Investigating the Function of TaUBX57 in Enhancing Abiotic Stress Tolerance in Wheat

Abstract

Wheat (Triticum aestivum L.), a crucial global food crop, provides approximately 20% of daily protein and caloric intake globally. However, its production is increasingly threatened by abiotic stressors, especially drought and salinity, which are exacerbated by climate change. These stressors adversely affect plant growth, development, and yield, necessitating the development of stress-tolerant varieties. This study investigated the role of TaUBX57, a U-box E3 ubiquitin ligase, in enhancing stress tolerance in wheat. Functional domain analysis of TaUBX57 confirmed the presence of a conserved U-box and a protein kinase domain, suggesting its involvement in protein ubiquitination and stress responses. TaUBX57-overexpressing transgenic Arabidopsis lines exhibited significantly improved germination rates, root growth, and survival under drought and salt stress conditions. TaUBX57 overexpression enhanced antioxidant enzyme activities and reduced the expression of oxidative stress markers, such as malondialdehyde. These findings highlight the potential role of TaUBX57 in modulating stress-responsive pathways and enhancing abiotic stress tolerance, offering a promising avenue for developing more resilient wheat varieties through genetic engineering.

Keywords: TaUBX57; U-box protein; drought and salt stress; wheat.

Figure 1

Domain and functional characterization of the wheat U-box domain protein TaUBX57. (A) Domain structure of TaUBX57 (TraesCS4D02G095000) was identified using InterProScan. The analysis revealed a conserved USP7_ICP0-binding domain at the N-terminal region (black line), U-box domain at the C-terminal region (amino acids 667–741, red line) and a protein kinase domain spanning amino acids 404–660 (green line). The domain structure was compared across several species, including XP_044980591 (Hordeum vulgare), XP_020149914 (Aegilops tauschii), and XP_010230025 (Brachypodium distachyon), highlighting conserved sequences indicative of similar functional roles. (B) A phylogenetic tree of TaUBX57 with other U-box proteins containing a protein kinase domain. The gene IDs included are XP_020149914 (A. tauschii), AT2G45910 (Arabidopsis thaliana), TaUBX57 (Triticum aestivum), XP_044980591 (H. vulgare), XP_047087049 (Lolium rigidum), XP_010230025 (B. distachyon), XP_021308267 (Sorghum bicolor), and NP_001404628 (Oryza sativa japonica). The analysis shows that TaUBX57 (red circle) is most closely related to XP_020149914 and AT2G45910, suggesting functional similarities in ubiquitin-mediated processes. (C) Subcellular localization of TaUBX57 fused with GFP in tobacco leaf epidermal cells. The TaUBX57-GFP fusion protein was transiently expressed under the CaMV 35S promoter. GFP fluorescence was predominantly observed along the plasma membrane. The red signal corresponds to the intrinsic autofluorescence of chloroplast. Confocal images were taken 72 h after infiltration. (D) In vitro ubiquitination assay demonstrating the E3 ligase activity of TaUBX57. Using recombinant TaUBX57 and E2 conjugating enzymes, polyubiquitin chains were formed, indicated by a smear of high-molecular-weight ubiquitinated proteins on the gel. This result confirms that TaUBX57 functions as an E3 ubiquitin ligase that can tag target proteins for degradation through the ubiquitin–proteasome system.

Figure 2

(A) Comparison of seed germination on 1/2 MS medium supplemented with 0, 100, 200, and 300 mM mannitol (top), and comparison of germination rates over 4 d (bottom). (B) Comparison of seed germination on 1/2 MS medium supplemented with 0, 0.5, 1.0, and 1.5 μM ABA (top), and comparison of germination rates over 7 d (bottom). (C) Comparison of seed germination on 1/2 MS medium supplemented with 0, 100, 150, and 200 mM NaCl (top), and comparison of germination rates over 7 d (bottom). All measured experiments were conducted with 50 replicates, and statistical analysis was performed using t tests. Error bars represent standard error (SE). Asterisks indicate levels of statistical significance compared with WT (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 3

Four-day-old seedlings were transferred to 1/2 MS medium with various concentrations of (A) mannitol, (B) ABA, and (C) NaCl. Status of root growth (top) and comparison of root lengths after 7 d (bottom). The images show root development in wild-type (WT) plants and TaUBX57-overexpressing lines (#2–4) under stress, highlighting differences in root elongation between the genotypes. All experiments were conducted with 10 replicates, and statistical analysis was performed using t tests. Error bars represent standard error (SE). Asterisks indicate levels of statistical significance compared with WT (* p < 0.05, ** p < 0.01).

Figure 4

Phenotypic and physiological responses of control (wild-type [WT]) and transgenic Arabidopsis plants under drought and salt stress conditions. (A) Comparison of growth status between control (wild-type [WT]) and transgenic Arabidopsis plants (#2–4) at 2 and 5 d after rewatering, following 14 d of no watering (drought stress). Scale bar = 0.25 cm (blue). (B) Survival rate of WT and transgenic lines under drought stress. (C) Comparison of malondialdehyde (MDA) content, an indicator of lipid peroxidation and oxidative stress, before and after drought treatment. (D) Comparison of growth status between control and transgenic plants before NaCl treatment, after treatment with 300 mM NaCl for 7 d, and 3 d after the 7 d treatment. Scale bar = 0.2 cm (green). (E) Survival rate under salt stress. (F) Comparison of MDA content before and after salt treatment. (G) DAB staining assay for detecting damage due to reactive oxygen species (ROS) under control, drought, and salt stress conditions. Scale bar = 0.5 cm (black). All experiments were conducted with 12 replicates, and statistical analysis was performed using t tests. Error bars represent standard error (SE). Asterisks indicate levels of statistical significance compared with WT (* p < 0.05, ** p < 0.01).

Figure 5

Antioxidant enzyme activity assays in TaUBX57-overexpressing Arabidopsis lines under drought (top panels) and salt (bottom panels) stress conditions. (A,G) Total antioxidant capacity (TAC) and the activities of (B,H) peroxidase (POD), (C,I) ascorbate peroxidase (APX), (D,J) superoxide dismutase (SOD), (E,K) catalase (CAT), and (F,L) glutathione (GSH) were measured. Each experiment was conducted with three biological replicates. Statistical significance was assessed using t tests, with error bars representing standard error of the mean (SE). Asterisks indicate significant differences between overexpressing lines and wild-type (WT) plants (* p < 0.05, ** p < 0.01).