Metabolite Responses to Exogenous Application of Nitrogen, Cytokinin, and Ethylene Inhibitors in Relation to Heat-Induced Senescence in Creeping Bentgrass

<div><p>The exogenous application of ethylene inhibitors, cyotkinins, or nitrogen has previously been shown to suppress heat-induced senescence and improve heat tolerance in cool -season grasses. The objectives of this study were to examine metabolic profiles altered by exogenous treatment of creeping bentgrass with an ethylene inhibitor, cytokinin or nitrogen under heat stress and to determine metabolic pathways regulated by those compounds in association with their effectiveness for improving heat tolerance. Creeping bentgrass (<i>Agostis stolonifera</i>) plants (cv. Penncross) were foliar sprayed with 18 mM carbonyldiamide (N source), 25μM aminoethoxyvinylglycine (AVG, ethylene inhibitor), 25μM zeatin riboside (ZR, cytokinin), or a water control, and then exposed to 20/15°C (day/night) or 35/30°C (heat stress) in growth chambers. All three exogenous treatments suppressed leaf senescence, as manifested by increased turf quality and chlorophyll content, and reduced electrolyte leakage under heat stress. Polar metabolite profiling identified increases in the content of certain organic acids (i.e. citric and malic acid), sugar alcohols, disaccharides (sucrose), and decreased accumulations of monosaccharides (i.e. glucose and fructose) with exogenous treatment of N, AVG, or ZR at the previously mentioned concentrations when compared to the untreated control under heat stress. Nitrogen stimulated amino acid accumulation whereas AVG and ZR reduced amino acid accumulation compared to the untreated control under heat stress. These results revealed that the alleviation of heat-induced leaf senescence by N, AVG, and ZR could be due to changes in the accumulation of metabolites involved in osmoregulation, antioxidant metabolism, carbon and nitrogen metabolism, as well as stress signaling molecules.</p></div

Principle component plot of AVG, ZR, N or water treatment groups during heat stress.

<p>Principle component analysis based on metabolite levels during 28 d heat stress for plants treated with AVG, ZR, N, or the water control. Component one is represented on the X-axis and accounted for 44.2% of variance, and component 2 is represented on the Y axis and accounted for 32.4% of variance.</p

Heat map of changes in metabolite levels in AVG, ZR, and N-treated plants at 28-d heat stress compared to the control.

<p>Heat map showing the fold change of each metabolite for AVG, ZR, and N-treated plants when compared against the control at 28-d Heat stress. Green indicates an increase in metabolite fold number or up-regulation, and red indicates a down-regulation of a specific metabolite compared to the control.</p

Sugar alcohol levels during heat stress for AVG, ZR, and N-treated plants, and the water-treated control.

<p>Relative quantities of sugar alcohols at 28-d heat stress for AVG, ZR, and N-treated plants, and the water-treated control. Error bars indicate standard deviations and letters are statistical groups according to Fisher’s protected LSD (p = 0.05), with groups not containing the same letter being significantly different. Only metabolites which had at least one group significantly different from the others are presented.</p

Amino acid levels during heat stress for AVG, ZR, and N-treated plants, and the water-treated control.

<p>Relative quantities of amino acids at 28-d heat stress for AVG, ZR, and N-treated plants, and the water-treated control. Error bars indicate standard deviations and letters are statistical groups according to Fisher’s protected LSD (p = 0.05), with groups not containing the same letter being significantly different. Only metabolites which had at least one group significantly different from the others are presented.</p

Physiological effects of AVG, CK, and N in creeping bentgrass.

<p>Changes to turf quality (A), chlorophyll content (B), and electrolyte leakage (C) during 28 d of 20/15 C or 35/30 C when treated with AVG, ZR, N or water as a control. Vertical bars represent least significance difference values between exogenous treatments at p = 0.05.</p

Monosaccharide levels during heat stress for AVG, ZR, and N-treated plants, and the water-treated control.

<p>Relative quantities of monosaccharides at 28-d heat stress for AVG, ZR, and N-treated plants, and the water-treated control. Error bars indicate standard deviations and letters are statistical groups according to Fisher’s protected LSD (p = 0.05), with groups not containing the same letter being significantly different. Only metabolites which had at least one group significantly different from the others are presented.</p

Variable importance plot of metabolites during heat stress.

<p>Variable importance projection plot generated from the partial least squares regression highlighting which metabolites may potentially play a larger role in heat tolerance as predicted by the model. Wold’s citerion is denoted by a horizontal line.</p

The 41 metabolites identified by GC-MS at 28 d heat stress and there respective retention times (RT).

<p>The 41 metabolites identified by GC-MS at 28 d heat stress and there respective retention times (RT).</p

Disaccharide levels during heat stress for AVG, ZR, and N-treated plants, and the water-treated control.

<p>Relative quantities of disaccharides at 28-d heat stress for AVG, ZR, and N-treated plants, and the water-treated control. Error bars indicate standard deviations and letters are statistical groups according to Fisher’s protected LSD (p = 0.05), with groups not containing the same letter being significantly different. Only metabolites which had at least one group significantly different from the others are presented.</p