The process of acclimating to winter conditions and overwintering are both important to the survival of perennial plants in temperate areas. Despite appearances that plants are doing nothing during winter dormancy, limited physiological processes such as respiration are still occurring (Ogren, 2000). Those physiological processes need to be altered during acclimation and protected during winter to maintain functionality under harsh winter conditions for spring regrowth. Plants can protect overwintering tissues via several mechanisms including sugar concentration gradients, changes in fatty acid profiles, and antioxidants (Samala et al., 1998). Evaluating how cellular-protection mechanisms may be affected by winter stresses in species sensitive to winter and determining whether management practices can improve cellular-protection mechanisms are important for developing strategies to reduce winter associated damages.
Annual bluegrass (Poa annua var. reptans), when maintained in a perennial growth habit typical in turfgrass management, is an ice-encasement sensitive species. It has been reported to survive an average of 60 d of ice cover (Beard, 1964; Tompkins et al., 2004; Vargas and Turgeon, 2004), which contrasts to creeping bentgrass (Agrostis stolonifera), which can survive under ice cover for up to 120 d (Beard, 1964). Annual bluegrass and creeping bentgrass are typically found as pure or mixed stands on golf course putting greens and fairways. Previously, we have found that treatments that inhibit ethylene improved annual bluegrass recovery following low temperature and ice conditions; and ethylene-promotive treatments reduced or had no effect on winter recovery, depending on the duration (Laskowski and Merewitz, 2020). As inhibiting ethylene may be a viable management practice that could be used to protect annual bluegrass or other turfgrass species during winter dormancy, it is important to understand the mechanism associated with improved tolerance. Additionally, how ethylene regulates acclimation and overwintering is still not fully understood.
Ethylene, the plant hormone commonly associated with stress responses and signaling, is primarily understood for plant stresses such as flooding or submergence (Fukao et al., 2006), thigmomorphogenesis (Biro and Jaffe, 1984), and wounding or pathogen responses (Lund et al., 1998). How ethylene plays a role in acclimation and overwintering is less well understood. Ethylene effects on cold or other winter stress tolerances may be based on plant species or environmental conditions because contrasting results have been found in various studies (Munshaw et al., 2010; Shi et al., 2012; Szalai et al., 2000; Yu et al., 2001; Zhao et al., 2014). It is clear ethylene has a major effect on plant acclimation and tolerance of winter conditions, but a better understanding of the physiological mechanisms associated with ethylene regulation is needed.
Perennial plant species contain different organs such as leaves, roots, and crowns that may have different survival or stress-protection mechanisms while acclimating to cold, surviving ice encasement, or overwintering. Here, we investigate several physiological responses such as apoplastic protein content, antioxidant enzymes, and fatty acid changes in response to chemical treatment, cold, and ice encasement in these different plant organs. Measuring apoplastic protein concentration can indicate plant cold and freeze tolerance, because more proteins in the apoplast could reduce ice crystal formation, particularly if they have antifreeze properties (Griffith et al., 1992). In barley (Hordeum vulgare), apoplastic proteins and antioxidants were found to play an important role in cold tolerance associated with salicylic acid treatment (Mutlu et al., 2013). Few studies of turfgrass cold or winterkill responses have investigated all three of these organs simultaneously, and little information exists about antioxidant and apoplastic protein content of important turfgrass species, particularly in response to ethylene regulatory treatments.
Although it sounds counterintuitive when first thinking about ice-encasement stress (a stress that can often result in anoxic conditions under the ice), oxidative stress-protective mechanisms can play an important role in plant survival of ice encasement. This is because a plant can experience a dramatic and rapid shift in oxygen concentration following ice melt. This stress is often referred to as post-anoxic aeration or reaeration stress. The lipid peroxidation and other anomalies associated with lipid damage that result from reaeration can differentiate between anoxia tolerant and intolerant species (Blokhina et al., 1999). Plants also respond to post-anoxic reaeration by modulating their antioxidant system to combat reactive oxygen species that can form, such as hydrogen peroxide (Blokhina et al., 2001). Post-anoxic reaeration has largely been studied with simulated anoxic stress using inert gasses or under flooding conditions, which means the interactions of cold temperatures and real ice encasement on antioxidant systems has not been thoroughly investigated.
Another major change that can occur during acclimation to cold is the alteration of cell membrane composition (Shang et al., 2006). Generally, plants more tolerant of winter conditions, including ice encasement, accumulate more unsaturated fatty acids than saturated fatty acids in their membranes (Dalmannsdottir et al., 2001; Heatherington et al., 1987; Shang et al., 2006). Shifts in fatty acid profiles occurred in annual bluegrass plants under different plant growth regulator treatments during the fall acclimation period (Laskowski et al., 2019). Thus, measurement of fatty acid profiles may be a good indicator for the effects of ethylene regulatory treatments on annual bluegrass acclimation and survival of ice stress.
We hypothesized that ethylene inhibition-induced improvements in annual bluegrass recovery from ice stress could be associated with cellular-protection mechanisms such as a promotion or maintenance of antioxidant enzyme activities, increased unsaturated fatty acid contents, and/or accumulations in apoplastic proteins. Therefore, the objectives of this study were to evaluate ethylene-promotive treatments [ethephon (2-chloroethyl phosphonic acid) and aminocyclopropane-1-carboxylic acid (ACC)] and ethylene inhibition treatment [aminoethoxyvinylglycine (AVG)] effects on annual bluegrass responses to winter stresses.
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