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Title: Generation of Reactive Oxygen and Anti-Oxidant Species by Hydrodynamically-Stressed Suspensions of Morinda citrofolia

Author
item JEFFERS, PAUL - DUBLIN IRELAND
item KERINS, SINEAD - DUBLIN IRELAND
item Baker, Con
item KIERAN, PATRICIA - DUBLIN IRELAND

Submitted to: Biotechnology Progress
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/3/2006
Publication Date: 2/15/2007
Citation: Jeffers, P., Kerins, S., Baker, C.J., Kieran, P.M. 2007. Generation of Reactive Oxygen and Anti-Oxidant Species by Hydrodynamically-Stressed Suspensions of Morinda citrofolia. Biotechnology Progress. p 138-145.

Interpretive Summary: Plant diseases cause major losses to farmers each year. Better understanding of the biochemical basis for plant resistance to disease will lead to improved strategies to improve plant health and reduce losses. To do this we need model systems that will allow us to monitor rapid changes in plant cell biochemistry as they encounter germs that cause disease. One such model system involves growing plant cells in a solution or ‘suspension’ so that a large number of cells undergo the same changes and can be analyzed quickly since they are already in solution. In this paper we report that the physical stress caused by shaking these cell suspensions in order to give them air, can cause biochemical changes in plant cells similar to those caused by germs. The report cautions researchers that some of the biochemical changes in plant cell suspensions that appear to be due to germs may be partially due to physical stress. This information will be of use to scientists who use plant cell suspensions to study biochemical basis for plant resistance to disease.

Technical Abstract: The generation of reactive oxygen species (ROS) by plant cell suspension cultures, in response to the imposition of both biotic and abiotic stress, is well-documented. This study investigated the generation of hydrogen peroxide by hydrodynamically-stressed cultures of Morinda citrifolia, over a 5-hour period poststress imposition. Suspensions were exposed to repeated passages through a syringe. Extracellular hydrogen peroxide was detected using a luminol-based, chemiluminescence assay. The addition of exogenous hydrogen peroxide facilitated the detection of low levels of hydrogen peroxide in the presence of anti-oxidants. Immediately after shear exposure, there was evidence of significant anti-oxidative capacity in the sheared cell cultures, which potentially masked any oxidative burst (OB), but which decreased over the following 40 minutes. This anti-oxidative capacity was determined to derive from the shearing process and was seen to interfere with the enzyme catalysing the luminescence reaction, horseradish peroxidase, resulting in a shift in the luminescence maxima. Trials in which ground cellular debris was added to control suspensions suggested that some of the antioxidative capacity observed in stressed suspensions was directly associated with debris generated by the shearing process. Additionally, using UV-Vis spectrophotometry and HPLC, phenolic compounds, which can act as anti-oxidants, were detected in the filtrates of stressed suspensions. Under the stress conditions investigated, maximum hydrogen peroxide levels of 11.5 'M were observed, 5 hours after shear exposure. This study emphasises the importance of considering both oxidative and anti-oxidative capacities as part of a holistic approach to the determination of the OB in hydrodynamically-stressed plant cell suspension cultures.