Polyamine Content Changes in Creeping Bentgrass Exposed to Salt Stress

Salt stress is a major problem in turfgrass management. Investigation of metabolites, such as polyamines (PAs) that may improve salt tolerance of turfgrass species, is needed. Two independent growth chamber studies were conducted to evaluate physiological characteristics and changes in PAs, such as putrescine (Put) spermidine (Spd) and spermine (Spm) in response to salt stress in 'Penncross' and 'PsgSLTZ' creeping bentgrass (Agrostis stolonifera) The study also aimed to determine a method of PA extraction to improve PA yields from creeping bentgrass. Salt solutions were drench applied to plants growing in pure sand daily in a stepwise manner for'70 days in both studies. For both cultivars, salt stress caused an increase in leaf Na content, percent of electrolyte leakage (EL) and canopy temperature depression (CTD) while it caused a decrease in turf quality (TQ) osmotic potential (Cs) and K D and Ca content compared with controls. In the early stages of salt stress, Put content increased in salt stressed plants compared with controls. Spd content did not change significantly while a transient increase in Spm was observed in the later stage of salt stress. The PA quantification method used in this study included using formic acid during the extraction process, which exhibited enhanced quantification of PAs from creeping bentgrass compared with other methods previously published. Salinity stress upregulated the content of Put and Spm in leaf tissue, which may be involved in salinity tolerance in creeping bentgrass, while Spd accumulation may not be a major salt tolerance mechanism; supplementation with these biochemical compounds could be an alternative to improve creeping bentgrass salt tolerance. Salt stress is a major issue for turfgrass management around the world. Use of reclaimed, nonpotable water that may contain high levels of salts is becoming a common management practice (Duncan et al. 2008) Additionally, numerous turf areas are located in salt prone environments, such as shorelines or areas with salt afflicted soils. The primary damage to plants caused by salinity is due to toxic ion accumulation of Na in plant tissues and water deprivation from decreased external Ys in the soil solution (Hasegawa et al. 2000) Creeping bentgrass does not possess major salt resistance mechanisms to rid plants of salt, such as salt glands, that are found in other grass species within Poaeceae (Marcum, 2001) Since adequate avoidance mechanisms are not possessed by creeping bentgrass, the species must rely on other salt tolerance mechanisms for survival under salt stress conditions. Evaluating mechanisms that may promote tolerance to salt stress is necessary to develop salt tolerant creeping bentgrass germplasm. Cultivar variation in salt tolerance of creeping bentgrass does exist, but the major metabolite pathways that may play a role in differential salt tolerance mechanisms between cultivars have not yet been fully elucidated. Additionally, knowledge of plant metabolites important in salt stress tolerance could lead to the development of new plant protective chemical products. A relatively unexplored biochemical process in turfgrass species is the PA biosynthetic pathway. The common PAs associated with growth regulation and abiotic stresses are Put, Spd, and Spm. PAs are synthesized from arginine or ornithine decarboxylation pathways (Janowitz et al. 2003) PAs may respond differently to abiotic stress based on plant species, plant tissue type, and developmental stage (Capell et al. 2004) In arabidopsis (Arabidopsis thaliana) there was a shift in PA biosynthesis pathways toward accumulating Put, whereas in the resurrection plant (Craterostigma plantagineum) this shift was toward accumulating Spm (Alcazar et al. 2011) Conversely, after salt stress in broad bean [Vicia faba (Sadak andAbdelhamid, 2015) and foxtail millet [Setaria italica (Sudhakar et al. 2015) all three types of endogenous PA concentration increased significantly compared with the control. In a salt tolerant rice (Oryza sativa) cultivar, Put and Spm increased more compared with the sensitive one (Do et al. 2014) In maize (Zea mays) leaf blade elongation zone (Rodriguez et al. 2009) an increase of Spd content was observed in response to salt treatment. Recently, Li et al. (2015a) found that PAs were generally upregulated in white clover (Trifolium repens) under artificially induced drought stress in hydroponic cultures. In creeping bentgrass, Spd content decreased due to drought stress, whereas Put and Spm content increased compared with control plants (Li et al. 2015b) All these results show the importance of PA in salt stress responses. However, it is not yet fully clear whether differences in PA fluctuation during stress is due to species differences, plant developmental stage, or difficulties in comparing across studies. Compared with other crop species, little information is available regarding the function, content, or regulation of PAs in creeping bentgrass or other cool season turfgrass species, particularly during long term salt stress conditions. For utility as an agricultural technology, determining how the common PAs, such as Put, Spd, and Spm, may respond to stress or play Received for publication 1 June 2016. Accepted for publication 14 July 2016. We thank AgBioResearch of Michigan State University, Project GREEEN, and The Michigan Turfgrass Foundation for funding support of this work. We also thank Arthur Danial Jones and Lijun Chen of Michigan State University Mass Spectrometry and Metabolomics Core for technical assistance and immense help with method modification of PA quantification. Corresponding author.  2016. a role in turfgrass tolerance to stress deserves investigation. In addition, current PA quantification methods used in other crop species and turfgrasses did not prove to provide adequate recovery of free PAs from creeping bentgrass tissue in our preliminary tests. Therefore, the objectives of the study were to improve the methods of extraction and quantification of PAs from creeping bentgrass tissues, evaluate creeping bentgrass physiological health under salt stress, and quantify PAs from creeping bentgrass to achieve a better understanding of PA content regulation due to prolonged salt stress conditions.  Two independent growth chamber studies (Expts. 1 and 2) were conducted. For both experiments, creeping bentgrass 'PsgSLTZ' and 'Penncross' were used. Seeds of both cultivars were obtained from the Seed Research of Oregon (Tangent, OR) These cultivars showed good performance under salt conditions in their breeding efforts; however, PsgSLTZ rated higher in salt tolerance (Seed Research of Oregon, unpublished data) Seeds were directly sown into pots (12 cm) at a rate of 4.88 g m into pure sand. Expt. 1 was seeded on 18 Aug. 2013 and Expt. 2 on 14 Mar. 2014. The seedlings for both experiments were established in the greenhouse under 900 mmol m s of photosynthetically active radiation (PAR) for 14 h of daylength under 65% to 75% humidity and 24/18 C (day/night) temperature for 2 months to attain dense and healthy turf canopy. Then, the fully established plants were transferred to an environmentally controlled growth chamber with 700 mmol m s PAR for 14 h of daylength, 65% to 70% humidity, and 23/18 C (day/ night) temperatures for both experiments. Treatments were arranged in a randomized complete block design with four biological replications. All salt treated plants were watered daily each week with 100 mL of salt solution at 0 21 d) 16 (35 d) 16 (42 d) 20 (56 d) and 24 (70 d) dS m for Expt. 1. The salt solution (Instant Ocean Aquarium Salt; Spectrum Brands, Blacksburg, VA) was made with half strength Hoagland solution (Hoagland and Arnon, 1950) twice per week. The salt solution was made from deionized water the rest of the 5 d within the week. The salt solution included 10.78 g L Na, 0.42 g L K, 1.32 g L Mg, 0.4 g L Ca, 0.008 g L Sr, 19.29 g L Cl, 2.66 g L SO4, 0.2 g L HCO3, 0.56 g L Br, and 0.001 g L F. The percentage of NaCl in the salt solution was 84% The same amounts of salt solution were used in Expt. 2, which were applied at 0 (6 May 2014) 4 (28 d) 8 (35 d) 12 (49 d) 16 (63 d) and 20 (77 d) dS m. PHYSIOLOGICAL TRAIT EVALUATION.TQ ratings were based on color, density, and uniformity of the grass canopy using a scale of 1 9 with 1 being dead plants and 9 being healthy and green plants (Beard, 2001) Percent EL was measured to estimate cell membrane stability of the leaves based on the method of Blum and Ebercon (1981) About 10 leaves were cut, briefly washed, and immersed in a 15 mL centrifuge tube with 7.5 mL of deionized water. They were then placed on a shaker for 24 h at 144 rpm. The conductivity of the solution containing the living tissue was measured using an electric conductivity meter (3200 conductivity meter; YSI, Yellow Springs, OH) as the initial conductivity (Ci) after shaking. The leaf tissues were boiled in a water bath for 5 h and then were placed on a shaker with the same speed for another 24 h. The electric conductivity of the solution containing dead tissues was measured as the maximum conductivity (Cmax) The percent EL was calculated as Ci/Cmax 100. The Ys was determined as in the method of Qian and Fry (1997) About 10 leaves were taken from each plant and were immersed in deionized water in a petri dish for 3 h. Leaves were blotted dry, transferred to a 2 mL microcentrifuge tube, immediately frozen in liquid nitrogen, and then stored at 80 C. Samples were ground with a micropestle and centrifuged at 20,817 gn (Eppendorf 5430 R; USA Scientific, Ocala, FL) for 15 min at room temperature. A 10 mL leaf extract was placed on a piece of 0.5 cm diameter filter paper, inserted into a vapor pressure osmometer