SummaryAlthough auxin and ethylene play pivotal roles in leaf abscission, the subsequent signaling molecules are poorly understood. This is mainly because it is difficult to effectively treat the intact abscission zone (AZ) with pharmacological reagents. We developed an in vitro experimental system that reproduces stress-induced leaf abscission in planta. In this system, 1-mm-thick petiole strips, encompassing the AZ, were separated within 4 days of abscission at the AZ through cell wall degradation in an auxin depletion-and ethylene-dependent manner. The system allowed us to show that hydrogen peroxide (H 2 O 2 ) is involved in abscission signaling. Microscopic analyses revealed continuous H 2 O 2 production by AZ cells. H 2 O 2 scavengers and diphenylene iodonium, an inhibitor of NADPH oxidase, suppressed in vitro abscission and cellulase expression. Conversely, the application of H 2 O 2 promoted in vitro abscission and expression of cellulase. Ethephon-induced abscission was suppressed by inhibitors of H 2 O 2 production, whereas the expression of ethylene-responsive genes was unaffected by both H 2 O 2 and an H 2 O 2 inhibitor. These results indicated that H 2 O 2 acts downstream from ethylene in in vitro abscission signaling. In planta, salinity stress induced the expression of genes that respond to ethylene and reactive oxygen species, and also induced H 2 O 2 production at the AZ, which preceded leaf abscission. These results indicate that H 2 O 2 has roles in leaf abscission associated with ethylene both in vitro and in planta.

The hypersensitive response (HR) is a powerful resistance system that plants have developed against pathogen attack. There are two major pathways for HR induction; one is through recognition of the pathogen by a specific host protein, and is known as the host HR. The other is through common biochemical changes upon infection--the nonhost HR. We previously demonstrated that hydrogen peroxide derived from polyamine degradation by polyamine oxidase triggers the typical host HR in tobacco plants upon infection with tobacco mosaic virus. However, it remains to be determined whether or not polyamines are involved in the nonhost HR in tobacco, and in the host HR in other plant species. When tobacco plants were infected with Pseudomonas cichorii, a representative nonhost pathogen, transcripts for six genes encoding enzymes for polyamine metabolism were simultaneously induced, and polyamines were accumulated in apoplasts. Hydrogen peroxide was concomitantly produced and hypersensitive cell death occurred at infected sites. Silencing of polyamine oxidase by the virus-induced gene silencing method resulted in suppression of hydrogen peroxide production and in disappearance of visible hypersensitive cell death with an increase in bacterial growth. Our results indicated that polyamines served as the source of hydrogen peroxide during the nonhost HR in tobacco plants. Further analysis revealed that polyamines were accumulated in apoplasts of Arabidopsis thaliana infected with Pseudomonas syringae, and of rice infected with Magnaporthe grisea, both causing the typical host HR. As in tobacco, it is conceivable that the same mechanism operates for nonhost HR in these plants. Our present observations thus suggested that polyamines are commonly utilized as the source of hydrogen peroxide during host- and nonhost HRs in higher plants.

We found that an L3 resistance-breaking field isolate of Pepper mild mottle virus (PMMoV), designated PMMoV-Is, had two amino acid changes in its coat protein (CP), namely leucine to phenylalanine at position 13 (L13F) and glycine to valine at position 66 (G66V), as compared with PMMoV-J, which induces a resistance response in L3-harboring Capsicum plants. The mutations were located to a CP domain corresponding to the outer surface of PMMoV particles in computational molecular modeling. Analyses of PMMoV CP mutants containing either or both of these amino acid changes revealed that both changes were required to efficiently overcome L3-mediated resistance with systemic necrosis induction. Although CP mutants containing either L13F or G66V could not efficiently overcome L3-mediated resistance, these amino acid changes had different effects on the elicitor activity of PMMoV CP. L13F caused a slight reduction in the elicitor activity, resulting in virus restriction to necrotic local lesions that were apparently larger than those induced by wild-type PMMoV, while G66V rendered wild-type PMMoV the ability to overcome L3-mediated resistance, albeit with a lower efficiency than PMMoV with both changes. These results suggest that a cooperative effect of the L13F and G66V mutations on the elicitor activity of CP is responsible for overcoming the L3-mediated resistance.

Reactive oxygen species (ROS) are produced in response to many environmental stresses, such as UV, chilling, salt and pathogen attack. These stresses also accompany leaf abscission in some plants, however, the relationship between these stresses and abscission is poorly understood. In our recent report, we developed an in vitro abscission system that reproduces stress-induced pepper leaf abscission in planta. Using this system, we demonstrated that continuous production of hydrogen peroxide (H(2)O(2)) is involved in leaf abscission signaling. Continuous H(2)O(2) production is required to induce expression of the cell wall-degrading enzyme, cellulase and functions downstream of ethylene in abscission signaling. Furthermore, enhanced production of H(2)O(2) occurs at the execution phase of abscission, suggesting that H(2)O(2) also plays a role in the cell-wall degradation process. These data suggest that H(2)O(2) has several roles in leaf abscission signaling. Here, we propose a model for these roles.

‘La France’ pear (Pyrus communis L.) fruit stored at 1°C for 1 month (short‐term storage) before transfer to 20°C softened and developed a melting texture during ripening, whereas fruit stored for 5 months (long‐term storage) before transfer to 20°C softened but did not develop a melting texture. To clarify the mechanisms involved in fruit softening and textural changes, the cDNAs encoding cell‐wall hydrolases were isolated by RT‐PCR, and their expression and localization were investigated in ‘La France’ pears. Genes encoding three polygalacturonases (PG; EC 3.2.1.15), four pectin methylesterases (PME; EC 3.1.1.11), one α‐arabinofuranosidase (ARF; EC 3.2.1.55), three β‐galactosidases (GAL; EC 3.2.1.23), and two endo‐1,4‐β‐d‐glucanases (Cel; EC 3.2.1.4) were isolated. Among these 13 isolated genes, PcPG1 was the only gene for which the mRNA expression levels increased in both the short‐ and long‐term stored fruits. This suggested that PcPG1 is involved in fruit softening rather than in the development of the melting texture. In contrast, the expression levels of PcPG3, PcPME1, PcPME2, PcPME3, PcGAL1, PcGAL2, and PcCel2 increased during ripening only in the short‐term stored fruit. These genes might thus be involved in the development of the melting texture.