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

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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 membrane protein 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.

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Authors and Affiliations

  1. MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, South China Normal University, Guangzhou, 510631, China

    Xuejun Liu, Da Xing, Lingling Li & Lingrui Zhang

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  1. Xuejun Liu
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  2. Da Xing
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  3. Lingling Li
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  4. Lingrui Zhang
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Correspondence to Da Xing.

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Liu, X., Xing, D., Li, L. et al. Rapid determination of seed vigor based on the level of superoxide generation during early imbibition. Photochem Photobiol Sci 6, 767–774 (2007). https://doi.org/10.1039/b704337f

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