Reactive oxygen species (ROS) play a crucial role in many cellular responses and signaling pathways, including the oxidative burst defense response to pathogens. We have examined very early events in cryptogein-induced ROS production in tobacco (Nicotiana tabacum) Bright Yellow-2 suspension cells. Using Amplex Red and Amplex Ultra Red reagents, which report realtime H 2 O 2 accumulation in cell populations, we show that the internal signal for H 2 O 2 develops more rapidly than the external apoplastic signal. Subcellular accumulation of H 2 O 2 was also followed in individual cells using the 2#,7#-dichlorofluorescein diacetate fluorescent probe. Major accumulation was detected in endomembrane, cytoplasmic, and nuclear compartments. When cryptogein was added, the signal developed first in the nuclear region and, after a short delay, in the cell periphery. Interestingly, isolated nuclei were capable of producing H 2 O 2 in a calcium-dependent manner, implying that nuclei can serve as a potential active source of ROS production. These results show complex spatial compartmentalization for ROS accumulation and an unexpected temporal sequence of events that occurs after cryptogein application, suggesting novel intricacy in ROS-signaling cascades.

Reactive oxygen species (ROS) can arise from normal metabolic activity such as organelle-based electron transport or be intermediates in signal transduction pathways activated by plant respiratory burst oxidase homologue (Rboh). UV-B exposure induces a pathogenesis-like response in leaves that can be abrogated by ROS scavengers [1]. A local signal is propagated by hydrogen peroxide and is sensitive to the application of catalase [2]. Similarly, local ROS production initiated by elicitors or pathogens can arise from stimulation of superoxide producing Rboh activity [3,4]. In this case as well, propagation of a ROS signal to adjoining cells is sensitive to catalase. Antisense of Rboh homologues in tomato lead to reduced ROS production [5]. The tomato plants show compromised wound-dependent responses. In addition, the antisense plants have a highly branched phenotype and fasciated-like reproductive organs. Transcriptome analysis of these plants revealed ectopic expression of homeotic MADS box genes that are normally expressed only in the reproductive organs. In addition, various applications of hormones were found to regulate Rboh levels. Thus, regulated ROS bursts and the general effect of Rboh activity on the steady state cellular redox milieu control short term physiological reactions and plant development. Divergent stress including temperature, drought and UV-B exposure yield overlapping transcriptome response profiles whose origin can be traced to the use of reactive oxygen signaling intermediates. Cellular scavenging systems and local production of NO are likely to temper these signalling properties by interacting with ROS and thus help to contribute to the specificity of particular responses.