors triggered cell division, and both Nod factors and ethylene induced cavities and cell death features in the root cortex. Thus, in S. rostrata, ethylene and reactive oxygen species act downstream from the Nod factors in pathways that lead to formation of infection pockets and initiation of nodule primordia.