In vivo imaging of an elicitor‐induced nitric oxide burst in tobacco

Abstract

SummaryA growing body of evidence suggests that nitric oxide (NO), an important signalling and defence molecule in mammals, plays a key role in activating disease resistance in plants, acting as signalling molecule and possibly as direct anti-microbial agent. Recently, a novel¯uorophore (diamino¯uorescein diacetate, DAF-2 DA) has been developed which allows bio-imaging of NO in vivo. Here we use the cellpermeable DAF-2 DA, in conjunction with confocal laser scanning microscopy, for real-time imaging of NO in living plant cells. Epidermal tobacco cells treated with cryptogein, a fungal elicitor from Phytophthora cryptogea, respond to the elicitor with a strong increase of intracellular NO. NO-induced uorescence was found in several cellular compartments, and could be inhibited by a NO scavenger and an inhibitor of nitric oxide synthase. The NO burst was triggered within minutes, reminiscent of the oxidative burst during hypersensitive response reactions. These results reveal additional similarities between plant and animal host responses to infection.

Nitric oxide production and its functional link with OIPK in tobacco defense response elicited by chitooligosaccharide

“…We found that NO generation took place in chloroplast, nucleus, and the whole cell. The results were similar with previous work performed by Foissner et al (2000) who studied NO generation in tobacco stimulated by a proteinaceous elicitor cryptogein, but COS could induce NO production more quickly compared to cryptogein. The results suggest that there may be a common mechanism for NO production in time and space in tobacco.…”

“…It was observed that NO first appeared in chloroplast at 5 s; then the nucleus was filled with green fluorescence at 30 s; 2 min later, NO green fluorescence emerged in the cytosol and probably covering cell membrane and cell wall. This result is similar to a previous study in tobacco stimulated by fungal elicitor cryptogein (Foissner et al 2000).…”

“…samsum NN). NO measurement was performed by using their fluorescent indicator dye DAF-2DA as described previously (Foissner et al 2000) with slight modifications. The epidermis was peeled carefully from leaves and cut into 5 mm length.…”

“…This fluorescent indicator allows direct and specific measurement of NO Plant Cell Rep (2011) 30:1153-11621155 generation in plant cells (Kojima et al 1998;Foissner et al 2000;Lamotte et al 2004). Epidermal sections from tobacco were loaded with DAF-2DA and analyzed with fluorescence microscopy.…”

“…The COS-elicited burst of DAF-2DA fluorescence in tobacco epidermal cells was reduced to control level by cPTIO and NOS inhibitor L-NAME, suggesting the occurrence of a NOS-like enzyme. L-NAME is a methyl ester derivate of the NOS substrate L-Arg, and several papers reports in vitro and vivo data on inhibition of plant NOS by L-NAME (Cueto et al 1996;Foissner et al 2000;Lamotte et al 2004). In addition to NOS, plant NR catalyzes the NAD(P)H-dependent reduction of nitrite to NO (Yamasaki and Sakihama 2000).…”