Molecular Characterization of U-box E3 Ubiquitin Ligases (TaPUB2 and TaPUB3) Involved in the Positive Regulation of Drought Stress Response in Arabidopsis

Abstract

Plant U-box E3 ubiquitin ligase (PUB) is involved in various environmental stress conditions. However, the molecular mechanism of U-box proteins in response to abiotic stress in wheat remains unknown. In this study, two U-box E3 ligase genes (TaPUB2 and TaPUB3), which are highly expressed in response to adverse abiotic stresses, were isolated from common wheat, and their cellular functions were characterized under drought stress. Transient expression assay revealed that TaPUB2 was localized in the cytoplasm and Golgi apparatus, whereas TaPUB3 was expressed only in the Golgi apparatus in wheat protoplasts. Additionally, TaPUB2 and TaPUB3 underwent self-ubiquitination. Moreover, TaPUB2/TaPUB3 heterodimer was identified in yeast and the cytoplasm of wheat protoplasts using a pull-down assay and bimolecular fluorescence complementation analysis. Heterogeneous overexpression of TaPUB2 and TaPUB3 conferred tolerance to drought stress. Taken together, these results implied that the heterodimeric form of U-box E3 ubiquitin ligases (TaPUB2/TaPUB3) responded to abiotic stress and roles as a positive regulator of drought stress tolerance.

Keywords: TaPUB2; TaPUB3; drought; plant U-box E3 ligase; ubiquitination; wheat.

Figure 1

Identification and expression patterns of TaPUB genes. (A) Phylogenetic tree showing deduced protein sequences of 15 TaPUB genes. (on the right) Schematic diagram of full-length TaPUB cDNA. (B) RT-PCR of 11 TaPUB genes in wheat leaf during drought stress treatment (polyethylene glycol (PEG) (20 %) with different times (0 and 6 h)). Red and green arrows represent the up and downregulation of genes. (C) RT-PCR and qRT-PCR analyses of TaPUB2 and TaPUB3 in wheat leaf tissues treated with drought stress (PEG (20%) in a time course experiment (0, 6, 12, and 24 h)). The relative expression levels of genes are presented as the mean ± SD of three experimental replicates. TaActin2 was used as the control. Asterisks represent statistically significant differences based on a two-tailed Student’s t-test when compared to controls (0 h); ** p < 0.01.

Figure 2

Gene expression patterns of TaPUB2 and TaPUB3 in response to different abiotic stresses (cold (4 °C), salt (200 mM NaCl), and abscisic acid (ABA) (0.1 mM)). (A,B) qRT-PCR analysis of TaPUB2 and TaPUB3 under different types of abiotic stresses in wheat leaf tissues. The TaPUB2 and TaPUB3 mRNA expression levels of the genes are presented as the mean ± SD of three biological replicates. TaActin2 was used as the control. Asterisks represent statistically significant differences based on a two-tailed Student’s t-test when compared to controls (0 h); * p < 0.05, ** p < 0.01.

Figure 3

Subcellular localization of TaPUB2 and TaPUB3. Subcellular localization of (A) 35S:GFP-TaPUB2 and (B) 35S:GFP-TaPUB3 in wheat protoplasts. 35S:GFP and G-rk-CD3-967 were used as controls. A confocal laser scanning microscope was used to detect the fluorescence signal.

Figure 4

In vitro E3 ligase activity of TaPUB2 and TaPUB3. (A) Schematic diagram of full-length TaPUB2 and TaPUB3 proteins. The solid bar depicts the protein length. The U-box and Armadillo repeat domain are shown as dark gray bars. (B) The ubiquitination reaction contains E1 (human), E2 (AtUBC1), E3 (MBP-TaPUB2, MBP-TaPUB2^C323A, MBP-TaPUB3, and MBP-TaPUB3^C325R), and ubiquitin. Polyubiquitin chains are visible by immunoblotting with a ubiquitin antibody. (+) means presence, and (-) means absence.

Figure 5

Heterodimeric complex formation of TaPUB2 and TaPUB3. (A) Yeast two-hybrid assay with TaPUB2 and TaPUB3. Full-length cDNA fragments encoding TaPUB2 and TaPUB3 were fused to sequences encoding GAL4 DNA-binding domain (BD) and GAL4 activation domain (AD) in pGBKT7 and pGADT7, respectively. Each construction was co-transformed into AH109 yeast strain in SD/-Leu/-Trp and SD/-Lue/-Trp/-His/-Ade/X-a-gal media. (B) In vitro pull-down assay of TaPUB3-His protein and MBP-TaPUB2 protein. (C) BiFC assay of TaPUB2 and TaPUB3 interactions using protoplast cells. TaPUB2 was fused into C-terminal YFP, and TaPUB3 was fused into N-terminal YFP. A confocal laser scanning microscope was used to detect the fluorescence signal.

Figure 6

Phenotypes of WT and 35S:TaPUB2 and 35S:TaPUB3 overexpression plants in response to drought treatment. (A,B) Root length of the indicated genotypes. Four-day-old seedlings were transferred from 1/2 MS to 1/2 MS medium previously infused with different concentrations of mannitol (200 and 400 mM). Photographs were taken 4 days after the transfer. (C,D) Germination rate of TaPUB2- and TaPUB3-overexpressing plants in MS and mannitol (200, 400 mM)-containing MS media. (E) TaPUB2, TaPUB3, and WT grown under drought stress. After planting in soil, seedlings were not watered for 2 weeks and then re-watered for 1 week. (F) Survival rates (%) under drought conditions were determined as the number of visibly green plants after rehydration. (G) Total chlorophyll content was measured in WT and overexpressing plants after recovery. (H) Comparison of the rate of water loss from detached rosettes between WT and overexpressing plants. (I) H₂O₂ content in Arabidopsis under normal conditions and drought treatment, with 3,3-diaminobenzidine staining assay of rosette leaves of WT and TaPUB2 and TaPUB3-overexpressing lines. Values are mean ± SD, n = 3. * Indicates significant difference between TaPUB2 or TaPUB3 plants and WT control based on the ANOVA (* 0.01 ≤ p < 0.05, ** p < 0.01), using the Duncan’s multiple range test (DMRT). Different letters indicate significant differences (p-value 0.05) among the genotypes under the same treatment.

Figure 7

Real-time qRT-PCR analysis of drought-stress related maker genes. For dehydration treatment, the lid of the dish was opened under light for 5 h at room temperature. The induction patterns of various ABA- and/or drought-responsive genes (RD29A, RD20, RD22, DREB2A, DREB1B, NCED3, ERD1, ABI5, ABF3, AFP1, PP2CA, HAI1, and KIN2) were analyzed using qRT-PCR. Data represent the fold induction of each gene under dehydration (5 h) relative to the gene level of the control treatment (0 h). The mean values from three independent technical replicates were normalized to the levels of the internal control, UBC10 mRNA. Different letters indicate significant difference at (p < 0.05) between the WT and 35S:TaPUB2 and 35S:TaPUB3 Arabidopsis transgenic plants.