Creeping bentgrass has been widely used as a cool-season turfgrass species for golf course greens, tees, and fairways in cool–humid and transitional climate zones due to its high density and tolerance to short mowing (Wang et al., 2012). It has excellent cold resistance and could tolerate temperatures as low as −35 °C (Gusta et al., 1980). However, creeping bentgrass has suffered winter-kill in some temperate regions such as in northern China. Winter-kill is a general term that is used to define turf loss during the winter and can be caused by a combination factors including desiccation, crown hydration, low temperatures, ice cover, and snow mold (Beard, 1973; Bhowmik et al., 2008). Winter-kill significantly damages the quality of sports surface, especially in golf greens, tees, and fairways.
In northern China such as Beijing area, the daily low temperatures are 8, 0, −6, −9, −6, and 0 °C and the monthly precipitations are 2.3, 0.8, 0.2, 0.3, 0.6, and 0.9 cm in October, November, December, January, February, and March, respectively, based on 30-year average. It appears that limited precipitation in late fall and winter may cause severe drought stress and could be a major factor causing winter-kill because the temperatures are well above killing temperature for creeping bentgrass in this area. Because of severe shortage of water resource and a high cost of irrigation water, golf course managers in this region are reluctant to water the grass in late fall through winter. Previous studies have shown that deficit irrigation could not only improve creeping bentgrass tolerance to stress but also significantly reduce cost of irrigation water (Ervin et al., 2009; Huang et al., 2014). However, little study has been reported on effect of deficit irrigation or mild drought stress before and during cold acclimation impacts on defensive metabolism and freezing tolerance of creeping bentgrass.
Creeping bentgrass undergoes cold acclimation, which is induced by a combination of reduced photoperiod and temperatures in late fall (Dionne et al., 2001a, 2001b; Hoffman et al., 2010). Cool-season turfgrasses require temperatures between 0 and 7 °C for 2–3 weeks to initiate the cold acclimation process. Second stage of cold acclimation is induced by subzero temperatures (Beard, 1973). Soil moisture status affects turfgrass cold acclimation process and thus cold tolerance (Beard, 1973). Previous studies have shown that mild drought stress can induce osmotic adjustment and antioxidant defenses in creeping bentgrass under normal temperature conditions (DaCosta and Huang, 2006, 2007; Fu and Dernoeden, 2009; McCann and Huang, 2007). Reduced soil water content may facilitate cold hardiness in st. augustinegrass [Stenotaphrum secundatum (Walt) Kuntze (Maier et al., 1994)] and arabidopsis [Arabidopsis thaliana (L.) Heynh (Mäntylä et al., 1995)]. This suggests preconditioning of the grass under mild drought stress before cold acclimation may improve plant defensive metabolism and freezing tolerance.
Environmental stress may damage plant cells by the accumulation of toxic reactive oxygen species (ROS), including O2−, H2O2, hydroxyl radicals (OH·), and singlet oxygen (1O2) (Apel and Hirt, 2004; Møller et al., 2007). The overproduction of ROS may damage lipids, proteins, and nuclei acids, resulting in plant senescence and death (Smirnoff, 1993). Plants have developed antioxidant defense mechanisms to eliminate ROS and prevent oxidative damage. Antioxidant enzymes, such as SOD (EC 22.214.171.124), CAT (EC 126.96.36.199), POD (EC 188.8.131.52), and ascorbate peroxidase [APX (EC 184.108.40.206)] protect plants against oxidative stress (Blokhina et al., 2003). SOD constitutes the first line of defense against ROS by dismutating the O2− to H2O2 (Apel and Hirt, 2004), which is finely regulated by CAT, POD, and APX (Wang et al., 2012). Various antioxidant metabolites and enzymes may work coordinately in suppressing ROS toxicity under stressful environments.
The level of antioxidant activity and gene expression is regulated in response to abiotic stresses such as drought and low temperature stresses (Jiang et al., 2010; Menezes-Benavente et al., 2004; Zhou et al., 2012). Catalase is induced by low temperature and an essential enzyme to protect mitochondria against chilling stress. The regulation in antioxidant enzyme activity and isozyme expression under adverse environments is closely related to plant tolerance to abiotic stresses (Allen, 1995; Mittler, 2002).
Plant may undergo osmotic adjustment in response to drought and cold stress (Hoffman et al., 2014; Huang et al., 2014). Accumulation of various osmoprotectants or compatible solutes, such as soluble proteins, simple sugars, and amino acids, in the cell during drought stress and cold acclimation improve plant tolerance to stresses (Ball et al., 2002; Blokhina et al., 2003; Hoffman et al., 2014; Shi et al., 2012).
Deficit or water-saving irrigation cannot only save water and reduce cost but also create mild drought stress which may improve defensive metabolism (Ervin et al., 2009; Huang et al., 2014). Severe drought stress occurred without irrigation in late fall in the region with limited precipitation may weaken defensive metabolism before and during cold acclimation and reduces winter survival of creeping bentgrass. Our hypothesis was that mild drought stress induced by deficit irrigation before and during cold acclimation would improve levels of osmoprotectants, antioxidant metabolism, and freezing tolerance when compared with severe drought stress (occurring in the region with limited precipitation and without irrigation in late fall and winter). In addition, mild drought stress treatment may improve osmorprectants, antioxidant metabolism, and freezing tolerance when compared with well-watered treatment.
The objectives of this study were to examine effects of three soil moisture levels (well-watered, mild drought stress, and severe drought stress) 14 d before and during 21 d of cold acclimation [2 °C (days 14 to 35)] on osmoprotectants, antioxidant metabolism, and freezing tolerance, and investigate if mild drought stress induced by deficit irrigation could improve plant freezing tolerance when compared with well-watered and severe drought stress regimes.
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