Diverse Subcellular Locations of Cryptogein-Induced Reactive Oxygen Species Production in Tobacco Bright Yellow-2 Cells

Abstract

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.

Unraveling the Tapestry of Networks Involving Reactive Oxygen Species in Plants

“…In addition, inhibition of the fusion of these ROScontaining endosomes with central vacuoles leads to increased salt tolerance. Intriguingly, a previously unidentified nuclear source of ROS production has been observed during elicitor treatment (Ashtamker et al, 2007). These reports add two extra subcellular sources of ROS production in plant cells, thereby adding to the complexity of the ROS signaling network.…”

“…Allan and Fluhr, 1997) and supports a model in which multiple sources of ROS production are involved in the response of plants to pathogen or insect interactions. Interestingly, ROS accumulation in response to elicitation was observed in nuclei (Ashtamker et al, 2007), chloroplasts , and mitochondria (Vidal et al, 2007). In addition, the distribution of iron within the different subcellular compartments was linked to ROS production and activation of plant defenses during the oxidative burst (G. .…”

“…Dichlorofluoroscein dyes have been widely used to monitor ROS levels in plants. Recently, the dyes Amplex Red and Amplex Ultra Red were added to the toolbox for detection/imaging of H 2 O 2 and successfully used to demonstrate ROS production in nuclei (Ashtamker et al, 2007). An interesting application for the measurement of ROS and photosynthesis was reported by Hideg and Schreiber (2007), who used 3-(N-dansyl)aminomethyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole, coupled with fluorescence imaging, to monitor in parallel in vivo photosynthesis and ROS formation.…”

Singlet Oxygen Signatures Are Detected Independent of Light or Chloroplasts in Response to Multiple Stresses

“…Nonetheless, the accumulation of superoxide and H 2 O 2 is readily detected in local or systemic wounded tissue (Sagi et al, 2004;Warwar et al, 2011) and in the immune responses (Dubiella et al, 2013). A possible source could be NADPH oxidase-like and superoxide dismutase activity, as has been shown for elicitor and salt-induced accumulation of ROS (Leshem et al, 2006;Ashtamker et al, 2007;Suzuki et al, 2011). Similarly, biotic elicitors are known to initiate ROS, likely through activation of calcium and phosphorylation cascades (Sagi and Fluhr, 2001;Fluhr, 2009;Dubiella et al, 2013).…”

The AtrbohD-Mediated Oxidative Burst Elicited by Oligogalacturonides in Arabidopsis Is Dispensable for the Activation of Defense Responses Effective against Botrytis cinerea

“…However, intracellular generation of ROS has mainly been studied in relation to abiotic stress (Asada, 1999;del Río et al, 2002). There are reports of intracellular accumulation of ROS in response to elicitors, such as cryptogein (Ashtamker et al 2007), although its role in plant defense response has not been assessed.…”