Reactive oxygen metabolism in mycorrhizal and non-mycorrhizal citrus (Poncirus trifoliata) seedlings subjected to water stress
Introduction
When higher plants are subjected to environmental stresses, such as water stress and salinity, a variety of reactive oxygen species (ROS) such as superoxide anion radical (O2−), hydrogen peroxide (H2O2), hydroxyl radicals (OH) and singlet oxygen (1O2) are induced (Elstner, 1982; Jung, 2004). The ROS may initiate destructive oxidative processes such as chlorophyll bleaching, lipid peroxidation, protein oxidation, and damage to nucleic acids (Herbinger et al., 2002). As a consequence, higher plants induce efficient antioxidant systems to protect them against oxidative injury (Asada, 1999). The antioxidant systems consist of antioxidant enzymes including superoxide dismustase (SOD), guaiacol peroxidase (G-POD), catalase (CAT), glutathione reductase (GR), ascorbate peroxidase (APX), and non-enzymatic antioxidants including ascorbate (ASC) and glutathione (GSH), which are designed to minimize the concentrations of O2− and H2O2.
Ninety percent of the earth's land plant species form symbiotic associations with arbuscular mycorrhizal fungi (AMF) (Gadkar et al., 2001). Arbuscular mycorrhizal (AM) symbiosis can affect the water relations of many plants (Auge, 2001). The effect is often more pronounced in plants grown under water stressed (WS) conditions than under well-watered (WW) conditions (Sanchez-Diaz and Honrubia, 1994). Stomatal conductance, transpiration rates, hydraulic conductivity and leaf water potential were usually higher in AM plants under WS condition, indicating that AM plants maintained more normal water relations (Auge et al., 2004; Sanchen-Blanco et al., 2004).
The mechanisms by which AMF have enhanced the water relations of host plants are often not clear. Potential mechanisms included enhanced absorption of water by external hyphae (Faber et al., 1991; Ruiz-Lozano and Azcon, 1995), stomatal regulation through hormonal signals (Goicoechea et al., 1997), the indirect effect of improved phosphorus nutrition (Nelsen and Safir, 1982; Fitter, 1988), and greater osmotic adjustment in AM plants (Auge et al., 1986; Ruiz-Lozano, 2003). In addition, Auge (2004) suggested that mycorrhizal soil itself could somehow directly influence the water relations of plants growing in them.
AM symbiosis might increase the drought tolerance of plants by promoting antioxidant enzymes (Ruiz-Lozano, 2003). However, information about activities of antioxidant enzymes in AM vs. non-AM plants is scarce. Host plants that have been examined include bean (Lambais et al., 2003), some shrub species (Alguacil et al., 2003), soybean (Porcel et al., 2003; Porcel and Ruiz-Lozano, 2004) and lettuce (Ruiz-Lozano et al., 1996). These studies focused mainly on antioxidant enzymes of leaves, including SOD, G-POD, CAT and APX. Non-enzymatic antioxidants and ROS of AM plants are not well understood. Moreover, little is known about the effects of mycorrhizal infection on antioxidant enzymes and non-enzymatic antioxidants of roots. The AM fungus, Glomus versiforme, has not been examined on citrus in regard to AMF and water stress.
The present study was undertaken to thoroughly evaluate reactive oxygen metabolism, including ROS, antioxidant enzymes and non-enzymatic antioxidants, in leaves and roots of trifoliate orange inoculated with G. versiforme under WW and WS conditions. Trifoliate orange is a major citrus rootstock used in China, and it has not been examine before when colonized by G. versiforme under WS conditions.
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Biological materials and material treatments
Seeds of trifoliate orange (Poncirus trifoliata (L.) Raf.) were surface-sterilized with 70% alcohol for 5 min and germinated on wet filter paper in Petri dishes in darkness at 28 °C. Six 7-day-old seedlings were transplanted into a plastic pot (15×20 cm) and were inoculated with G. versiforme (Karsten) Berch or with a non-AM fungal control. Both AM and non-AM treatments received 30 g of their respective inocula. For each AM treatment, 30 g AM inoculum (approx. 2233 spores) were placed 5 cm below
Results
Colonization of seedlings by G. versiforme varied from 22% to 37% (Table 1). The WS treatment notably decreased root colonization, with WW seedlings having 66% higher root colonization than WS seedlings. No mycorrhizal colonization was observed in the roots of non-AM seedlings.
Water stress significantly decreased shoot dry weight, root dry weight, plant height and stem diameter (Table 1). AM inoculation markedly increased shoot dry weight, plant height and stem diameter of trifoliate orange
Discussion
AM symbiosis increased growth of trifoliate orange seedlings in both the presence and absence of water stress, confirming earlier findings (Graham and Timmer, 1985; Shrestha et al., 1995; Fidelibus et al., 2001; Wu and Xia, 2004; Wu et al., 2005). AM effect on host plant growth during water stress has often been related to improved phosphorus nutrition (Nelsen and Safir, 1982; Johnson and Hummel, 1985), which though not measured, was probably the case with our plants, as well.
In higher plants,
Acknowledgements
This research was funded by Ministry of Science and Technology, P.R. China (2001EP090010; 2002EP090016; 2003EP090018; 2004EP090019). We are grateful to Prof. R.M. Auge (Department of Plant Science, University of Tennessee, USA) for two critical reviews of the manuscript.
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