INVESTIGATION OF POLYAMINES AND HORMONAL RESPONSES DURING ABIOTIC STRESS EPISODES IN COOL SEASON GRASS SPECIES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1000637
• Project Start Date: Aug 12, 2013
• Project End Date: Jul 31, 2018
• Program Code: [(N/A)]- (N/A)

Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824

Performing Department
Plant, Soil and Microbial Science

Non Technical Summary
The U.S. turfgrass industry generated $124.6 billion in 2005 (Haydu et al., 2012) and continues to be a major source of revenue and jobs for communities. Turfgrasses, particularly those used on golf courses and athletic fields, are a major high value commodity, particularly in Michigan. A significant amount of monetary, labor, water, and chemical inputs are required to maintain turfgrasses to an acceptable level of quality for functionality as a playing surface. To a large degree, many of the inputs are needed due to the susceptibility of turfgrasses to abiotic and biotic stresses. The abiotic stresses drought and salt stress are of particular importance to the turfgrass industry (Huang, 2006). Drought stress readily occurs even on irrigated areas in times of limited rainfall, due to water use restrictions, on areas with poor irrigation instrumentation, and due to the high demand for firm dry conditions for adequate playability of golf courses and other fields. Salt stress is an emerging stress as it is desirable to used reclaimed, saline water as a cheaper source of water than potable sources. There is also a significant amount of golf courses and athletic fields in naturally salt prone environments such as on shorelines. Therefore, the overall goals of the research are to identify mechanisms of stress tolerance in various commonly grown cool-season turfgrass species such as creeping bentgrass (Agrostis stolonifera), Annual bluegrass (poa annua), perennial ryegrass (Lolium perenne), and Kentucky bluegrass (Poa pratensis). Specifically, we will look for changes in metabolite (polyamines) and hormone compounds (jasmonic acid, salicylic acid, and ethylene). Little is known about how these compounds relate to abiotic stresses of turfgrass species. Basic methods such as determination of content and activity of enzymes related to their biosynthesis will be used to understand how these compounds change in grasses Through this research a greater understanding of the mechanisms of stress tolerance of turfgrasses will be achieved allowing for the development of germplasm with increased stress resistance in the future. Increased stress resistance will allow for reduced inputs and therefore benefit the turfgrass industry as a whole.

Animal Health Component

25%

Research Effort Categories

Basic

75%

Applied

25%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
203	1629	1020	100%

Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
1629 - Perennial grasses, other;

Field Of Science
1020 - Physiology;

Keywords

cool-season turfgrass

polyamines

abiotic stress tolerance

plant hormones

Goals / Objectives
Goal1 - Understand the Effects of Higher Polyamines on Abiotic Stress Tolerance of Creeping Bentgrass 1. Determination of the effects of abiotic stresses on higher polyamine content, biosynthetic enzyme content and activity 2. Effects of exogenous application of higher ordered polyamines on stress tolerance 3. Evaluation of the changes in major plant hormones in response to higher polyamine application and stress incidence 4. Development of genetically modified creeping bentgrass to increase endogenous spermidine content 5. Evaluation of the changes in the transcriptome, proteome, and metabolome in transgenic and non-transgenic plants differing in the accumulation of spermidine during drought and salt stresses 6. Determine the effects of commonly usedplant growthregulatorson polyamine accumulation and regulation Goal 2 - Determine how abiotic stresses and turf management practices alter the response of plant hormones involved in the systemic aquired resistance (SAR) and induced systemicresistance (ISR)pathways such as ethylene, jasmonic, and salicylic acid 1. Evaluate physiological and hormonal responses between Creeping bentgrass (Agrostis stolonifera) and Annual bluegrass (Poa annua) in response to drought and traffic stress 2. Identify hormone changes following plant growth regulator application and during drought stress conditions 3. Develop predictions regarding disease susceptibility during abiotic stress and management practices based onSAR and ISR pathway indicators

Project Methods
Project 1 - A total of 6 cultivars of creeping bentgrass that are known to differentially respond to abiotic stresses will be tested. These will include a stress tolerant and sensitive type for each drought and salt. 'Mariner' will be the more salt tolerant cultivar, 'L93' will represent the more drought tolerant cultivar, and 'Penncross' will serve as the stress sensitive cultivar for both stress treatments. Creeping bentgrass plants will be exposed to optimal conditions (control), salt stress (instant ocean, Spectrum Brands, Inc. at 12 decisiemens; dS), and drought stress (water withheld completely). The exogenous application of polyamines prior to stress incidence (objective 2) will include two concentrations of spermidine (500uM, 750uM) and spermine (500uM), one positive control [proline for drought 10mM; (Ashraf and Foolad, 2007) glycine betaine for salt 20mM; (Hu et al., 2012)], and one negative control (water). Four replications will be used for each plant treatment. Therefore, a total of 120 plants will be evaluated. Higher polyamine content will be determined with an acid extraction, spectrophotometric method as in He et al (2002) and biosynthetic enzyme accumulation and activity assays will be determined by western analysis and HPLC analysis as in Kasukabe et al. (2004). Physiological measurements will be conducted throughout the duration of all trials. Plant measurements will include leaf relative water content (Barrs and Weatherly, 1962), photochemical efficiency (CID biosciences), photosynthesis rates (LICOR 6400), osmotic adjustment (Wescor osmometer), cellular membrane stability (Blum and Ebercon, 1979), chlorophyll content (Arnon, 1949), antioxidant enzyme responses and lipid peroxidation (Dhindsa et al, 1981). Quantitative measurements that indicate turfgrass uniformity, density, and Merewitz et al., 2010) and turf quality ratings (Beard, 2006) will be determined. The aforementioned traits are widely used physiological parameters that give an indication of stress tolerance. In addition, destructive sampling will occur at the beginning of the study (pre-stress) and at the completion of the study by destructive sampling for determination of leaf and root number, leaf and root viability by TTC and dye exclusion methods, biomass, and area. Plant samples will be taken throughout the duration of the study, frozen in liquid nitrogen, and stored at -80°C for evaluation of the changes in major plant hormones in response to higher polyamine application and stress incidence (objective 3). For plant hormone analysis ultra-fast liquid chromatography-electrospray ionization tandem mass spectrometry (UFLC-ESI-MS) for methods will be performed as appropriate to identify the content of abscisic acid, indole -3-acetic acid, jasmonic acid, salicylic acid (and glycoside derivatives), cytokinins (Liu et al., 2012). Ethylene content will be monitored on intact plants using a portable ethylene gas analyzer (CID biosciences). Development of multiple lines of transgenic creeping bentgrass that constitutively (cauliflower mosaic virus 35S promoter) or have stress regulated expression (a wound inducible promoter from the proteinase inhibitor II-K (PI-II) gene; Smigocki, 1995) stress promoter (PR AlSAP; Saad et al., 2011) of spermidine synthase (SPDS, EC 2.5.1.16) or S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50), two of the major enzymes involved in higher polyamine biosynthesis (Kasukabe et al., 2004). As turfgrasses are consistently mowed or wounded, we propose to test the effectiveness of a wound inducible promoter on turfgrass stress tolerance, as to our knowledge this has not yet been tested in turfgrasses. A new, rapid protocol for transforming creeping bentgrass via stolons has been identified, that will allow us to quickly generate transgenic plants (Wang et al., 2005). The plant transformation facility at Michigan State University and the post-doctoral researcher to be involved in this project have expertise in the methods of creation of transgenic grasses and detection of transgene presence and activity (http://www.ptc.msu.edu/index.html). The development of creeping bentgrass transgenic for a gene promoting the synthesis of polyamines may be the most viable way to detect the effects of polyamine accumulation on bentgrass responses. After generation of these plants, we will be able to further characterize the effects of increased polyamine accumulation on physiological attributes and stress tolerance of creeping bentgrass. However, the best genes to target for manipulation of higher ordered polyamine accumulation will be differential between plant species. Therefore, the research proposed here will provide us with a significant amount of evidence validating the importance of the investigation of higher polyamines and enhanced direction for future investigations of higher polyamines in turfgrass species. To get a comprehensive evaluation of how the transgenic grasses are responding to increased polyamine content we will perform transcriptome, proteomic, and metabolite evaluations. For transcriptomic analysis suppression subtractive hybridization (SSH) will be performed (Xiong et al., 2001). Proteomic evaluation will be performed by two-dimensional gel electrophoresis followed by hand selection of differential protein spots and protein identification by MALDI-TOF/MS/MS (Merewitz et al., 2011a) and metabolomics analysis of primary and secondary metabolites will be performed by GC-MS techniques (Merewitz et al., 2011b). Project II - The field plan, watering treatments, and experimental design will be as follows. 9 plots each of P. annua and creeping bentgrass (36 x 36 ft) will be maintained with typical nutrient and cultural processes adequate for desirable turfgrass growth. The irrigation system will be controlled to provide optimal soil moisture levels (12% for creeping bentgrass and 15% for P. annua). Water deficit to cause drought stress symptoms will be imparted to simulate a gradual dry down of the grasses. The irrigation system (Rain Bird ISS Integrated Sensor System) will be set to reduce weekly water inputs so that a 3% less of the total soil moisture will be maintained each week. An attempt to achieve drying in a stepwise fashion from (approximately 25% soil moisture to 5%) will be imparted to half of the plots to serve as the drought stress treatment. A Rain Bird ISS Integrated Sensor System will be used to measure soil moisture, soil temperature, and salt levels at two depths, 1 and 5.5 inch. The sensors are able to provide real-time data through wireless communication that makes the data available on the internet. A portable TDR will also be used to measure volumetric soil moisture content in the rootzone at two depths, surface to 2 inches, and surface to 4 inches. Each 36 ft. by 36 ft. putting green contains three buried lysimeters. Strip plots (12 x 36 ft) of low and moderate traffic stress will be imparted to the plots above the three buried lysimeters to create subplots that will create a total of 54 total research plots. Project III - Recent evidence suggests that significant cross-talk exists between giberellic acid (GA) and systemic and induced resistance (SARS and ISR) pathways, which involve JA and SA and lead to stress resistance. In order to evaluate the effects of inhibition of GA synthesis by TE on SARS and IRS, two cultivars, 'Kenblue' and 'A06-46', were treated with three foliar application of TE [1.95 ml l-1 (v: v); a.i. TE = 0.113%] at one week intervals and then subjected to drought or water treatments for 15 days in growth chamber. Determination of phytohomones jasmonic acid (JA), indole acetic acid (IAA), salicylic acid (SA), abscisic acid (ABA) and zeatin riboside (ZR) following TE treatment and during stress in leaf tissues will be done using liquid chromatography mass spectrometry (LC-MS).

Progress 08/12/13 to 07/31/18

Outputs
Target Audience: The target audiences include turfgrass scientists, plant physiologists, turfgrass managers (primarily golf-course, lawn care, athletic field), Agri-business representatives, turfgrass consultants, and turfgrass education specialists in Michigan and the world. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The opportunities that all of the projects described above provided within the last year include training of three graduate students and one post-doctoral researcher in turfgrass physiology research. How have the results been disseminated to communities of interest?Results from all years of this project have been disseminated by publishing in peer-reviewed journals and/or disseminated via a local industry conference (Michigan Turfgrass Conference), presentations to industry representatives (via the Michigan chapter of the Golf Course Superintendents Association, MiGCSA), field day presentations to local turf managers (MSU Turfgrass Field Day), presentations to corporate functions (such as Residex Co.), a webinar for the Golf Course Superintendents Association of America,video for the Canadian Turfgrass Research Foundation, and academic conference presentations (ASA-CSSA-SSSA international meeting). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? As reported last year, in the past several years there has been a renewed interest in the turfgrass industry at finding solutions and management strategies to minimize winter related stresses of turfgrasses. Thus, the stress incidence of the overall goals and objectives of the projects were focused primarily on winterkill issues and a bacterial pathogen. All of the objectives listed above were satisfied in 2017 with the exception of objectives 4 and 5 in goal 1, but based on results of previous objectives our interest and priorities changed from the original objectives outlined. Our publications for 2018 have major impact on the turfgrass industry through better ways to manage winterkill, bacterial pathogens, and a better understanding of plant-bacteria interactions.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Laskowski K, Frank K, and Merewitz E. 2018. Effects of chemical plant protectants and plant growth regulators on annual bluegrass survival of ice cover. Journal of Agronomy and Crop Science. 00:1-11. DOI: 10.1111/jac.12309
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Liu S, Vargas J, and Merewitz E. 2018. Temperature and hormones associated with bacterial etiolation symptoms of creeping bentgrass and annual bluegrass. J. Plant Growth Regul. https://doi.org/10.1007/s00344-018-9837-4
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Liu S, Vargas J, and Merewitz E. 2018. Jasmonic and salicylic acid treatments reduce bacterial etiolation disease of creeping bentgrass in optimal and high temperatures. Crop protection 109:916.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Laskowski K, Frank K, and Merewitz E. 2018. Surfactant effects on creeping bentgrass and annual bluegrass exposed to different irrigation and traffic stress treatments. Agronomy J 110:1-7.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The target audiences include turfgrass scientists, plant physiologists, turfgrass managers (primarily golf-course, lawn care, athletic field), Agri-business representatives, turfgrass consultants, and turfgrass education specialists in Michigan and the world. Changes/Problems:In the past several years there has been a renewed interest in the turfgrass industry at finding solutions and management strategies to minimize winter related stresses of turfgrasses. Damages associated with winterkill can cause major expenditures for rennovation and economic hardship for golf courses. Going forward this will be a major goal of our research and we are also interested in determining physiological consequences of ice stress, low temperature stress, and practices associated with winter preparation. We have already performed a significant amount of work this reporting year in this area. What opportunities for training and professional development has the project provided?The opportunities that all of the projects described above provide within the last year include training of three graduate students and one post-doctoral researcher in turfgrass physiology research. How have the results been disseminated to communities of interest?The results to date have been published in peer-reviewed journalsand/or disseminated via a local industry conference (Michigan Turfgrass Conference), presentations to industry representatives (via the Michigan chapter of the Golf Course Superintendents Association, MiGCSA), field day presentations to local turf managers (MSU Turfgrass Field Day), presentations to corporate functions (such as Residex Co.), a webinar for the Golf Course Superintendents Association of America, and academic conference presentations (ASA-CSSA-SSSA international meeting). What do you plan to do during the next reporting period to accomplish the goals?Goal 2 will be expanded to include winter related stress, other hormones, and metabolites. We are going to focus on this goal over the next reporting period. We hope to also get a better understanding of other physiological changes associated with abiotic and biotic stress tolerance of turfgrasses. For instance, changes in fatty acids, carbohydrates and other factors will be evaluated.

Impacts
What was accomplished under these goals? Abiotic stresses cause major damage to high value turfgrass species grown in Michigan and around the world. The central theme of the umbrella project is to identify abiotic and biotic stress tolerance mechanisms to utilize this knowledge for development of stress tolerant grass germplasm, protective products, or turf management strategies that can reduce stress incidence. For the first goal of the project, last reporting period we have indicated that we have satisfied many of the objectives (1, 2, and 6) or do not plan to pursue certain objectives (objective 4 and 5) due to results of previous objectives and due to research interest changes. For objective 2, we have published a transcriptome analysis of creeping bentgrass treated or not treated with a polyamine under drought stress or control conditions (Ma et al., 2017). Much of our findings and the current state of polyamine research has been summarized in a published review paper (Ma and Merewitz, 2016b). For objective 3, understanding how plant hormones may respond to polyamine application and stress incidence, we have published a journal article on our results (Krishnan and Merewitz, 2017). The second goal is to determine how abiotic stresses and turf management practices alter the response of plant hormones involved in the systemic acquired resistance (SAR) and induced systemicresistance (ISR)pathways such as ethylene, jasmonic, and salicylic acid. Our accomplishments within objective 1 have been accepted for publication (Laskowski et al., 2017a). We have reported on the second objective last year. For the third objective, we have published how a bacterial pathogen of creeping bentgrass can produce phytohormones both in vitro and in vivo to elicit disease symptoms (Liu et al., 2017a). We have also shown that salicylic acid and jasmonic acid applied exogenously are effective in mitigating bacterial disease symptoms in creeping bentgrass. This work is currently under review (Liu et al., 2017b). We are also investigating whether chemical priming using jasmonic and salicylic acid is an effective method to alleviate drought stress in creeping bentgrass under field conditions, which is central to goal 2. The student working on this project has just completed year two of field research and is currently writing a master's thesis and publications of this work. As discussed in the changes section, a significant amount of work this year has been done to better understand turfgrass responses to ice stress and winter conditions. One journal article regarding this work is currently under review (Laskowski et al., 2017b). Krishnan SK and Merewitz E. 2017. Polyamine application effects on gibberellic acid content in creeping bentgrass during drought stress. J. Amer. Soc. Hort. Sci. JASHS 142 (2) 2135-142. doi: 10.21273/JASHS03991-16. Laskowski K, Frank K, and Merewitz E. 2017a. Surfactant effects on creeping bentgrass and annual bluegrass exposed to different irrigation and traffic stress treatments. Agronomy J Accepted Laskowski K, Frank K, and Merewitz E. 2017b. Annual bluegrass responses to ice stress and plant growth regulator treatment. Journal of Agronomy and Crop Science. Under Review. Liu S, Vargas J, and Merewitz E. 2017a. Phytohormones associated with bacterial etiolation disease in creeping bentgrass. Environmental and Experimental Botany 133:35-49. Liu S, Vargas J, and Merewitz E. 2017b. Jasmonic and salicylic acid treatments reduce bacterial etiolation disease of creeping bentgrass in optimal and high temperatures. Crop Protection. Under Review.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Ma Y and Merewitz E. 2016. Role of polyamines in stress responses. CAB Reviews. 11 (003):1-11.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Liu S, Vargas J, and Merewitz E. 2017. Phytohormones associated with bacterial etiolation disease in creeping bentgrass. Environmental and Experimental Botany 133:35-49.
• Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Liu S, Vargas J, and Merewitz E. 2017. Jasmonic and salicylic acid treatments reduce bacterial etiolation disease of creeping bentgrass in optimal and high temperatures. Crop Protection. Under Review.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Krishnan SK and Merewitz E. 2017. Polyamine application effects on gibberellic acid content in creeping bentgrass during drought stress. J. Amer. Soc. Hort. Sci. JASHS 142 (2) 2135-142. doi: 10.21273/JASHS03991-16.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Ma Y, Shukla V, and Merewitz E. 2017. Transcriptome analysis of creeping bentgrass exposed to drought stress and polyamine treatment. PLoS ONE 12(4): e0175848. https://doi.org/10.1371/journal.pone.0175848
• Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Laskowski K, Frank K, and Merewitz E. 2017. Annual bluegrass responses to ice stress and plant growth regulator treatment. Journal of Agronomy and Crop Science. Under Review.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The target audiences include turfgrass scientists, plant physiologists, turfgrass managers (primarily golf-course, lawn care, athletic field), Agri-business representatives, turfgrass consultants, and turfgrass education specialists in Michigan and the world. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The opportunities that all of the projects described above provide within the last year include training of four graduate students, one post-doctoral researcher, and two undergraduate students in the methods of turfgrass physiology research.? How have the results been disseminated to communities of interest?The results to date have been disseminated via a local industry conference (Michigan Turfgrass Conference), presentations to industry representatives (via the Michigan chapter of the Golf Course Superintendents Association, MiGCSA), field day presentations to local turf managers (MSU Turfgrass Field Day), presentations to corporate functions (such as Residex Co.), and academic conference presentations (ASA-CSSA-SSSA international meeting). The PI has also written a book chapter related to both goals 1 and 2 (Merewitz, 2016).Much of our findings from goal 1 and the current state of polyamine research has been summarized in a published review paper (Ma and Merewitz, 2016b). What do you plan to do during the next reporting period to accomplish the goals?Goal 1 objective 6 and goal 2 objective 3 will be satisfied in the next reporting period. Experiments will be performed to better understand salicylic acid, jasmonic acid and ethylene in turfgrass species in order to determine whether these hormones can be manipulated for stress tolerance of turfgrasses.

Impacts
What was accomplished under these goals? Abiotic stresses cause major damage to high value turfgrass species grown in Michigan and around the world. The central theme of the umbrella project is to identify abiotic and biotic stress tolerance mechanisms in order to utilize these mechanisms toward development of stress tolerant grass germplasm or protective products. Within the first goal of the project, we now have significant data to support conclusions for each objective. For objective 1, we have a paper published that describes how salt stress effects polyamine content in creeping bentgrass (Ma and Merewitz, 2016a) and a paper published on how mechanical wounding and heat stress affect polyamine and phytohormone content in creeping bentgrass (Krishnan et al., 2016). For objective 3, we have a paper in preparation that will be submitted for review in late Sept 2016. We have decided not to pursue objective 5 due to the results of our above objectives. Experiments are being conducted to satisfy Objective 6.For objective 5, we have conducted transcriptome analysis of thenon-transgenic plants exposed to polyamine treatmentand have submitted the paper for review in August 2016 (Ma et al., 2016). The second goal is to determine how abiotic stresses and turf management practices alter the response of plant hormones involved in the systemic acquired resistance (SAR) and induced systemicresistance (ISR)pathways such as ethylene, jasmonic, and salicylic acid. The results of objective 1 are in the final stages of preparation for submission to a journal for review (Laskowski et al., 2016). We have reported on the second objective last year. For the third objective, we have submitted a paper on this subject. It is currently undergoing minor revisions (Liu et al., 2016). Liu is also evaluating whether SAR and ISR hormones can reduce bacterial disease incidence, as a way to naturally manage this disease. This work will be submitted for publication in fall 2016. A new graduate student with a Fulbright scholarship began in the Fall 2015. He is investigating whether chemical priming using jasmonic and salicylic acid is an effective method to alleviate drought stress in creeping bentgrass, which is central to goal 2. The student has conducted one-year of field work which will be repeated in the summer of 2017. We also have published a paper with objectives that are central to this goal regarding phytohormone responses to salt stress (Krishnan and Merewitz, 2015).

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Ma Y and Merewitz E. 2016a. Polyamine content changes in creeping bentgrass exposed to salt stress. JASHS. In press.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Ma Y and Merewitz E. 2016b. Role of polyamines in stress responses. CAB Reviews. In press.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Krishnan S, Ma Y, and Merewitz E. 2016. Leaf trimming and high temperature regulation of phytohormones and polyamines in creeping bentgrass leaves. J. Amer. Soc. Hort. Sci. 141:66-75.
• Type: Journal Articles Status: Under Review Year Published: 2016 Citation: Liu, S., J Vargas, and E. Merewitz. 2016. Phytohormones associated with bacterial etiolation disease in creeping bentgrass. Env and Exp Bot Minor Revisions
• Type: Book Chapters Status: Published Year Published: 2016 Citation: Merewitz, E. 2016. Priming-induced drought stress tolerance in plants. In: Drought Tolerance in Plants. Editors: A. Hossain, et al. Springer, New York, NY. ISBN: 9783319288970
• Type: Journal Articles Status: Other Year Published: 2016 Citation: Ma Y, Shukla V, and Merewitz E. 2016. Transcriptome analysis of creeping bentgrass exposed to drought stress and polyamine treatment. In preparation for Frontiers Plant Sci.
• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Laskowski K and Merewitz E. 2016. Irrigation rate and surfactant effects on creeping bentgrass and poa annua exposed to traffic stress. In preparation Agron J

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:The target audiences include turfgrass scientists, plant physiologists, turfgrass managers (primarily golf-course, lawn care, athletic field), Agri-business representatives, turfgrass consultants, and turfgrass education specialists in Michigan and the world. Changes/Problems:We have added a major component to goal 1 that was not specifically described in the objectives but is related to objective 1. Transcriptome analysis of creeping bentgrass plants in response to drought and polyamine application was performed.The work has been completed and we arecurrently analyzing the data. We expect that it will be published in 2016.This data will provide highly valuable information to turfgrass scientists and breeders since creeping bentgrass is not a species with a sequenced genome and gene expression datafor the species is limited. What opportunities for training and professional development has the project provided? The opportunities that all of the projects described above within the last year include training of three graduate students, one post-doctoral researcher, and four undergraduate students in the methods of turfgrass physiology research. The project has allowed me to develope professionally by gaining furtherexperience in graduate student instruction, manuscript preparation, and analysis of large quantity of transcriptome data that I had not previously had the experience of working with. How have the results been disseminated to communities of interest?The results have largely been disseminated or are in the process of being disseminated via a local industry conference (Michigan Turfgrass Conference), presentations to industry representatives (via the Michigan chapter of the Golf Course Superintendents Association, MiGCSA), field day presentations to local turf managers (MSU Turfgrass Field Day), presentations to corporate functions (such as Rainbird Co.), and academic conference presentations (ASA-CSSA-SSSA international meeting). What do you plan to do during the next reporting period to accomplish the goals?The major goals listed will continue to be investigated based on the methods provided in the initial report. We expect to satisfy several of the objectives within the next reporting period.

Impacts
What was accomplished under these goals? Abiotic stresses cause major damage to high value turfgrass species grown in Michigan and around the world. The central theme of the umbrella project is to identify abiotic stress tolerance mechanisms in order to utilize these mechanisms toward development of stress tolerant grass germplasm or protective products. Within the first goal of the project, we now have significant data to support conclusions for each objective. For objective 1, we have a paper now under review that describes how salt stress effects polyamine content in creeping bentgrass. For objective 2, we have a paper published Shukla, Ma, and Merewitz (2015) that documents that exogenous application of relatively low concentrations of some polyamines were effective in mitigating drought damage to creeping bentgrass. We have also conducted transcriptome analysis of the plants in this study, which was not originally documented in the objectives. Thus, we will publish work that discusses gene changes associated with drought stress and polyamine application in 2016. We have performed work subsequent to this to evaluate higher rates of polyamines, which prove to be more effective in promoting drought tolerance. For objective 3, we have analyzed some hormone changes in response to salt stress and polyamine application but this work still needs to be repeated in time. We have yet to complete work for objectives 4 and 5 since we are still evaluating whether a transgenic approach is warranted. Objective 6 will be satisfied in 2016. The second goal is to determine how abiotic stresses and turf management practices alter the response of plant hormones involved in the systemic aquired resistance (SAR) and induced systemicresistance (ISR)pathways such as ethylene, jasmonic, and salicylic acid. The results of objective 1 are currently being processed by the graduate student working on the project. This objective was being satisfied by a two year field study. We have concluded the two years of the study and plan to publish the results by the end of 2015 or in 2016. The student currently writing a master's thesis from this project. For the second objective, we have published a paper on this in 2014 (Krishnan and Merewitz, 2014). For the third objective, a new graduate student is looking at a bacterial disease to better understand turfgrass-pathogen interactions central to goal 2. This student has completed one year of work and we will publish the results in 2016. We have found that the bacterial pathogen is able to produce phytohormones and is thereby having a significant effect on phytohormones profiles in the plants. The bacteria may be using these hormones to cause disease symptoms, particularly symptoms of etiolation.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Krishnan SK and Merewitz E. 2015. Phytohormone Responses and cell viability during salinity stress in two creeping bentgrass cultivars differing in salt tolerance. JASHS 140:346-355.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Shukla V, Yingmei Ma, and Merewitz E. 2015. Creeping bentgrass responses to drought stress and polyamine application. JASHS 140:94-101.
• Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Ma Y and Merewitz E. 2015. Polyamine content changes in creeping bentgrass exposed to salt stress. Under Review Crop Sci.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: The target audiences include turf managers (primarily golf-course, lawn care, athletic field), Agri-business representatives, turf consultants, and turf education specialists in Michigan and the world. Changes/Problems: Additional areas of interest that are related to the central theme of the umbrella project have been developed this year as a result of extreme weather and disease incidences in the state of Michigan and other areas of the US. We have begun to investigate physiological responses of creeping bentgrass and poa annua to ice cover and other winterkill related issues. A post-doctoral researcher and graduate student already employed in our lab have begun this project this fall. Additionally, a project to investigate bacterial-turf plant relations has begun. A new graduate student, Sha Liu, has arrived this fall in order to conduct experiments on this project. What opportunities for training and professional development has the project provided? The opportunities that all of the projects described above have provided include training of two graduate students, two post-doctoral researchers, and two undergraduate students in the methods of turfgrass physiology research. A workshop on turfgrass physiology and abiotic stress was also given at the Michigan Turfgrass Conference for continuing education credits for certification of local turfgrass managers. How have the results been disseminated to communities of interest? The results to date have been disseminated via a local industry conference (Michigan Turfgrass Conference), presentations to industry representatives (via the Michigan chapter of the Golf Course Superintendents Association, MiGCSA), field day presentations to local turf managers (MSU Turfgrass Field Day), presentations to corporate functions (such as Rainbird Co.), and academic conference presentations (ASA-CSSA-SSSA international meeting). What do you plan to do during the next reporting period to accomplish the goals? The major goals listed will continue to be investigated based on the methods provided in the initial report. We plan to publish all results listed as unpublished or in progress within this progress report as well as publish new results that are yet to be generated.

Impacts
What was accomplished under these goals? Abiotic stresses cause major damage to high value turfgrass species grown in Michigan and around the world. The central theme of the umbrella project is to identify abiotic stress tolerance mechanisms in order to utilize these mechanisms toward development of stress tolerant grass germplasm or protective products. Several overall goals and specific projects are being conducted that attempt to address this central theme. The first goal is to understand the effects of metabolites known as higher polyamines on abiotic stress tolerance of creeping bentgrass. Within this goal we have currently accomplished results, either published or not yet published, for three of the six listed objectives (see initial report for full list). The objective in which we have made significant progress in are as follows:1) Determination of the effects of abiotic stresses on higher polyamine content, biosynthetic enzyme content and activity 2) Effects of exogenous application of higher ordered polyamines on stress tolerance 3) Evaluation of the changes in the transcriptome due to drought stress and polyamine application. The result of objective two has generated two papers (one submitted for publication and one published; Krishnan et al., 2013) the results of objectives 1 and 3 are in preparation for submission. The second goal is to determine how abiotic stresses and turf management practices alter the response of plant hormones involved in the systemic aquired resistance (SAR) and induced systemicresistance (ISR)pathways such as ethylene, jasmonic, and salicylic acid. The objectives are as follows: 1) evaluate physiological and hormonal responses between Creeping bentgrass (Agrostis stolonifera) and Annual bluegrass (Poa annua) in response to drought and traffic stress 2) Identify hormone changes following plant growth regulator application and during drought stress conditions 3) Develop predictions regarding disease susceptibility during abiotic stress and management practices based onSAR and ISR pathway indicators. The results of objective 1 are currently being processed by the graduate student working on the project. These results will be published within the coming year. For the second objective, we have published a paper that describes our results in detail and indicates future research directions for the goal (Krishnan and Merewitz, 2014).

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Krishnan SK and Merewitz E. 2014. Drought stress and trinexapac-ethyl modify phytohormone content within Kentucky bluegrass leaves. J PGR. DOI:10.1007/s00344-014-934-0
• Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Krishnan SK, Laskowski K, Shukla V, and Merewitz EB. 2013. Mitigation of drought stress damage by exogenous application of non-protein amino acid gamma-aminobutyric acid on perennial ryegrass. JASHS 138:358-366.

Progress 08/12/13 to 09/30/13

Outputs
Target Audience: The primary target audiences of the efforts reported for this period are the turfgrass science research community and turfgrass industry professionals. Other disciplines within plant biology may also be interested in the reported efforts. Changes/Problems: We are experiencing difficulty with polyamine analysis as they are low in content and diffiult to isolate. Currently we are attempting to perfect the protocol for extraction and analysis. This has caused a setback in the expected timeline of resuls however major changes are not expected. What opportunities for training and professional development has the project provided? Profssionals working on the project have learned new techniques including hormone extraction and detection methods. Experience working on plant care, maintenance and sampling for physiological methods were also obained as well as general laboratory skills. How have the results been disseminated to communities of interest? The research has been published and the results have beennorally presented at the joint CSSA-SSSA-ASA international meeting in Tampa Fla in November 2013 (outside of reporting period) What do you plan to do during the next reporting period to accomplish the goals? We are currently working on all parts1 and 2of Goal 1 and parts1 and 3 of Goal 2..

Impacts
What was accomplished under these goals? Part 2 of goal 2 was accomplished. We have completed the growth chamber study described in the project description.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Krishnan S, Laskowski K, Shukla V, and Merewitz E. 2013. Mitigation of drought stress damage by exogenous application of a non-protein amino acid gamma amino butyric acid on perennial regress. JASHS 138:358-366.