Elevated cytokinin content in ipt transgenic creeping bentgrass promotes drought tolerance through regulating metabolite accumulation

Increased endogenous plant cytokinin (CK) content through transformation with an adenine isopentyl transferase (ipt) gene has been associated with improved plant drought tolerance. The objective of this study is to determine metabolic changes associated with elevated CK production in ipt transgenic creeping bentgrass (Agrostis stolonifera L.) with improved drought tolerance. Null transformants (NTs) and plants transformed with ipt controlled by a stress- or senescence-activated promoter (SAG12-ipt) were exposed to well-watered conditions or drought stress by withholding irrigation in an environmental growth chamber. Physiological analysis confirmed that the SAG12-ipt line (S41) had improved drought tolerance compared with the NT plants. Specific metabolite changes over the course of drought stress and differential accumulation of metabolites in SAG12-ipt plants compared with NT plants at the same level of leaf relative water content (47% RWC) were identified using gas chromatography–mass spectroscopy. The metabolite profiling analysis detected 45 metabolites differentially accumulated in response to ipt expression or drought stress, which included amino acids, carbohydrates, organic acids, and organic alcohols. The enhanced drought tolerance of SAG12-ipt plants was associated with the maintenance of accumulation of several metabolites, particularly amino acids (proline, γ-aminobutyric acid, alanine, and glycine) carbohydrates (sucrose, fructose, maltose, and ribose), and organic acids that are mainly involved in the citric acid cycle. The accumulation of these metabolites could contribute to improved drought tolerance due to their roles in the stress response pathways such as stress signalling, osmotic adjustment, and respiration for energy production

Protein accumulation in leaves and roots associated with improved drought tolerance in creeping bentgrass expressing an ipt gene for cytokinin synthesis

Cytokinins (CKs) may be involved in the regulation of plant adaptation to drought stress. The objectives of the study were to identify proteomic changes in leaves and roots in relation to improved drought tolerance in transgenic creeping bentgrass (Agrostis stolonifera) containing a senescence-activated promoter (SAG12) and the isopentyl transferase (ipt) transgene that increases endogenous CK content. Leaves of SAG12-ipt bentgrass exhibited less severe senescence under water stress, as demonstrated by maintaining lower electrolyte leakage and lipid peroxidation, and higher photochemical efficiency (Fv/Fm), compared with the null transformant (NT) plants. SAG12-ipt plants had higher root/shoot ratios and lower lipid peroxidation in leaves under water stress than the NT plants. The suppression of drought-induced leaf senescence and root dieback in the transgenic plants was associated with the maintenance of greater antioxidant enzyme activities (superoxide dismutase, peroxidase, and catalase). The SAG12-ipt and NT plants exhibited differential protein expression patterns under well-watered and drought conditions in both leaves and roots. Under equivalent leaf water deficit (47% relative water content), SAG12-ipt plants maintained higher abundance of proteins involved in (i) energy production within both photosynthesis and respiration [ribulose 1,5-bisphosphate carboxylase (RuBisCO) and glyceraldehyde phosphate dehydrogenase (GAPDH)]; (ii) amino acid synthesis (methionine and glutamine); (iii) protein synthesis and destination [chloroplastic elongation factor (EF-Tu) and protein disulphide isomerases (PDIs)]; and (iv) antioxidant defence system (catalase and peroxidase) than the NT plants. These results suggest that increased endogenous CKs under drought stress may directly or indirectly regulate protein abundance and enzymatic activities involved in the above-mentioned metabolic processes, thereby enhancing plant drought tolerance