Butanediol-enhanced heat tolerance in Agrostis stolonifera in association with alteration in stress-related gene expression and metabolic profiles

Accepted Manuscript Title: Butanediol-enhanced heat tolerance in Agrostis stolonifera in association with alteration in stress-related gene expression and metabolic profiles Authors: Yi Shi, Jing Zhang, Huibin Li, Mingna Li, Bingru Huang PII: DOI: Reference:

S0098-8472(18)30381-2 https://doi.org/10.1016/j.envexpbot.2018.06.002 EEB 3460

To appear in:

Environmental and Experimental Botany

Received date: Revised date: Accepted date:

10-3-2018 30-5-2018 4-6-2018

Please cite this article as: Shi Y, Zhang J, Li H, Li M, Huang B, Butanediol-enhanced heat tolerance in Agrostis stolonifera in association with alteration in stress-related gene expression and metabolic profiles, Environmental and Experimental Botany (2018), https://doi.org/10.1016/j.envexpbot.2018.06.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Butanediol-enhanced heat tolerance in Agrostis stolonifera in association with alteration in stress-related gene expression and metabolic profiles

1 College

of Grassland Science, Gansu Agricultural University, Lanzhou 730070, China;

Department of Plant Biology and Pathology, Rutgers, the State University of New Jersey, New

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2

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Authors: Yi Shi1,2†, Jing Zhang3†, Huibin Li2,4, Mingna Li2,5, Bingru Huang2*

Brunswick, NJ 08901, USA; 3 College

Laboratory of Crop Growth Regulation of Hebei Province, Agricultural University of Hebei,

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4 Key

of Agro-grassland Science, Nanjing Agricultural University, Nanjing 210095, PR China;

Science Department, College of Animal Science and Technology, China Agricultural

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5 Grassland

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Baoding 071001, China;

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University, Beijing 100193, P.R. China

authors

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*Corresponding

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†These authors contributed equally to this work.

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Bingru Huang, [email protected]; Tel: +1 848 932 6390 Fax: +1 732 932 9441

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Email addresses: YS: [email protected] JZ: [email protected] HL: [email protected] ML: [email protected]

BH: [email protected]

Highlights

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Foliar application of 2,3 –butanediol improved heat tolerance of creeping

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bentgrass. BD up-regulated genes related to cell elongation, metabolism, and stress responses in plants exposed to heat stress.

BD enhanced metabolite accumulation involved in energy metabolism and

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

stress signaling.

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Abstract

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Butanediol (BD) is a bacterial volatile compound which can activate induced-systemic

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resistance to diseases in plants, but its effects on abiotic stress tolerance are not well-known. The objectives of this study were to examine physiological effects of BD

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on heat tolerance in creeping bentgrass and to identify BD-responsive metabolites and genes contributing to effects of BD on heat tolerance. Creeping bentgrass plants

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(cv. ’PennA4’ and ’Penncross’) were treated with 2,3 -butanediol or water through foliar spray and were exposed to heat stress (35/30 oC, day/night) or optimal

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temperature (20/15 oC) in growth chambers. Creeping bentgrass plants treated with BD exhibited improved heat tolerance, demonstrated by higher visual quality and leaf photochemical efficiency when compared with the untreated control plants. Real-time

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PCR revealed that BD application resulted in up-regulation of genes related to cell elongation, metabolism, and stress responses in plants exposed to heat stress. Metabolite profiling identified a number of organic acids, sugars and sugar acids that accumulated due to BD treatment under heat stress. Results of the current study suggest that BD is effective in improving heat tolerance in creeping bentgrass, mainly

through the enhancement of gene expression and metabolite accumulation involved in energy metabolism and stress signaling.

Keywords: Creeping bentgrass, 2, 3-butanediol, Heat stress, Gene expression, Metabolites

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1. Introduction

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There is an increasing use of bacterial volatile compounds (BVCs) secreted by plant growth-promoting rhizobacteria (PGPR) for promoting plant growth and resistance to biotic and abiotic stresses ( Kazan, 2015; Nadeem et al., 2014). 2, 3-butanediol, a

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major component of BVCs secreted by some PGPR, has been found to promote plant

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growth and activate induced-systemic resistance (ISR) to biotic stresses in Arabidopsis

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thaliana (Ryu et al., 2004). Cho et al. (2008) reported that exogenously applying

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2R,3R-butanediol (an isomer of 2,3-butanediol) or colonizing Arabidopsis with BD-producing bacteria induced stomatal closure, which led to reduced water use in

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Arabidopsis, suggesting that BD may also affect drought tolerance; however, little information is available regarding the physiological, biochemical, and molecular

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effects on abiotic stress tolerance.

Heat stress is a major abiotic stress limiting the growth of temperate plant species.

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Whether 2, 3-butanediol may affect plant tolerance to heat stress and the manner in which it may do so have not yet been reported. Our previous study (Shi et al., 2017) found that applying 2, 3-butanediol as a foliar spray led to the activation of ISR in

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creeping bentgrass, which functions in resistance against Rhizoctonia solani through regulation of the plant transcriptome, including the up-regulation of genes involved in cell elongation, carbon metabolism, nitrogen metabolism, cytokinin metabolism and stress responses. Some BD-responsive genes identified in the transcriptome of creeping bentgrass infected with diseases (Shi et al., 2017), including expansin 1, expansin 3, isocitrate lyase, 3-Ketoacyl-CoA synthase, beta-glucosidase, nitrate reductase-like

gene, ARR-A two-component response regulator, adenine phosphoribosyl-transferase, phytoene synthase, lipoxygenase, hydroperoxide dehydratase, chitinase, and NAC transcription factors. Those BD-responsive genes may also play roles in regulating abiotic stress tolerance. Therefore, we hypothesized that BD application may alter the expression of the aforementioned genes, thereby positively affecting plant tolerance to

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heat stress. Stress defense also involves metabolomic changes, which can be quantified through metabolic profiling (Arbona et al., 2013). Alteration of metabolomic profiles or

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reprogramming of metabolomes by exogenous application of various plant growth regulators have been reported to be related to improved stress tolerance. For example,

Li et al. (2016) reported that exogenous application of γ-aminobutyric acid (GABA)

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resulted in improvement of heat tolerance in creeping bentgrass, which was associated

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with the accumulation of amino acids, organic acids, sugars, and sugar alcohols.

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Jespersen et al. (2015) reported enhanced heat tolerance of creeping bentgrass by the

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application of ethylene inhibitors and cytokinins in association with the increased content of certain organic acids, sugar alcohols, disaccharides, and decreased content

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of monosaccharides. As BD exhibits properties similar to plant growth regulators (Ryu et al., 2003), we hypothesize that BD may alter the expression of stress-defense

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proteins and metabolic profiles, contributing to its promotive effects on plant tolerance to heat stress.

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Heat stress is the primary factor causing the decline in visual quality of cool-season

grass species during summer months in many areas of the world, and therefore, improving heat tolerance of these species is of great priority. Creeping bentgrass

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(Agrostis stolonifera) is a cool-season grass species widely used as forage and turf, which is sensitive to high temperature and often suffer from heat stress damages during summer months (Fry and Huang, 2004). The objectives of this study were to examine the physiological effects of BD on heat tolerance in creeping bentgrass and to identify BD-responsive metabolites and genes that may contribute to BD-induced

heat stress tolerance.

2. Materials and methods 2.1 Plant materials and growth conditions Sod plugs of creeping bentgrass (Cv. ’PennA4’ and ’Penncross’) were collected from

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field plots at the Rutgers University Horticultural Farm II research facility in North Brunswick, NJ. ‘PennA4’ is more heat tolerant relative to ‘Penncross’ (Fry and Huang,

2004). Plants were transplanted into plastic pots (11 cm in diameter and 20 cm deep)

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filled with fritted clay and allowed to establish in a greenhouse for 30 d. During the

establishment period, plants were irrigated every other day each week, fertilized with Hoagland’s nutrient solution once weekly (Hoagland and Arnon, 1950), and were

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trimmed every 3 days to maintain a 2-cm canopy height. Following the establishment

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period, plants were transferred to growth chambers (Environmental Growth Chamber,

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Chagrin Falls, Ohio, USA) maintained at a temperature of 20/15 °C (day/night), 70%

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relative humidity, a 14 h photoperiod, 610 μmol m-2s-1 photosynthetically active radiation (PAR) and were allowed to acclimate for 7 d before initiation of treatments.

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2.2 Treatments and experimental design Creeping bentgrass plants were treated with BD (Sigma-Aldrich, St. Louis, MO,

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USA) at a 300 µM concentration or with water as untreated control daily for 3 d prior to exposure of plants to heat stress (35/30 oC, day/night) or optimal air temperature (20/15 o

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C) conditions. Both treatments were applied as foliar spray at a volume which

saturated the canopy (approximately 15 ml per pot). Treatments were applied at 10 d intervals for the remainder of the 40 d of heat treatment, after which stressed plants

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were maintained in growth chambers at optimal temperature (20/15 oC) for 14 d to examine recovery following heat stress. The 300 µM concentration of BD was observed to be most effective for enhancing heat tolerance in creeping bentgrass in a preliminary study which tested sveral concentrations of BD (150, 200, 250, 300, 350µM).

The experiment was arranged to a split-plot design with temperature treatments (heat stress and optimal temperature) as the main plots and chemical treatments (with BD or without BD) and cultivars (‘PennA4’ or ‘Penncross’) as the sub-plots. Each treatment had four replicates. Each stress treatment was repeated in four growth chambers using the environmental conditions described above.

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2.3 Physiological measurements Physiological measurements were conducted at 7, 20, 30 and 40 d of heat stress, and at 14 d of recovery. Each parameter was measured on four replicates for each treatment.

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Leaf senescence and whole-plant heat tolerance were evaluated using four

commonly-used indicators, including turf quality (TQ), leaf photochemical efficiency (Fv/Fm), chlorophyll content (Chl) and cell membrane stability. Turf quality was

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evaluated using SigmaScan Pro (v. 5.0, SPSS Inc., Chicago, IL) software to quantify

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percent green leaves. A higher percent green leaves indicated a better turf quality. Leaf

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photochemical efficiency (Fv/Fm) was measured as the ratio of variable (Fv) to

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maximum (Fm) fluorescence using a fluorescence induction meter (Fim 500; Bio-Scientific Ltd., Herts, UK). Fv/Fm of leaves was measured following a 30 min

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dark-adaptation period. For Chl quantification, fresh leaves (0.1 g) were excised and extracted with 10 ml dimethyl sulfoxide in the dark for 72 h. Chl content was measured

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on a spectrophotometer at wavelengths of 663 nm and 645 nm according to the procedure of Arnon(1949). Membrane stability was estimated by electrolyte leakage

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(EL) using the methods described by Blum(1981). Approximately 0.1g of fresh leaf tissue was placed in a conical tube containing 35 ml de-ionized water and incubated on an orbital shaker for 16 h. Initial conductance readings (Ci) of the incubated solutions

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were taken using a conductivity meter (YSI Incorporated, Yellow Springs, OH). Tubes were autoclaved at 121 °C for 20 min to kill all leaf tissue, then placed back on the shaker for an additional 16 h and a final conductance reading was measured (Cmax). EL was calculated using a percentage of Ci/Cmax. Leaf growth rate was evaluated by measuring canopy by diameter and height. Canopy diameter was measured every

sampling day. The turfgrass canopy was trimmed to a 2 cm height following application of the first chemical treatment and each subsequent sampling day. 2.4 Analysis of gene expression levels by real-time PCR Several functional categories of genes that were previously reported by Shi et al. (2017) were selected as candidate genes for analysis of BD effects related to heat

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tolerance. The literature analyzed the transcriptome of creeping bentgrass treated with BD by different times, but without biotic or abiotic stresses. So the gene expression

trend from the transcriptome analysis can be instructive for the current research

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related with heat. We assume that BD induced plant stress related-gene will show more remarkable expression change with heat stress following the BD treatment.

Gene expression was measured at 30 d of heat stress, when plants treated with BD

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exhibited significant physiological effects. The gene names and primer designs are

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shown in Supplemental Table S1.

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Total RNA was isolated from ground leaves using Trizol-reagent (Gibco BRL, Life

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Technologies, Grand Island, NY) according to manufacturer’s instructions and DNA contamination was removed using the Turbo DNA-Free kit (Ambion, Austin, TX).

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RNA was reverse-transcribed in a 20 ml reaction using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies, Grand Island, NY) according to

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manufacturer’s instructions. cDNA was amplified using Power SYBR Green PCR Master Mix (Life Technologies, Grand Island, NY) on the StepOnePlus Real-Time

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PCR System (Applied Biosystems, Foster City, CA, USA). 2.5 Metabolite extraction, separation, and quantification Metabolomic profiling was analyzed at 40 d of heat stress and 14 d of recovery

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when plants treated with BD showed significant physiological effects. Before quantifying the content of metabolites, leaves were washed thoroughly with deionized and distilled water to avoid the potential interference of microorganisms on the leaf surface. . Extraction of metabolites was conducted according to the methods of Roessner et al. (2000) and Rizhsky et al. (2004). For each sample, frozen leaves were

ground to a fine powder with liquid nitrogen, and 25 mg leaf tissue powders were transferred into 1.5 mL centrifuge tubes and extracted with 1.4ml of 80% (v/v) aqueous methanol at 23 oC for 2 h. A 10 μl ribitol solution (2mg·ml−1) was added to the samples as an internal standard prior to incubation in a water bath at 70 oC for 15 min. After being centrifuged at 12 000 rpm for 30min, supernatant from each sample was pipetted

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into new tubes, and 1.4ml of water and 0.75 ml of chloroform were added to each new tube. The resultant mixture was thoroughly vortexed and centrifuged for 5 min at 5000

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g. Polar phase (methanol/water) at 2 ml was decanted into 1.5 ml high-performance liquid chromatography (HPLC) vials and dried in a centrivap benchtop centrifugal

concentrator (Labconco, Kansas City, MO). The dried polar phase was methoximated

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with 80 μl methoxyamine hydrochloride (20 mgml−1) at 30 oC for 90 min and was

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trimethylchlorosilane) for 60 min at 70 oC.

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trimethylsilylated with 80 μl N-methyl-N-trimethylsilyltri-fluoroacetamide (with 1%

Analysis conducted using the gas chromatography-mass spectrometer (GC-MS) was

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performed according to methods described by Qiu et al.(2007). The extracts were

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analyzed with a PerkinElmer gas chromatograph coupled with an Autosystem XL TurboMass mass spectrometer (PerkinElmer Inc., Waltham, MS). Equal extracts (1 μl) were injected into a DB-5MS capillary column (30m× 0.25mm× 0.25 μm, Agilent

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J&W Scientific, Folsom, CA). The inlet temperature was maintained at 260 oC. After a

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5 min solvent delay, the initial GC oven temperature was set at 80 oC, and 2 min after injection, the GC oven temperature was raised to 280 oC at a rate of 5 oC min−1 and finally held at 280 oC for 15 min. The injection temperature was set to 280 oC and the

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ion source temperature was adjusted to 200 oC. Helium was used as the carrier gas with a constant flow rate set at 1 ml·min−1. The measurements were made with electron impact ionization (70 eV) in the full scan mode (m/z 30–550). The metabolites were identified using the TURBOMASS 4.1.1 software (PerkinElmer Inc.) coupled with commercially available compound libraries: NIST 2005 (PerkinElmer Inc.), Wiley 7.0 (John Wiley & Sons Ltd., Hoboken, NJ).

2.6 Statistical analysis All data were subjected to analysis of variance according to the general linear model of SAS v9.0 (SAS Institute Inc., Cary. NC). Treatment means were separated using Fisher’s protected least significant difference (LSD) test at P < 0.05.

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3. Results 3.1 Effects of butanediol on growth and physiological traits

Heat stress inhibited shoot growth and induced leaf senescence, whereas heat stress

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was alleviated in both cultivars treated with BD. BD-treated plants appeared to recover

to a greater extent than untreated plants following heat stress (Fig. 1A). The canopy diameters and heights of BD-treated plants were significantly higher than those of

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untreated plants during and after heat stress for ‘PennA4’ and following heat stress for

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‘Penncross’ (Fig. 1B).

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The percent green leaves declined significantly with heat stress for both cultivars, and

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BD treatment resulted in greater percent green leaves compared to untreated ‘Penncross’ plants (Fig. 2A). Heat stress caused significant reduction in photochemical efficiency

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for both cultivars, while BD-treated plants showed significantly higher photochemical efficiency than untreated plants at 40 d of heat stress and following recovery (Fig. 2B).

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3.2 Effects of butanediol on the expression of cell elongation-related and metabolism-related genes expression

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The

levels

of

four

expansin

genes

and

six

xyloglucan

endotransglycosylase (XET) genes regulating cell wall loosening were examined to determine whether the shoot growth enhanced by BD treatment could be associated

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with changes in expansin and XET expression under prolonged periods (30 d) of heat stress. EXP1 and EXP3 exhibited increased expression in BD-treated plants compared to untreated controls (Fig.3A-1, Fig.3A-2, Fig.3B-1, Fig.3B-2), while other expansin and XET genes examined in the study did not show clear patterns in terms of up- or down-regulation in response to either heat stress or BD treatment (data not shown).

Several genes related to carbon and nitrogen metabolism (isocitrate lyase, 3-ketoacyl-CoA synthase, and beta-glucosidase) and nitrate reductase-like gene, which was previously found to be upregulated in the creeping bentgrass transcriptome due to application of BD, were detected by qRT-PCR in this study (Fig. 3A-3, 4, 5, 6; Fig. 3B-3, 4, 5, 6). The increased expression levels of isocitrate lyase, 3-ketoacyl-CoA

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synthase, beta-glucosidase, and nitrate reductase-like gene responded to BD treatment in both cultivars at 30 d of heat stress.

Two cytokinin-related genes (Type-A Arabidopsis Response Regulator (ARR-A) and

Fig.3B-7,

8).

The

expression

levels

of

the

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adenine phosphoribosyltransferase) were also analyzed by qRT-PCR (Fig.3A-7, 8, ARR-A

and

adenine

phosphoribosyltransferase genes were up-regulated by BD treatment in both cultivars.

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BD significantly increased the expression levels of the GABA biosynthesis related

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gene-phytoene synthase gene and the jasmonic acid biosynthesis gene-lipoxygenase

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gene in ‘PennA4’ at 30 d of heat stress (Fig.3A-9, 10). The hydroperoxide dehydratase

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gene was upregulated by BD treatment in both cultivars (Fig.3B-11). BD increased the expression of chitinase and NAC transcription factor at 30 d of heat stress in both

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cultivars (Fig.3A-12, 13, Fig.3B-12, 13).

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3.3 Effects of butanediol on metabolomic profiles Based on the GC-MS analysis, more than 500 peaks and 450 putative metabolites

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were found in leaves of creeping bentgrass. A total of 47 metabolites were identified and quantified, including 12 organic acids, 14 sugars, 5 sugar alcohols, 5 glucosides, 11 sugar acids, and other metabolites. The overall changes in 47 metabolites are shown in

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Fig. 4. BD treatment induced a number of changes in metabolite accumulation which were unique or common to both non-stress and high temperature conditions. Not all of the identified organic acids were significantly induced by BD treatment. Six of them exhibited significantly higher levels after BD treatment for ‘PennA4’ and ‘Penncross’ exposed to heat stress (Fig. 5). Under optimal temperature conditions, the

content of most organic acids was not enhanced by BD treatment for either cultivar under heat stress. Aconitic acid, pentonic acid, glycolic acid, malic acid, methylmaleic acid, and palmitic acid showed significantly higher contents in BD-treated plants than in untreated plants for both cultivars, however, the increase was to a greater extent in ‘Penncross’.

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Most sugars did not respond to BD treatment under optimal temperature conditions. The contents of talose, tagatose, and maltose were increased in ‘PennA4’ under heat stress, while lyxopyranose was enhanced following heat stress recovery. For

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‘Penncross’ treated with BD, the contents of talose, tagatose, galactose, ribofuranose, maltose, and lyxopyranose all increased under heat stress (Fig. 6).

BD treatment also affected the contents of other metabolites (methyl galactoside,

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threonic acid, eicosatriynoic acid, silanol, and uridine) under optimal temperature and

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heat stress conditions (Fig.7). Methyl galactoside contents were significantly increased

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in both of the cultivars under stress conditions. BD treatment induced increases in

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threonic acid and eicosatriynoic acid contents in stressed plant in the two cultivars. The contents of silanol and uridine were significantly enhanced by BD treatment in

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stressed plants of each cultivar during both heat stress and recovery.

4. Discussion The current study is the first to find positive effects associated with the application of BD promoting heat tolerance in plants. The effects of the compound to mitigate heat stress is demonstrated by significant increases in shoot growth, turf quality, and photochemical efficiency in creeping bentgrass exposed to prolonged high temperature

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stress. This study also identified genes, metabolites, and metabolic processes associated with BD-induced heat tolerance. The alteration in expression of selected genes and the accumulation of metabolites with important biological functions may contribute to

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BD-enhanced heat tolerance in creeping bentgrass, as discussed below.

A previous transcriptomic analysis has found a large number of genes related to cell elongation, carbon metabolism, nitrogen metabolism, cytokinin metabolism, and stress

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response that are abundantly up- or down-regulated in response to BD treatment in

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creeping bentgrass (Shi et al., 2017). In this study, 13 BD-upregulated genes identified

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through transcriptomic profiling, including expansin 1, expansin 3, isocitrate lyase,

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3-Ketoacyl-CoA synthase, beta-glucosidase, nitrate reductase-like gene, ARR-A two-component response regulator, adenine phosphoribosyltransferase, phytoene

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synthase, lipoxygenase, hydroperoxide dehydratase, chitinase and NAC transcription factors were examined to determine whether changes in the expression levels of those

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genes could be related to BD-enhanced heat tolerance in creeping bentgrass. Cell expansin and elongation are directly controlled by cell wall loosening proteins,

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including expansins and XETs. Isocitrate lyase and 3-ketoacyl-CoA synthase genes are key enzymes in glyoxylate cycle in respiratory metabolism (Lorenz and Fink, 2002). Beta-glucosidase is associated with the release of glucose in carbon metabolism (Chan

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et al., 2018; Lorenz and Fink, 2002). Nitrate reductase-like gene is related to nitrogen and protein metabolism (Das et al., 2017). ARR-A and adenine phosphoribosyl transferase are related to cytokinin metabolism and regulating cell growth and development (Abdelrahman et al., 2017; Liu and Huang, 2002). Phytoene synthase is a transferase enzyme involved in the biosynthesis of carotenoids. Flowerika et al. (2016)

reported that the transcript of TaPSY3, which is one of the phytoene synthase genes, was upregulated during drought and heat stress in wheat. The higher expression level of TaPSY3 was correlated with the multiple stress responsive cis-regulatory elements in the promoter region, which suggests that it has a role in stress adaptation. Lipoxygenases and hydroperoxide dehydratase are involved in the biosynthesis of

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lipid-derived signaling molecules such as jasmonic acid (JA) (Bell et al., 1995; Sivasankar et al., 2000). Clarke et al. (2009) showed that the JA and salicylic acid

mutants of Arabidopsis thaliana were sensitive to heat stress, which suggests that JAs

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provide thermotolerance to basal cells. It is reported that chitinase and NAC

transcription factor are involved in plant biotic and abiotic stress responses (Kasprzewska, 2003; Nuruzzaman et al., 2013). In this study, qPCR analysis showed

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that all 13 genes exhibited up-regulation by BD treatment in one or both cultivars of

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creeping bentgrass exposed to heat stress, suggesting those genes play positive roles in

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BD-enhanced heat tolerance. ARR-A-two-component response regulator gene (7),

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phytoene synthase gene (9) and lipoxygenase gene (10) were up-regulated in PennA4 and down-regulated in Penncross after BD treatment with heat stress. The differential

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responses of gene expression for ARR-A-two-component response regulator, phytoene synthase, and

lipoxygenase gene between the two cultivars may be

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associated with the genotypic variations in heat tolerance, as PennA4 was more heat-tolerant than Penncross based on the physiological traits.

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Primary metabolites, including organic acids and sugars, play essential roles in

regulating plant growth and stress tolerance (Lopez-Bucio et al., 2000; Sweetlove et al., 2010). In the current study, treatment with BD resulted in higher accumulations of

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organic acids (aconitic acid, pentonic acid, glycolic acid, malic acid, methylmaleic acid and palmitic acid) in both cultivars of creeping bentgrass exposed to heat stress. Among those metabolites, aconitic acid and malic acid are the major intermediates of TCA cycle, which is involved in energy metabolism via respiration (As et al., 2007). Increases in aconitic acid and malic acid content due to acibenzolar-S-methyl were

suggested to be related to improved heat tolerance in creeping bentgrass (Jespersen et al, 2017). Elevated levels of aconitic acid and malic acid in this study may indicate that BD-treated creeping bentgrass had higher respiratory activity in order to help maintain repair and defense mechanisms during heat stress. Although the contents of many sugars were affected by heat stress, only three sugars, talose, tagatose and

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maltose, exhibited differential responses to BD treatment under heat stress. Application of BD resulted in increases in the contents of these three sugars under high temperature conditions. Apart from organic acids and sugars, several other

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metabolites were also regulated by BD treatment under heat stress. The content of

threonic acid, a sugar acid derived from threose, was decreased under heat stress when creeping bentgrass of the two cultivars was not treated with BD but increased

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following treatment with BD under high-temperatures. Increased accumulation of

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talose, maltose, and threonic acid in response to γ-aminobutyric acid treatment was

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suggested to be associated with improved heat tolerance in creeping bentgrass (Li et

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al., 2016). The enhanced contents of organic acids, sugars, or sugar acids due to BD may contribute to BD-enhanced heat tolerance in creeping bentgrass through

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activation or maintenance of active respiratory activities, carbon metabolism, and stress protective compounds.

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In conclusion, foliar application with BD effectively improved heat tolerance in creeping bentgrass. The enhanced heat tolerance may be related to the up-regulation of

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genes related to cell elongation, metabolism, and stress response, and to the accumulation of organic acids, sugars, and sugar acids involved in respiration, carbon metabolism, and stress protection. BD could be used as a potential growth-regulating

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or stress protective agent in improving turfgrass heat tolerance, although further research is needed to understand the underlying molecular factors associated with the improvement of heat tolerance in creeping bentgrass and other plant species.

Author contribution: YS and JZ performed the experiment and analyzed the data. HB and ML helped with the experiment. BH provided all financial support and design of this study. YS and BH wrote and revised the manuscript. All authors read and approved the final manuscript. The authors declare no competing financial interests.

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Acknowledgements: This study was supported by the Center for Turfgrass Science at Rutgers University and the Program of Study Abroad for Young Teachers by Agricultural University of Hebei. We thank the Chinese Scholarship Council for

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providing a stipend during Yi Shi’s study at Rutgers University. Thanks also go to

Xiqing Ma, Yi Xu, and Xiaxiang Zhang for helping with the experiment and to

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Stephanie Rossi for editing the manuscript.

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Figure 1. Effects of butanidiol (BD) on canopy growth for two cultivars of creeping bentgrass exposed to heat stress. A: photos of plants. B: turf canopy diameter and

p=0.05 for comparison between BD treatment and untreated control.

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height. The vertical bars indicate the values of least significant difference (LSD) at

Figure 2. Effects of butanidiol (BD) on leaf photochemical efficiency (Fv/Fm) for two

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cultivars of creeping bentgrass exposed to heat stress. The vertical bars indicate the

values of least significant difference (LSD) at p=0.05 for comparison between BD treatment and untreated control.

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Figure 3. Effects of butanidiol (BD) on the transcript levels of cell elongation-related

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and metabolism-related genes for two cultivars of creeping bentgrass exposed to 30 d

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of heat stress. The different lowercase letters over the columns indicate significant

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differences between treatments based on the least significant difference (LSD) at p=0.05. 1- Expansin 1 gene; 2- Expansin 3 gene; 3- Isocitrate lyase gene; 4-

gene;

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3-Ketoacyl-CoA synthase gene; 5- Beta-glucosidase gene; 6- Nitrate reductase-like ARR-A-two-component

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phosphoribosyl-transferase gene; 9- Phytoene synthase gene; 10- Lipoxygenase gene; 11- Hydroperoxide dehydratase gene; 12- Chitinase gene; 13- NAC transcription

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Figure 4. Heat map of changes in 47 metabolites in creeping bentgrass as affected by butanidiol (BD) at 40 d of heat stress and 14 d of recovery. The log2-fold change

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ratios are shown in the results. Green indicates up-regulation and red indicates down-regulation. Figure 5. Effects of butanidiol (BD) on the contents of organic acids for two cultivars of creeping bentgrass at 40 d of heat stress and 14 d of recovery. The different lowercase letters over the columns indicate significant differences between treatments

based on the least significant difference (LSD) at p=0.05.

Figure 6. Effects of butanidiol (BD) on the contents of sugars for two cultivars of creeping bentgrass at 40 d of heat stress and 14 d of recovery. The different lowercase letters over the columns indicate significant differences between treatments based on

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the least significant difference (LSD) at p=0.05. Figure 7. Effects of butanidiol (BD) on the contents of sugar acids and sugar alcohols for two cultivars of creeping bentgrass at 40 d of heat stress and 14 d of recovery. The

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