Rapid determination of seed vigor based on the level of superoxide generation during early imbibition

Abstract

It has been reported that a large amount of reactive oxygen species (ROS) is produced during seed imbibition and this ROS is related to seed vigor. To make this physiological mechanism clear, we have used 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo(1,2-α)pyrazin-3-one (MCLA) as a sensitive and physiologically compatible probe for the determination of superoxide anion (O₂˙^–) production in vivo. Our results showed that dry rice (Oryzae sativa L.) seed embryo cells possessed the capacity to generate O₂˙^–. Conversely, the O₂˙^– production of seed embryo cells was inhibited by quinacrine (QA) and diphenylene iodonium (DPI), two specific inhibitors of NADPH oxidase, and O₂˙^– induced MCLA-mediated chemiluminescence was also blocked by superoxide dismutase (SOD). Additionally, O₂˙^–-production ability increased dramatically in a NADPH-dependent way in the plasma membraneprotein abstract from rice seed embryo cells, whereas SOD and the inhibitors mentioned above suppressed O₂˙^– production. These preliminary results suggested that rice seeds contained intrinsic NADPH oxidase activity. To validate this conclusion, dichlorofluorescein (DCF) fluorescence staining was used (observed under a laser scanning microscope, LSM) to reflect the in situ assessment of O₂˙^–-generation. The position of O₂˙^– production located at the plasma membrane. Additionally the ability to synthesize O₂˙^– was activated directly by calcium ions. These observations are in accord with the character of NADPH oxidase catalyzed O₂˙^–-generation. All these results indicated that NADPH oxidase contribute to O₂˙^– production and release to the outside. We concluded that NADPH oxidase plays an intrinsic role as an NADPH sensor, so, measuring the O₂˙^– one can monitor the NADPH concentration, which is an index of seed vigor. Therefore the O₂˙^– generation during early imbibition can serve as a rapid measurement of seed vigor.