Submitted to: Journal of the American Society for Horticultural Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/15/2004
Publication Date: 2/15/2005
Citation: Wang, Y., Wisniewski, M.E., Meilan, R., Webb, R., Fuchigami, L., Boyer, C. 2005. Overexpression of cytosolic ascorbate peroxidase in tomato (lycopersicon esculentum) confers tolerance to chilling and salt stress. Journal of the American Society for Horticultural Science 130(2):167-173. 2005. Interpretive Summary: Unfavorable environmental conditions result in reduced crop yields and loss of vigor for perennial crops such as fruit crops. Exposure of plants to environmental stress results in the formation of reactive forms of oxygen, such as hydrogen peroxide and superoxide radicals. Collectively, these types of oxygen molecules are referred to as Reactive Oxygen Species (ROS). ROS can be very toxic and result in injury to plant membranes, proteins, and DNA. Understanding how plants manage and deal with oxidative stress is a key objective of the ARS National Program 302 which addresses Plant Molecular and Biological Processes. Increased resistance to the effects of ROS could result in plants being less injured by many different types of environmental stress. The current research examined whether or not the overexpression of ascorbate peroxidase (APX) gene in tomato could improve its resistance to chilling and salt stress. APX is part of an antioxidant system in plants that has evolved to help the plant cope with oxidative stress. This enzyme is responsible for reducing hydrogen peroxide (a toxic ROS) to water and ascorbate. We isolated the APX gene from spinach and transformed tomato to overexpress this gene. Results indicated that higher levels of APX were present in the transgenic lines of tomato compared to the non-transformed wild-type (WT) tomatoes. The transgenic tomato lines were less injured compared to WT plants when exposed to chilling and salt stress, and transgenic seeds had greater levels of germination when placed at chilling temperatures. These results indicate that overexpression of APX is an approach that can be used to produce plants more resistant to environmental stress. Alternatively, this information can be used as a breeding tool, i.e. plants can be screened for APX activity. Those plants with naturally higher levels of APX could be used as breeding material to develop new cultivars of crops that have increased resistance to environmental stress. Further research will explore the effect of overexpression of APX on other antioxidant enzymes and whether or not the overexpression of other antioxidant genes also result in improved resistance to environmental stress.
Technical Abstract: Ascorbate peroxidase (APX) plays an important role in the metabolism of hydrogen peroxide (H2O2) in higher plants, affording them protection against oxidative stress. We studied the effect of overexpressing a cytosolic ascorbate peroxidase (cAPX) gene'derived from pea (Pisum sativum)'in transgenic tomato plants (Lycopersicon esculentum). Transformants were selected in vitro using kanamycin resistance and confirmed by PCR and northern analyses. An APX native-gel assay indicated that, in the absence of stress, APX activity in transgenic plants was several times greater than that measured in wild-type (WT) plants. Several independently transformed lines were propagated and evaluated for resistance to chilling and salt stress. After placing seeds at 9 °C for five weeks, percent germination was greater for seeds obtained from transgenic lines (26% - 37%) compared to the WT (3%). Plants from transgenic lines also had lower electrolyte leakage (20% - 23%) than WT (44%) after exposure to 4 °C. Transgenic seedlings also showed enhanced tolerance to NaCl stress (200 or 250 mM). Moreover, APX activity was nearly 25-fold and 10-fold higher in the leaves of transgenic plants in response to chilling and salt stresses, respectively. Our results substantiate that increased levels of APX may play an important role in ameliorating oxidative injury induced by chilling and salt stress.