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

Abstract

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 (F(v)/F(m)), 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.

Fig. 1.

Soil volumetric water content (SWC, %) measured using buried time domain reflectometry probes (20 cm) during the 14 d duration of water treatment of well-watered and drought-stressed SAG12-ipt and NT plants. Vertical bars indicate LSD values where significant differences were detected (P ≤0.05) for comparison between plant lines on a given treatment day.

Fig. 2.

Leaf physiological responses of SAG12-ipt and NT leaves to drought stress conditions evaluated by measurement of (A) turf quality (TQ; 1–9 scale, with 1=completely desiccated and 9=healthy, turgid), (B) photochemical efficiency (F_v/F_m), (C) relative water content (RWC, %), and (D) electrolyte leakage (EL, %). Vertical bars indicate LSD values where significant differences were detected (P ≤0.05) for comparison between plant lines at a given soil water content (SWC %).

Fig. 3.

Leaf antioxidant activity responses of (A) superoxide dismutase (SOD), (B) peroxidase (POD), and (C) catalase (CAT) in ipt transgenic creeping bentgrass (SAG12-ipt) compared with null transformant (NT) lines under drought stress. Vertical bars indicate LSD values where significant differences were detected (P ≤0.05) for comparison between plant lines at a given soil water content (SWC %).

Fig. 4.

Root characteristics and enzyme activity assays of ipt transgenic creeping bentgrass (SAG12-ipt) compared with null transformant (NT) lines under drought stress as measured by (A) root:shoot ratio, (B) root viability, (C) superoxide dismutase (SOD), and (D) peroxidase (POD). Vertical bars indicate LSD values where significant differences were detected (P ≤0.05) for comparison between plant lines at a given soil water content (SWC %).

Fig. 5.

Representative gel image following two-dimensional PAGE analysis of leaf protein extracts of null transformant (NT) and ipt transgenic creeping bentgrass (SAG12-ipt) exposed to water stress. Protein spots circled had differential accumulation due to water stress relative to the respective non-stressed control plant line (blue, greater accumulation; red, lower accumulation) (P ≤0.05).

Fig. 6.

Four-way Venn diagram comparing the number of proteins that exhibited a significant (P ≤0.05) increase or decrease due to water stress in ipt transgenic creeping bentgrass (SAG12-ipt) compared with null transformant (NT) plant lines relative to the protein content of the respective well-watered control plants for leaves and roots. Overlapping regions of the circles indicate proteins that were regulated in either the same or the opposite manner in the respective treatment, whereas non-overlapping circles indicate proteins regulated in only that treatment.

Fig. 7.

Percentages of proteins exhibiting significant differential expression (P ≤0.05) due to transgene expression or drought stress of ipt transgenic creeping bentgrass (SAG12-ipt) compared with null transformant (NT) lines within each functional category for (A) leaves and (B) roots.