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

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.


Introduction
Plants are consistently exposed to adverse environmental conditions throughout their life cycle. They are regularly subjected to harmful environmental cues that affect their growth, development, and yield. Therefore, plants have evolved various physiological, biochemical, and molecular strategies to cope with these extreme environmental conditions [1,2]. Although the metabolic response to drought stress in various crops has been widely studied, little is known about the molecular mechanisms underlying drought tolerance, especially in wheat. Therefore, the study of stress responsive genes in wheat would clarify its response to various conditions. Wheat (Triticum aestivum L.) is an economically important cereal crop in the developing world, as an estimated 80 million farmers depend on wheat for their livelihood [3]. Recurrent droughts caused by climate change are a major constraint on the global wheat productivity [4]. Recently, numerous efforts have been made to mitigate drought stress by breeding drought-resistant varieties. However, wheat has a large and structurally complex genome, and drought tolerance in wheat is a complex trait that is controlled by many genes. To date, several candidate genes for drought stress tolerance in wheat have been reported. TaMYB31-B overexpression in Arabidopsis has been shown to regulate the drought stress response in an abscisic acid (ABA)-dependent manner [5]. TaAREB3 (ABA response element binding 3), a new member of the AREB transcription factor family, not only enhances drought tolerance, but also strengthens ABA sensitivity and cold tolerance [6]. Moreover, wheat E3 ligase (TaSAP5) is highly induced by drought stress and ubiquitinates defective

Isolation of Drought-Induced PUB Genes
In a previous report, 213 PUB genes were identified and classified in wheat, according to their functional domain. Among them, 118 genes showed drought-and heat-stressspecific expression patterns [18]. Based on these results, we selected 15 genes that were highly expressed under drought stress conditions to identify the involvement of TaPUBs in the metabolism of drought stress responses ( Figure 1A). Phylogenetic analysis revealed five groups, of which two groups consisted of three genes from each subgenome-A, B, and D. Furthermore, RT-PCR was used to examine the expression patterns of 11 U-box genes that were expressed under drought conditions, and the results indicated that the expression of two genes (TraesCS5A02G198800.1 and TraesCS2B02G499300.1) was upregulated, while the expression of two genes (TraesCS5D02G210500.1 and TraesCS4B02G371600.1) was downregulated after 6 h of drought treatment ( Figure 1B). Therefore, TraesCS5A02G198800.1 and TraesCS2B02G499300.1, named TaPUB2 and TaPUB3, respectively, were selected for further studies. Furthermore, we tried to confirm the transcript levels of TaPUB2 and TaPUB3 in wheat leaf tissues treated with drought stress for different durations (0, 6, 12, and 24 h) ( Figure 1C). Compared to TaPUB2 and TaPUB3 levels in the control (0 h), TaPUB2 and TaPUB3 were significantly induced in the leaves after 6 to 24 h of drought stress treatment. expression of two genes (TraesCS5A02G198800.1 and TraesCS2B02G499300.1) was upregulated, while the expression of two genes (TraesCS5D02G210500.1 and TraesCS4B02G371600.1) was downregulated after 6 h of drought treatment ( Figure 1B). Therefore, TraesCS5A02G198800.1 and TraesCS2B02G499300.1, named TaPUB2 and Ta-PUB3, respectively, were selected for further studies. Furthermore, we tried to confirm the transcript levels of TaPUB2 and TaPUB3 in wheat leaf tissues treated with drought stress for different durations (0, 6, 12, and 24 h) ( Figure 1C). Compared to TaPUB2 and TaPUB3 levels in the control (0 h), TaPUB2 and TaPUB3 were significantly induced in the leaves after 6 to 24 h of drought stress treatment.  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 < 0.05, ** p < 0.01.

TaPUB2 and TaPUB3 in Response to Different Abiotic Stresses
We analyzed the transcript levels of TaPUB2 and TaPUB3 in response to cold stress (4 °C), salt stress (200 mM NaCl), and ABA (0.1 mM) at 0, 6, 12, and 24 h to determine whether they were upregulated ( Figure 2). The expression levels of TaPUB2 and TaPUB3 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.

TaPUB2 and TaPUB3 in Response to Different Abiotic Stresses
We analyzed the transcript levels of TaPUB2 and TaPUB3 in response to cold stress (4 • C), salt stress (200 mM NaCl), and ABA (0.1 mM) at 0, 6, 12, and 24 h to determine whether they were upregulated ( Figure 2). The expression levels of TaPUB2 and TaPUB3 were highly increased under cold treatment at 6 h and decreased afterwards. TaPUB2 and TaPUB3 were highly expressed between 6 and 24 h under salt treatment. However, they were highly expressed only at 12 h after ABA treatment.
were highly increased under cold treatment at 6 h and decreased afterwards. TaPUB2 and TaPUB3 were highly expressed between 6 and 24 h under salt treatment. However, they were highly expressed only at 12 h after ABA treatment. TaActin2 was used as the control. Asterisks represent statistically significant differences based on a two-tailed Student's ttest when compared to controls (0 h); * p < 0.05, ** p < 0.01.

Subcellular Localization of TaPUB2 and TaPUB3 Proteins in Wheat Protoplast
To examine the transient expression of TaPUB2 and TaPUB3, the fluorescence signals of 35S:TaPUB2-GFP and 35S:TaPUB3-GFP were detected, respectively, in wheat protoplast cells using the polyethylene glycol (PEG) transfection system ( Figure 3). The 35S:GFP-fused TaPUB2 was targeted to the cytosol and Golgi apparatus ( Figure 3A). The 35S:GFP and Golgi apparatus marker (G-rk-CD3-967) were used to confirm the localization of TaPUB2 ( Figure 3A,B) [27]. Similarly, the 35S:GFP-fused TaPUB3 was also targeted to the Golgi apparatus ( Figure 3B).  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.

Subcellular Localization of TaPUB2 and TaPUB3 Proteins in Wheat Protoplast
To examine the transient expression of TaPUB2 and TaPUB3, the fluorescence signals of 35S:TaPUB2-GFP and 35S:TaPUB3-GFP were detected, respectively, in wheat protoplast cells using the polyethylene glycol (PEG) transfection system ( Figure 3). The 35S:GFP-fused TaPUB2 was targeted to the cytosol and Golgi apparatus ( Figure 3A). The 35S:GFP and Golgi apparatus marker (G-rk-CD3-967) were used to confirm the localization of TaPUB2 ( Figure 3A,B) [27]. Similarly, the 35S:GFP-fused TaPUB3 was also targeted to the Golgi apparatus ( Figure 3B).

In Vitro Ubiquitination Assay of TaPUB2 and TaPUB3
TaPUB2 (TraesCS5A02G198800.1) and TaPUB3 (TraesCS2B02G499300.1) are homologous genes that encode putative E3 ligases with 61% amino acid sequence similarity in wheat ( Figure 4A). Both proteins contain a U-box and an Armadillo (ARM) repeat domain in the central regions. Moreover, most U-box-containing proteins have been shown to possess E3 ligase activity. We performed an ubiquitination assay using MBP-tagged recombinant proteins (pMAL-c5x-TaPUB2 and pMAL-c5x-TaPUB3). The analysis showed that MBPfused TaPUB2 and TaPUB3 generated poly ubiquitinated chains in the presence of E1, E2, and Ub ( Figure 4B). Furthermore, we generated mutants-MBP-TaPUB2 C323A and MBP-TaPUB3 C325R to confirm the ubiquitination activity of the U-box domain, where cysteine-323 and cysteine-325 were replaced by alanine and arginine, respectively. However, no poly ubiquitinated chains were detected in the absence of E1, E2, E3, or mutated TaPUB2 and TaPUB3.

In Vitro Ubiquitination Assay of TaPUB2 and TaPUB3
TaPUB2 (TraesCS5A02G198800.1) and TaPUB3 (TraesCS2B02G499300.1) are homologous genes that encode putative E3 ligases with 61% amino acid sequence similarity in wheat ( Figure 4A). Both proteins contain a U-box and an Armadillo (ARM) repeat domain in the central regions. Moreover, most U-box-containing proteins have been shown to possess E3 ligase activity. We performed an ubiquitination assay using MBP-tagged recombinant proteins (pMAL-c5x-TaPUB2 and pMAL-c5x-TaPUB3). The analysis showed that MBP-fused TaPUB2 and TaPUB3 generated poly ubiquitinated chains in the presence of E1, E2, and Ub ( Figure 4B). Furthermore, we generated mutants-MBP-TaPUB2 C323A and MBP-TaPUB3 C325R to confirm the ubiquitination activity of the U-box domain, where cysteine-323 and cysteine-325 were replaced by alanine and arginine, respectively. However, no poly ubiquitinated chains were detected in the absence of E1, E2, E3, or mutated Ta-PUB2 and TaPUB3.

TaPUB2 and TaPUB3 Form Heterodimers
The U-box/RING type E3 ligase often has a heterodimeric form, which has significant E3 ligase activity [28,29]. We performed yeast co-transformation to determine whether TaPUB2 and TaPUB3 form a complex. The full-length ORFs of TaPUB2 and TaPUB3 were cloned into the pGBKT7 (BD) and pGADT7 (AD) vectors, respectively, and co-transformed into AH109 yeast strain cells. Transformed yeast cells were placed onto D-aspartate oxidase DDO (SD/-Lue/-Trp) and QDO/X (SD/-Lue/-Trp/-His/-Ade/X-a-gal) growth

TaPUB2 and TaPUB3 Form Heterodimers
The U-box/RING type E3 ligase often has a heterodimeric form, which has significant E3 ligase activity [28,29]. We performed yeast co-transformation to determine whether TaPUB2 and TaPUB3 form a complex. The full-length ORFs of TaPUB2 and TaPUB3 were cloned into the pGBKT7 (BD) and pGADT7 (AD) vectors, respectively, and co-transformed into AH109 yeast strain cells. Transformed yeast cells were placed onto D-aspartate oxidase DDO (SD/-Lue/-Trp) and QDO/X (SD/-Lue/-Trp/-His/-Ade/X-a-gal) growth media. The results showed that co-transformed TaPUB2 and TaPUB3 could interact with each other and form a heterodimer in quadruple dropout QDO/X media ( Figure 5A). Next, we performed an in vitro pull-down assay using His-tagged TaPUB2 and MBP-tagged TaPUB3. The in vitro pull-down assay detected TaPUB3 protein, which was pulled down by TaPUB2 ( Figure 5B). Furthermore, we examined the possible mechanisms by which TaPUB2 interacts with TaPUB3 using BiFC assays in planta. Both nYFP-TaPUB3 and cYFP-TaPUB2 were co-localized in the cytoplasm of the wheat protoplast cells ( Figure 5C). These results implied that TaPUB2 and TaPUB3 bonded to each other to form a heterodimeric complex in yeast and plants.

Overexpression of TaPUB2 and TaPUB3 Confers Tolerance to Drought Stress in Arabidopsis Plants
To evaluate the molecular function of TaPUB2 and TaPUB3 under drought conditions, we developed TaPUB2-and TaPUB3-overexpressing Arabidopsis plants, respectively ( Figure S1). Heterologous overexpression of TaPUB2 and TaPUB3 led to a higher percentage of seed germination rate than that of the wild type (WT) upon treatment with different concentrations of mannitol (200 and 400 mM) on a half-strength MS medium ( Figure  6C,D). Similarly, we measured the root lengths in transgenic and WT plants that had been exposed to different concentrations of mannitol (200 and 400 mM) for 4 days after transferring 4-day-old seedlings onto 1/2 MS media. The root lengths of TaPUB2 (OE1 and OE2) and TaPUB3 (OE1 and OE2) transgenic plants significantly increased compared with that of the WT, while the root length under normal growth conditions did not differ clearly between the two groups ( Figure 6A,B). 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.

Overexpression of TaPUB2 and TaPUB3 Confers Tolerance to Drought Stress in Arabidopsis Plants
To evaluate the molecular function of TaPUB2 and TaPUB3 under drought conditions, we developed TaPUB2and TaPUB3-overexpressing Arabidopsis plants, respectively ( Figure S1). Heterologous overexpression of TaPUB2 and TaPUB3 led to a higher percentage of seed germination rate than that of the wild type (WT) upon treatment with different concentrations of mannitol (200 and 400 mM) on a half-strength MS medium ( Figure 6C,D). Similarly, we measured the root lengths in transgenic and WT plants that had been exposed to different concentrations of mannitol (200 and 400 mM) for 4 days after transferring 4-day-old seedlings onto 1/2 MS media. The root lengths of TaPUB2 (OE1 and OE2) and TaPUB3 (OE1 and OE2) transgenic plants significantly increased compared with that of the WT, while the root length under normal growth conditions did not differ clearly between the two groups ( Figure 6A,B). The performance of soil grown TaPUB2 and TaPUB3 transgenic seedlings was examined under water deprivation conditions in the soil. Under normal conditions, plants did not exhibit any morphological differences between the WT and OE lines. After 15 days of normal growth conditions, the plants were deprived of water for 14 days and then watered normally ( Figure 6E). TaPUB2 and TaPUB3-overexpressing plants showed a significantly higher survival rates (by 2.3-fold) compared with that of the WT plants ( Figure  6F). Furthermore, under drought stress conditions, the chlorophyll content of the transgenic plants was 1.5-fold higher than that of the WT plants ( Figure 6G). In contrast, Ta-PUB2 and TaPUB3-overexpressing plants were found to have a lower water loss rate than that of the WT plants ( Figure 6H).
Additionally, the accumulation of ROS in TaPUB2 and TaPUB3 transgenic lines and WT plants was analyzed using 3,3-diaminobenzidine (DAB) staining and nitroblue tetrazolium (NBT) analysis. Under drought stress conditions, tthe WT plants accumulated The performance of soil grown TaPUB2 and TaPUB3 transgenic seedlings was examined under water deprivation conditions in the soil. Under normal conditions, plants did not exhibit any morphological differences between the WT and OE lines. After 15 days of normal growth conditions, the plants were deprived of water for 14 days and then watered normally ( Figure 6E). TaPUB2 and TaPUB3-overexpressing plants showed a significantly higher survival rates (by 2.3-fold) compared with that of the WT plants ( Figure 6F). Furthermore, under drought stress conditions, the chlorophyll content of the transgenic plants was 1.5-fold higher than that of the WT plants ( Figure 6G). In contrast, TaPUB2 and TaPUB3-overexpressing plants were found to have a lower water loss rate than that of the WT plants ( Figure 6H).
Additionally, the accumulation of ROS in TaPUB2 and TaPUB3 transgenic lines and WT plants was analyzed using 3,3-diaminobenzidine (DAB) staining and nitroblue tetrazolium (NBT) analysis. Under drought stress conditions, tthe WT plants accumulated more ROS than the OE lines ( Figure 6I and Figure S2). These results strongly suggested that TaPUB2 and TaPUB3 function in the same signaling pathway in their plant responses to drought stress.

Discussion
Although the role of PUB E3 ligases has been reported in many abiotic and biotic stress conditions in various plant species, their molecular and metabolic functions in wheat remain unclear, particularly under abiotic stress conditions [19,35,36], since their biological and molecular study has only been in the initial stages in wheat. In this study, we identified functional analysis of two PUB E3 ligases, TaPUB2, and TaPUB3, in wheat ( Figures 1A and 4A). The transcriptional profiles of TaPUB2 and TaPUB3 were confirmed by the results of their response to abiotic stresses in wheat; the expression of both TaPUB2 and TaPUB3 was highly upregulated under abiotic stresses-drought, cold, salt, and ABA (Figures 1 and 2). Furthermore, the sequence similarity index (61%) and domain structure analysis revealed the presence of a conserved domain (ARM and U-box) and other residues, indicating that TaPUB2 and TaPUB3 were highly conserved ( Figure 4A). Subcellular localization analysis revealed that TaPUB2 and TaPUB3 were localized in the Golgi apparatus ( Figure 3A), indicating that they may have been involved in various intercellular mechanisms or functions in wheat.
Several studies have reported that wheat U-box proteins have multiple functions in response to abiotic stresses. For example, the U-box protein TaPUB1 improved salt and drought stress tolerance in Nicotiana benthamiana [13]. TaPUB15 overexpression resulted in enhanced root growth and development and an increased resistance to salt stress in rice [37]. E3 ubiquitin ligase functions in response to abiotic stress and is dependent on the ABA pathway [38]. Similarly, TaPUB1 and TaPUB15 E3 ligases were shown to be highly expressed in transgenic plants in response to ABA signaling and enhance tolerance to salt stress [13,37]. In our study, the transcription of TaPUB2 and TaPUB3 was significantly induced by drought stress and highly upregulated by ABA, cold, and salt stresses (Figures 1 and 2), suggesting that TaPUB2 and TaPUB3 may be involved in drought, cold, and salt stress responses in an ABA-dependent manner. The bZIP transcription factor (TFs) are well known drought stress response pathways. bZIPs members are related in post-translational modification, such as phosphorylations/de-phosphorylation events [39]. bZIP TFs, including nine subfamily member ABREs/ABFs, have existed in Arabidopsis. Among them, ABF1, AREB1/ABF2, AREB2/ABF3, and ABF4 have been phosphorylated by SnRK2s (Snf1-related protein kinase) and regulate the expression of ABA-dependent drought signaling pathways [40]. In this study two ABA-dependent drought induced genes, such as AtABI5 and AtABF3, were up-regulated in response to the TaPUB2 and TaPUB3-overexpressing in plants growing drought condition (Figure 7). Moreover, when there was a water deficit condition, ABA caused stomatal closure, reducing water loss through transpiration [41]. These results were consistent with reducing the water loss rate in TaPUB2 and TaPUB3-overexpressing plants ( Figure 6H). However, further work is needed to understand the ABA-dependent response and other abiotic stress mechanisms in transgenic plants.
A previous study [26] demonstrated that heterologous overexpression of TaPUB1 positively regulates post-germination plant growth under drought stress (PEG 6000). To study the physiological function of TaPUB2 and TaPUB3 in plant growth as well as drought tolerance, we generated transgenic Arabidopsis plants using the floral dipping method ( Figure S1). Water deficit significantly delayed the onset of seed germination rate. Some of E3 ligase, which contains the U-box domain, have important roles in seed germination under the drought stress [42]. Similarly, under drought stress conditions, the seed germination rate of TaPUB2 and TaPUB3 overexpression plants was much greater than that of WT plants ( Figure 6C,D). Moreover, the root development of the TaPUB2 and TaPUB3 transgenic lines have also longer than WT under osmotic stress conditions ( Figure 6A,B). Water deficiency has a considerable impact not only during the seedling stage but also during the vegetative stages [26]. Under drought stress, TaPUB2 and TaPUB3 transgenic plants showed improved survival rates and robust phenotypes, such as green leaves and high chlorophyll content ( Figure 6). These findings indicate that U-box type E3 ligases were involved in the regulation of plant adaptation mechanisms during harsh environments, such as drought.
When the amount of available soil water is severely limited, the first option for plants induced accumulation of ROS and close their own stomata [8]. Drought stress causes oxidative stress by increasing ROS, such as 1 O 2 (singlet oxygen), O 2 − (superoxide radical), HO• (hydroxyl radical), and H 2 O 2 (hydrogen peroxide), all of which are highly reactive and contribute to drought stress [43]. Excess ROS are scavenged or detoxified by an efficient antioxidative system that includes both nonenzymic and enzymic antioxidants, such as superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) [44]. Previously, [45] it was demonstrated that drought stress resistance in VyP5CR transgenic Arabidopsis revealed less efficient scavenging of H 2 O 2 as compared WT lines. In the present study, we observed that TaPUB2 and TaPUB3 transgenic line have a more effective antioxidant defense system and less accumulation of ROS ( Figure 6I and Figure S2). These findings revealed that TaPUB2 and TaPUB3 were involved in mechanisms that protect cells during drought stress.
The predicted U-box protein has a domain similar to that of the RING-finger protein, despite the loss of hallmark and histidine residue [46,47], and is found in combination with other domains, such as ARM (armadillo) repeats, Ser/Thr kinase domain, WD40 repeats, or tetratricopeptide (TPR) domain, or peptidyl-prolyl isomerase [48]. E3 ligase, which contains a U-box domain, performs ubiquitination upon forming a complex with the activating enzyme E1 and conjugating enzyme E2 [49]. We incorporated a single amino acid mutation in TaPUB2 and TaPUB3 by substituting cysteine at the 323rd position with alanine and cysteine at the 325th position with arginine, respectively, in the U-box domain. A previous report demonstrated that PUB E3 ligase activity depends on cysteine residues [15]. Thus, the mutations in TaPUB2 and TaPUB3 result in catalytically inactive forms in the presence of E1, E2, and ubiquitin ( Figure 4B). These findings showed that TaPUB2 and TaPUB3 have E3 ligase activities, and they require the U-box domain to perform ubiquitination.
Recent studies have shown that several E3 ligases play combinatory roles, suggesting that they form heterodimers [39,40]. Furthermore, [50] identified homologous OsPUB2 and OsPUB3 proteins degrade rapidly upon forming a heterodimer. Therefore, we tested whether TaPUB2 interacts with TaPUB3 using the yeast-two hybrid assay ( Figure 5A). Moreover, we confirmed the interaction between TaPUB2 and TaPUB3 using BiFC assays ( Figure 5B). Interestingly, the TaPUB2/TaPUB3 protein complex occurred in the cytoplasm, whereas TaPUB2 was transiently expressed, and TaPUB3 was localized in the Golgi apparatus ( Figure 3). The spatial differences in the expression of complex proteins could also be observed in other studies. For example, the cytoplasm and nucleus-localized Histone H2B mono-ubiquitination enzyme (TaHUB2) physically interacts with the nuclear proteins, such as TaELF7, in wheat [51]. OsSADR1 interacts with and regulates the chloroplast-localized protein OsPIPIN in the cytoplasm [8]. Therefore, TaPUB3 might be translocated from the Golgi apparatus to the cytoplasm, thus interacting with TaPUB2 to form a heterodimeric complex.

Plant Material and Growth Conditions
The seeds of a wheat variety [Triticum aestivum L. 'Keumkang' (Korea RDA Accession No. IT213100)] were germinated and grown in a growth chamber (25 • C with 60% relative humidity and photoperiod of 16 h light intensity) on mesh supported in plastic containers with one-half concentration of MS (Murashige and Skoog) liquid media. The 14-dayold plants were treated with abiotic stresses, including drought (20% PEG 6000), salt (200 mM), cold (4 • C), and ABA (0.1 mM). Leaf tissues were collected at 0, 6, 12, and 24 h following stress treatment. Samples were immediately frozen in liquid nitrogen for further experiment.

Gene Expression Studies
RNA extraction, cDNA conversion, primer design, and qRT-PCR was used to analyze the transcript expression patterns of the putative genes and the results were estimated using the delta-delta CT method [51,53]. qRT-PCR experiments were performed from three independent biological replicates.

Subcellular Localization
To examine the subcellular localization of TaPUB2 and TaPUB3, two genes were cloned into the GFP vector (pMDC43) and BiFC vector (pGTQL1211 and pGTQL1221) using LR reaction (Invitrogen, Cat: 11791020, Carlsbad, CA, USA), respectively. The resulting constructs were used for transient assays using wheat protoplasts transfected with polyethylene glycol [54].

Ubiquitination Assay
To perform the E3 ligase activity of TaPUB2 and TaPUB3, each gene was cloned into the MBP-tagged vector (pMAL-c5x). An in vitro ubiquitination experiment was carried out using a previously described protocol [10,55].

Yeast Two-Hybrid Assay
The Matchmaker GAL4 bases two-hybrid system was used for the yeast two-hybrid assays (Clontech, Cat: 630489, Palo Alto, CA, USA). The full-length sequences of TaPUB2 and TaPUB3 were recombined into the pGDAT7 (including activation domain (AD)) and pGBKT7 (including binding domain (BD)), respectively. TaPUB2, TaPUB3, and empty vector were co-transformed and grown in DDO medium after being transformed into the AH109 yeast strain. Each clone was cultured in DDO broth for 5 days before being spotted on a QDO medium to test for protein-protein interactions.

Pull-Down Assay
To determine the PUB2 physical interaction with TaPUB3, MBP-tagged TaPUB2 and His-tagged TaPUB3 proteins were expressed in Escherichia coli (strain BL21). The pull-down assay was used for His Protein Interaction Pull-Down Kit (Invitrogen, Cat: 21277, Carlsbad, CA, USA), as previously described by [54].

Generation of Transgenic Arabidopsis Plants
For overexpression of TaPUB2 and TaPUB3 in Arabidopsis, the ORF fragments that had been amplified were subcloned into pMDC43 vectors. The resulting plasmids were introduced into pMDC43 binary vectors to obtain the 35S:TaPUB2-GFP and 35S:TaPUB3-GFP constructs that were used for Agrobacterium (GV3101)-mediated transformation in Arabidopsis (ecotype Columbia-0) using the floral dipping method [56]. All experiments were carried out on homozygous T3 generation plants. For each assay, at least two independent transgenic lines were used.

Dehydration and Drought Tolerance
Overexpression lines and WT seeds were germinated and grown for 15 days at 25 • C in a growth chamber (25 • C with 60% relative humidity and photoperiod of 16 h light intensity). Some seedlings were dehydrated, while others were subjected to drought treatment by stopping irrigation for 14 days. The rosette leaves of 14-day-old seedlings of each transgenic line were placed on filter papers and allowed to dry for up to 5 h for dehydration analysis. To determine the rate of water loss, the fresh weight of each sample was measured every 20 min. To detect H 2 O 2 , the leaves were incubated for 4 h in a 1 mg/mL DAB solution (Sigma-Aldrich, Cat: D8001, St Louis, MO, USA). Following the incubation period, the leaf samples were immersed in a bleaching solution (ethanol: acetic acid: glycerol, 3:1:1) and boiled for 15 min at 95 • C [50].

Statistical Analysis
All experimental data were the means of at least three separate replicates, and statistical analysis was carried out with Microsoft Excel 2016. (Microsoft, Seattle, WA, USA). For comparisons between two analyzed populations and among three or more populations, the Student's t-test and Duncan's multiple range test from one-way and two-way analysis of variance (ANOVA) were used to determine if the differences were statistically significant at p-values < 0.05.

Conclusions
In the present study, the molecular function of TaPUB2 and TaPUB3, including their expression patterns, subcellular localization, and E3 ligase activity was elucidated. Two proteins directly interact with one another and form heterodimeric complexes (TaPUB2/TaPUB3). Furthermore, TaPUB2 and TaPUB3-overexpressing Arabidopsis displayed a resistance phenotype under the drought condition (media and soil) and a lesser rate of ROS accumulation, respectively. These findings suggest that the two putative U-box E3 ligases, TaPUB2 and TaPUB3, are involved in the response to abiotic stresses, especially drought. However, studying double mutants or transgenic wheat lines would be helpful to further understand the genetic mechanisms involved in drought tolerance.