Functional Identification Reveals That TaTGA16-2D Promotes Drought and Heat Tolerance

Abstract

The TGACG motif-binding factor (TGA) family is an important group of basic region/leucine zipper (bZIP) transcription factors in plants, playing crucial roles in plant development and stress responses. This study conducted a comprehensive genome-wide analysis of the TGA transcription factor (TF) family in common wheat (Triticum aestivum L.). A total of 48 wheat TGAs were identified and classified into four subgroups. Collinearity analysis of the TGAs between wheat and other species identified multiple duplicated gene pairs and highlighted the presence of highly conserved TGAs in wheat. Whole-genome and segmental duplications were identified as the primary drivers of TaTGA expansion. Expression pattern analysis indicated that TaTGAs are involved in plant development and responses to abiotic stresses, including drought, heat, and cold treatment. Among these, TaTGA16-2D was significantly upregulated under both drought and heat stresses, showing more than a five-fold increase in expression. Subcellular localization confirmed its nucleus localization. Functional validation through ectopic expression in Arabidopsis demonstrated that transgenic lines overexpressing TaTGA16-2D exhibited significantly improved stress tolerance. Under heat stress, the survival rates of transgenic lines exceeded 34%, compared to less than 18% in wild-type plants. Overall, this study provides valuable insights into the evolution and functional roles of TaTGAs and identifies TaTGA16-2D as a promising candidate to enhance abiotic stress tolerance in wheat via molecular breeding.

1. Introduction

Transcription factors (TFs) play essential roles in regulating gene expression [1]. TGACG motif-binding factor (TGA) TFs belong to clade D of the basic region/leucine zipper (bZIP) family and specifically recognize and bind to the TGACG motif [2,3]. TGA TFs primarily function in regulating various physiological and metabolic processes in plants, as well as in responses to biotic and abiotic stresses [4]. Members of the TGA family interact with a variety of regulatory proteins, such as NPR1 (nonexpressor of pathogenesis-related genes 1), GRX480 (glutaredoxin 480), ERF72 (ethylene response factor 72), ARR2 (Arabidopsis response regulator 2), and SCL14 (scarecrow-like protein 14), and are involved in multiple stress and disease resistance signaling pathways, through which TGAs contribute to enhancing plant resilience to environmental challenges [5,6,7].

In Arabidopsis, the TGA TFs have been extensively studied and shown to participate in various biological processes. Several members are involved in plant development: AtTGA1, AtTGA4, and AtTGA7 regulate flower development [8,9], while AtTGA2, AtTGA5, AtTGA8, and AtTGA16 promote root growth by modulating redox homeostasis and oxidative stress responses [10]. In rice (Oryza sativa), OsbZIP49—a TGA-related bZIP TF—controls the tiller angle and plant architecture by influencing auxin homeostasis through the activation of indole-3-acetamide synthase [11].

TGA TFs also play key roles in biotic stress responses. For instance, AtTGA9 and AtTGA10 are involved in both pathogen defense and anther development [12]. Homologs of Arabidopsis TGA genes have been identified in other species. BjCdR15 from Brassica juncea, a homolog of AtTGA3, is strongly induced by cadmium and other heavy metal stresses [13], while AtTGA3 itself has been shown to participate in detoxification responses [14].

In addition to biotic stress, TGA factors are increasingly recognized for their roles in abiotic stress responses. AtTGA1 enhances drought tolerance by promoting nitrate transport and uptake when overexpressed [15], and AtTGA7 is involved in the drought stress response by regulating its target gene, AtBG1 [16]. In soybean (Glycine max), the overexpression of GmTGA17 enhances both drought and salt tolerance by modulating stress-responsive gene expression in the roots [17]. Similarly, MhTGA2 from Malus hupehensis responds to low temperature, NaCl, and polyethylene glycol (PEG) treatment; the overexpression of MhTGA2 improves salt and osmotic stress tolerance in transgenic Malus hupehensis and Nicotiana tabacum [18,19].

TGA TFs play a central role in wheat’s responses to abiotic and biotic stresses, as well as in growth and development. In wheat, TaTGA2.1 was overexpressed in Brachypodium distachyon, and the transgenic plants showed enhanced resistance to Fusarium graminearum in both spikes and detached leaves [20]. Under drought and salinity stresses, as well as treatment with abscisic acid (ABA), TaTGA2.2 expression is upregulated [21]. Additionally, the genes TaTGA1a, TaTGA1b, and TaTGA4 have been successfully cloned using RT-PCR, and the expression pattern of TaTGA2.2 has been preliminarily investigated using virus-induced gene silencing (VIGS) [22]. Research on TGAs in wheat remains relatively limited.

In this study, forty-eight TaTGAs were identified in wheat, followed by a comprehensive phylogenetic analysis. A systematic analysis was conducted on the gene structures and conserved domains of the TaTGAs. In addition, cis-acting regulatory elements and expression patterns were analyzed to explore their potential regulatory roles. The expression profiles of TaTGAs under drought, heat, and cold stress treatments were investigated to elucidate their involvement in environmental stress responses. Among them, a significantly differentially expressed gene, TaTGA16-2D, was selected for further functional analysis. Its role was ultimately validated through the phenotypic characterization of transgenic Arabidopsis plants. We hypothesize that TaTGA16-2D positively regulates abiotic stress tolerance and that its overexpression would enhance stress resilience in plants, making it a promising candidate for wheat molecular breeding.

2. Results

2.1. Identification of TaTGA Gene Family Members

We conducted a comprehensive search of the wheat genome database using ten Arabidopsis and 16 riceTGA protein sequences as references. Based on the conserved domains and motifs, a total of 48 TaTGAs were identified through BLAST and HMMER analyses using TBtools-v2.119 (Figure 1; Table S1). The corresponding protein sequences ranged from 198 to 570 amino acids (aa) in length, with an average of 400 aa (Table S1). The molecular weight (kDa) values ranged from 21.66 kDa to 62.18 kDa, averaging 44.00 kDa. The isoelectric points (pI) ranged from 5.24 to 10.37, with 39.6% (19 out of 48) displaying alkaline characteristics. The instability index values ranged from 44.06 to 70.26, with all proteins exceeding 44, suggesting potential instability in vitro. The aliphatic index ranged from 66.70 to 96.86, with an average value of 78.68. The grand average of hydropathicity (GRAVY) values ranged from −0.74 to −0.10, all of which are negative, indicating that all TaTGA proteins are hydrophilic. Subcellular localization predictions showed that all TaTGA proteins were localized in the nucleus. To investigate their chromosomal distribution, the 48 TaTGAs were mapped onto the wheat genome. They were found to be unevenly distributed across all 21 chromosomes, with the number of TaTGAs per chromosome ranging from one to five (Figure S1).

2.2. Phylogenetic and Sequence Analyses of TaTGA Members

To understand the evolution of the TaTGA proteins, a phylogenetic analysis was conducted using a total of 79 TGA protein sequences, including 48 from Triticum aestivum (TaTGA), 10 from Arabidopsis, and 21 from rice (Figure 2; Table S2). The TGA proteins were classified into four groups (I–IV) based on their aa sequence similarity (Figure 2). Group I contained the largest number of TGA proteins across the three species. The analysis of the homoeologous relationships showed that the 48 TaTGAs corresponded to 48 homoeologs derived from 18 ancestral genes: 14 of these genes had all three homoeologs (one from each subgenome: A, B, and D), two had two homoeologs, and two had only a single homoeolog (Table S3).

2.3. Chromosomal Location and Duplication Events of TaTGA Genes

Chromosomes 3A, 3B, and 3D together harbored nearly 31.3% of all TaTGAs. An analysis of the chromosomal translocations and inversions revealed that two triads—TaTGA32-4A/TaTGA35-4B/TaTGA37-4D and TaTGA33-4A/TaTGA34-4B/TaTGA36-4D—were involved in an inversion of chromosome 4A (Table S4). To elucidate the mechanisms underlying gene family expansion, we performed a collinearity analysis of the TaTGAs within the wheat genome. A total of 44 genes were located within syntenic blocks, forming 58 duplicated gene pairs (Figure 3; Table S5). Furthermore, all TaTGAs were found to originate from whole-genome duplication (WGD) or segmental duplication events (Table S4). To further explore the evolutionary history and homology of the TGAs across species, a comparative collinearity analysis was conducted between wheat, Arabidopsis, and rice. No collinear gene pairs were identified between wheat and Arabidopsis, while 31 collinear gene pairs were detected between wheat and rice (Figure 4), indicating a closer evolutionary relationship between wheat and rice.

The Ka/Ks ratios ranged from 0 (TaTGA32-4A/TaTGA35-4B, TaTGA32-4A/TaTGA37-4D and TaTGA35-4B/TaTGA37-4D) to 0.56 (TaTGA23-3B/TaTGA28-3D) (Figure S2, Table S5). These results suggest that most duplicated TaTGAs have undergone purifying selection.

2.4. Analysis of Gene Structures and Conserved Motifs of TaTGA Members

The exon–intron structures and conserved motifs of the 48 TaTGAs were analyzed (Figure 5). Among the 48 genes, seven lacked introns, while the remaining TaTGAs contained between one and 11 introns (Figure 5A,B; Table S6). TaTGA2-1A, TaTGA4-1B, TaTGA7-1D, TaTGA21-3A, TaTGA33-4A, TaTGA34-4B, and TaTGA36-4D had the highest numbers of exons and introns (12 and 11, respectively). In terms of UTRs, seven genes lacked a 5′ UTR and five genes lacked a 3′ UTR. TaTGA32-4A, TaTGA35-4B, and TaTGA37-4D possessed the longest 5′ and 3′ UTRs, with three and one regions, respectively. In Arabidopsis, most TGA genes, including AtTGA1 through AtTGA6, shared a uniform structure of eight exons and seven introns. AtTGA9 had the most complex structure among them. Regarding UTRs, AtTGA2 and AtTGA7 had the highest numbers of 5′ UTRs (three each), while the other TGA genes had simpler UTR arrangements. In rice, the gene structures were similar to those of Arabidopsis in several TGA members. Os01t0279900, Os06t0265400, Os03t0318600, Os05t0492000, and Os07t0687700 all had eight exons and seven introns. However, Os01t0859500, Os12t0152900, and Os11t0152700 exhibited the most complex structures, with 12 exons and 11 introns—paralleling the complexity seen in certain wheat TaTGA genes. In terms of UTRs, Os06t0265400, Os07t0687700, and Os04t0637000 showed the most extended 5′ UTRs, each with three regions (Table S6).

Genes grouped in the same phylogenetic branch, typically homologous, generally exhibited similar gene structures. However, structural divergence was observed among some homologous genes; for example, TaTGA21-3A, TaTGA26-3B, and TaTGA31-3D differed in their exon, intron, and UTR compositions. A total of 20 conserved motifs were seen, each ranging from six to 50 aa in length (Figure 5C; Table S7). Among these, motif 1 contained highly conserved sequence domains corresponding to PF14144 and PF00170, indicating potential functional importance.

2.5. Analysis of Promoter Cis-Elements of TaTGA Members

In the promoter regions of the 48 TaTGAs, we identified a total of 528 potential cis-elements (Table S8). These elements were grouped into four primary categories: plant growth-related elements (12%), hormone-responsive elements (26%), environmental stress-responsive elements (34%), and light-responsive elements (28%) (Figure 3). Notably, the environmental stress-responsive category included 177 drought-responsive elements, while the hormone-responsive category contained 111 methyl jasmonate (MeJA)-responsive elements. These findings suggest that TaTGAs may play significant roles in abiotic stress responses, particularly in drought stress adaptation. Furthermore, meristem expression elements were the most abundant, totaling 63, suggesting a potential role for TaTGAs in regulating plant growth and development (Table S8).

2.6. Expression Pattern Analysis of TaTGA Genes

An analysis of the TaTGA expression patterns across various tissue types and developmental stages revealed that TaTGA8-2A and TaTGA13-2B exhibited extremely low transcription levels in the examined samples (Figure 6A; Table S9). Multiple genes exhibited distinct tissue-specific expression profiles. For example, TaTGA5-1B was highly expressed in the grain, while TaTGA4-1B, TaTGA7-1D, TaTGA22-3B, and TaTGA34-4B formed a distinct cluster with strong expression in the roots. In contrast, some genes were constitutively expressed across various tissue types. Notably, TaTGA12-2B, TaTGA9-2A, TaTGA15-2D, TaTGA32-4A, and TaTGA37-4D showed high transcription levels throughout plant development (Figure 6A). Overall, the TaTGAs exhibited pronounced tissue-specific expression, suggesting potential roles in diverse physiological processes and developmental regulation.

In the analysis of the TaTGAs’ expression patterns under abiotic stress conditions, thirteen genes were differentially expressed under the two stress conditions, including TaTGA9-2A, TaTGA11-2A, TaTGA12-2B, TaTGA14-2B, TaTGA15-2D, TaTGA16-2D, TaTGA17-3A, TaTGA18-3A, TaTGA23-3B, TaTGA28-3D, TaTGA32-4A, TaTGA35-4B, and TaTGA37-4D (Figure 6B; Table S9). Additionally, 10 genes were differentially expressed under cold treatment, namely TaTGA9-2A, TaTGA12-2B, TaTGA15-2D, TaTGA16-2D, TaTGA17-3A, TaTGA18-3A, TaTGA23-3B, TaTGA27-3D, TaTGA28-3D, and TaTGA37-4D, TaTGA27-3D. Notably, TaTGA27-3D exhibited the strongest response to cold stress among all genes analyzed (Figure 6C). Based on their responsiveness to abiotic stresses and their consistently high expression across developmental stages, six genes were selected for further analysis: TaTGA10-2A, TaTGA11-2A, TaTGA14-2B, TaTGA16-2D, TaTGA17-3A, and TaTGA27-3D.

2.7. TaTGAs Are Involved in Abiotic Stress Responses

The expression patterns of six TaTGAs were examined using qRT-PCR (Figure 7). Under drought treatment, TaTGA11-2A, TaTGA14-2B, and TaTGA16-2D were significantly upregulated (>five-fold), reaching their respective expression peaks at 24 h, 6 h, and 24 h (Figure 7A), indicating temporal differences in stress responsiveness. In contrast, the other three genes exhibited only slight responses to dehydration (Figure 7A). Following heat treatment, a similar expression trend was observed: TaTGA11-2A, TaTGA14-2B, and TaTGA16-2D were again significantly upregulated (>five-fold), all peaking at 6 h (Figure 7B), suggesting a more rapid transcriptional response to heat stress compared to drought. These results imply that, while the same genes respond to multiple stress conditions, their expression dynamics—such as the peak time and intensity—differ according to the type of abiotic stimulus. These findings demonstrate that, although some TaTGAs are repeatedly induced under various stresses, their differential expression patterns—both in magnitude and timing—reflect nuanced regulatory roles in distinct stress signaling pathways. Among them, TaTGA16-2D was selected for further functional analysis due to its robust and consistent response.

2.8. TaTGA16-2D Was Located in the Nucleus

The subcellular localization of the 48 TaTGAs was predicted using BUSCA (Table S1). To experimentally validate the localization of TaTGA16-2D, transient expression assays were conducted. TaTGA16-2D was predominantly localized to the nucleus (Figure 8).

2.9. TaTGA16-2D Enhances Drought Tolerance in Arabidopsis

The total root length and fresh weight were measured in root growth assays. Under control conditions, there were no significant differences in the root length or fresh weight between transgenic and wild-type (WT) plants. However, under 6% PEG treatment, although growth was inhibited in both genotypes, the growth inhibition was significantly reduced in the transgenic lines relative to the WT plants, as evidenced by longer roots and higher fresh weights. Under 8% PEG treatment, both WT and transgenic plants exhibited more severe growth inhibition; however, the transgenic lines maintained significantly greater root lengths and fresh weights compared to WT plants, demonstrating enhanced tolerance (Figure 9A,B).

To further investigate whether TaTGA16-2D influenced stomatal behavior under ABA treatment, we performed a stomatal aperture assay (Figure 9C,D). Leaves from the same position in the WT and TaTGA16-2D-overexpressing Arabidopsis were excised and incubated under strong light for 3 h in a stomatal opening buffer. The leaves were then transferred to a buffer containing either 0 μM or 5 μM ABA and incubated for an additional 2 h. In the absence of exogenous ABA, there was no significant difference in stomatal opening between the WT and TaTGA16-2D-overexpressing plants. However, after treatment with 5 μM ABA, the stomatal apertures decreased in both lines, but the reduction was significantly greater in the TaTGA16-2-overexpressing plants compared to the wild type. These results suggest that TaTGA16-2 enhances the sensitivity of Arabidopsis to ABA and plays a role in regulating ABA-mediated stomatal closure (Figure 9C,D).

2.10. TaTGA16-2D Increases Heat Tolerance in Arabidopsis

TaTGA16-2D was upregulated over 30-fold under heat stress. Transgenic Arabidopsis lines overexpressing TaTGA16-2D exhibited significantly higher survival rates under heat stress compared to WT (>34% vs. <18%), suggesting that the ectopic expression of TaTGA16-2D enhances heat tolerance (Figure 10).

3. Discussion

TGA TFs mediate various processes, including plant defense and growth, and have been proven to play crucial roles in regulating plant stress responses, as well as growth and development [23]. To date, the genomic identification of TGA TFs has primarily been conducted in Arabidopsis [24], rose (Rosa spp.) [25], common bean (Phaseolus vulgaris L.) [26], soybean [27], etc. The numbers of identified TGA TFs vary across species, from six to 40 [4]. However, few studies have been conducted on wheat, and the identification of TGAs remains particularly limited. Here, this study performed a comprehensive analysis of the 48 TaTGA TFs in wheat.

TGA TFs are more numerous in wheat than in Arabidopsis and rice [23]. The 48 TaTGA TFs are unevenly distributed across chromosomes (Figure 1); for instance, chromosome 1 harbors the most in rice, and chromosome 5 contains the most TGAs in Arabidopsis [28]. In wheat, chromosome 3 contains the highest number of TaTGAs (Figure 1). While gene family expansion is generally driven by genome polyploidy and gene duplication, an analysis of the wheat TGA gene family revealed 18 segmentally duplicated gene pairs but no tandem duplication events (Table S3). All wheat TaTGAs were determined to have originated from WGD/segmental duplication events, indicating that WGD/segmental duplication is the primary force driving expansion of TGAs in wheat (Figure 3). A phylogenetic analysis was conducted comparing TGAs from wheat, Arabidopsis, and rice (Figure 2). The results indicated that TGA sequences and functions are conserved across these species. However, wheat TGAs show a closer phylogenetic relationship and higher collinearity with rice TGAs than with those of Arabidopsis (Figure 4). This evolutionary divergence may help to explain why the TGAs are classified into five subgroups in Arabidopsis but only four in wheat. The 10 AtTGAs in Arabidopsis are divided into five groups, with AtTGA9 and AtTGA10 assigned to the same group [4]. However, in wheat, these two TGAs are separated into distinct groups due to the higher number of homologous genes corresponding to each (Figure 2).

A total of 20 conserved motifs were identified across the TaTGA proteins. Notably, motif 1 contained highly conserved sequences corresponding to the bZIP TF domain (PF00170) and the seed dormancy control domain (PF14144). As expected, proteins within the same phylogenetic branch typically shared similar motif compositions and arrangements (Figure 5). Nevertheless, some proteins in the same clade displayed variations in motif distribution, which may reflect differences in exon–intron organization and potential functional divergence.

Cis-elements in promoter regions play a critical role in regulating gene expression. Several stress-responsive cis-elements were identified in the promoter regions of TaTGAs that exhibited differential responses to abiotic stress (Figure 5), suggesting their potential involvement in stress adaptation mechanisms. These findings lay a foundation for the functional characterization of TaTGAs in wheat. Increasing evidence indicates that TaTGA TFs play important roles in plant responses to abiotic stresses. However, only a few stress-related TGA genes have been functionally characterized in wheat to date, such as TaTGA2.1, TaTGA2.2 [29], and TaTGA1 [22], etc.

The functional analysis of the TaTGAs expressed in different organs has provided novel insights into their regulatory roles in plant growth and organ-specific responses to drought, heat, and cold (Figure 6 and Figure 7). Through expression analysis, we selected six potential candidate genes that responded to various stresses. Among them, TaTGA27-3D showed no significant response to drought and heat stress but responded significantly to cold stress (Figure 6 and Figure 7); TaTGA11-2A and TaTGA16-2D exhibited the most significant responses to drought and heat stress. These genes will be the focus of our next study, and elucidating their response mechanisms will contribute to uncovering the complex pathways through which TGAs mediate responses to various stresses.

The transcriptomic analysis revealed that TaTGA16-2D was significantly upregulated in response to multiple abiotic stresses, prompting its selection for further functional analysis. Under drought and heat stress conditions, TaTGA16-2D transgenic Arabidopsis plants exhibited significantly greater root lengths and fresh weights or higher survival rates compared to controls (Figure 9 and Figure 10). Furthermore, under drought conditions, TaTGA16-2D enhances the sensitivity of Arabidopsis to ABA and plays a key role in regulating ABA-mediated stomatal closure (Figure 9). These results suggest that TaTGA16-2D plays a positive regulatory role in drought and heat stress responses. Arabidopsis TGA4, a homolog of TaTGA16-2D, is highly expressed in the roots and plays a crucial role in salicylic acid-mediated immune responses [30]. Additionally, TGA4 functions as a key TF in nitrate-mediated nitrogen signaling [31]. These findings highlight the important role of TGA4 in both biotic and abiotic stress responses, providing a valuable reference for further research on TaTGA16-2D. Given its strong stress-responsive expression and positive effects in Arabidopsis, TaTGA16-2D represents a promising candidate gene to enhance abiotic stress resistance in wheat. In summary, this study provides the first comprehensive genome-wide identification and characterization of the TGA transcription factor family in wheat. This study not only deepens our understanding of the evolutionary and functional diversity of TGAs in wheat but also identifies TaTGA16-2D as a promising gene to improve abiotic stress resistance in crops, thereby offering valuable resources for molecular breeding.

4. Materials and Methods

4.1. Identification of TGA Genes in Wheat

First, wheat reference sequences were downloaded from Ensembl Plants (http://plants.ensembl.org/index.html (accessed on 13 March 2025)). The TGA sequences of Arabidopsis and Oryza sativa L. were obtained from the TAIR (https://www.arabidopsis.org/ (accessed on 10 March 2025)) and RGAP databases (http://rice.uga.edu/ (accessed on 10 March 2025)), respectively. These sequences were then used as queries in BLASTP searches against the wheat protein database, with an E-value threshold of <1 × 10⁻¹⁰. Additionally, the Hidden Markov Model (HMM) profiles for the bZIP TF (PF00170) and seed dormancy control (PF14144) were downloaded from the Pfam database for the identification of TGA proteins (http://pfam.xfam.org/ (accessed on 8 April 2025)). We conducted an HMM search to build a wheat-specific HMM profile using the Simple HMM Search tool in TBtools v2.119 (https://github.com/CJ-Chen/TBtools (accessed on 15 April 2025)) [32], with an E-value threshold of <1 × 10⁻¹⁰. The results obtained from both BLAST and HMM analyses were integrated. Following validation, all putative TaTGAs were successfully identified (Table S1). Subcellular localizations were predicted through the Plant-mPLoc website (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/ (accessed on 26 April 2025)).

4.2. Phylogenetic Analysis of the TaTGA Gene Family

Multiple alignment of the conserved TaTGA protein sequences was conducted using Clustal X, and the optimal substitution model was applied to construct an interspecific phylogenetic tree using MEGA 11 [33]. The evolutionary tree was constructed using the neighbor-joining (NJ) method, with 1000 bootstrap replicates.

4.3. Analysis of Conserved Motifs and Cis-Acting Elements in Wheat TaTGA Genes

The MEME program (Multiple EM for Motif Elicitation; http://meme-suite.org/tools/meme (accessed on 27 April 2025)) was applied to analyze motifs; we set the motif number to 20 and downloaded the MAST XML output files from the analysis results. PlantCARE was used to analyze the cis-acting elements in the 2000-base-pair (bp) promoter region upstream of the ATG start codons of the TaTGAs (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 27 April 2025)) [34]. The Batch CD-Search tool on NCBI was used to analyze conserved domains. The results were subsequently visualized using the Gene Structure View (Advanced) module in TBtools v2.119 [32].

4.4. Chromosome Localization, Gene Duplication, and Collinearity Analysis of TaTGA Gene Family

Segmental and tandem duplication events within the TaTGAs were identified using the integrated MCScanX module in TBtools v2.119 [34]. The nonsynonymous substitution rate (Ka), synonymous substitution rate (Ks), and Ka/Ks ratios were subsequently calculated using the same software. In addition, homology relationships among TGAs from Arabidopsis, rice, and wheat were analyzed and visualized to explore evolutionary conservation across species.

4.5. Transcriptome Analysis of TaTGA Gene Family in Different Tissue Types and Under Abiotic Stress

Transcriptomic data from the Wheat Expression Browser (http://www.wheat-expression.com (accessed on 11 April 2025)) were used (choulet_URGI) [35,36]. Datasets SRP045409 and SRP043554 were obtained to investigate the expression levels of TaTGAs under drought, heat, and cold abiotic stresses [37,38]. Heatmaps were generated using the HeatMap tool in TBtools v2.119 [34].

4.6. TaTGA Expression Profiling and Real-Time PCR (qRT-PCR)

The wheat cultivar Xiaobaimai was used to analyze gene expression patterns. The plants were cultured under conditions of 16 h of light and 8 h of darkness, with temperatures of 22 °C and 60% humidity. Seven-day-old wheat seedlings were subjected to drought stress (10% PEG 200) and heat stress (42 °C). Samples were systematically collected at 0, 1, 2, 4, 6, 12, 24, and 48 h after treatment. The leaf samples were processed to extract RNA using the RNA plant extraction kit (TIANGEN, Beijing, China). cDNA was synthesized using the ReverTra Ace qPCR RT Master Mix (Toyobo, Kyoto, Japan). Six candidate genes, namely TaTGA10, TaTGA11, TaTGA14, TaTGA16, TaTGA17, and TaTGA27, were subjected to qRT-PCR analysis using Super Real PreMix Plus (SYBR Green) (TIANGEN, China). The thermal cycling conditions were as follows: denaturation at 95 °C for 2 min, followed by 45 cycles at 94 °C for 20 s, 60 °C for 15 s, and 72 °C for 20 s. The expression of genes was normalized to that of a housekeeping gene (TaACTIN, accession no. AB181991) [39]. Each experiment was performed with three biological replicates. Relative expression levels were calculated using the 2^−ΔΔCT method [40]. All primers are listed in Table S10.

4.7. Subcellular Localization

The coding sequence (CDS) of TaTGA16-2D was inserted into the p16318hGFP vector under control of the CaMV35S promoter. The p16318hGFP-TaTGA16-2D and control GFP plasmids were transformed into wheat protoplasts mediated by PEG4000. Wheat protoplasts were isolated from five-day-old seedlings [41]. The fluorescence signals were captured by a confocal laser scanning microscope (LSM700; Carl Zeiss, Jena, Germany). Primers used for amplification are shown in Table S10.

4.8. Generation of Transgenic Arabidopsis

The Arabidopsis cultivar Columbia-0 was used for transformation. The CDS excluding the termination codons of TaTGA16-2D was cloned into plant expression vector pCAMBIA2300 under the control of the CaMV35S promoter. After sequencing, the correct plasmid pCAMBIA2300-TaTGA16-2D was transformed into Arabidopsis by the Agrobacterium tumefaciens-mediated floral dip method [42]. The transformed seeds were surface-sterilized with sodium hypochlorite and selected on 1/2 MS medium containing 50 μg/mL Kanamycin and then transferred to soil (rich soil–vermiculite = 1:1). T₃ generation plants were grown at 22 °C with 60% relative humidity under a 16 h light/8 h dark photoperiod. Three homozygous T₃ lines were selected for the following phenotypic analysis.

4.9. Abiotic Stress Treatments of Transgenic Arabidopsis

For drought stress treatment, five-day-old transformed seedlings were transferred to MS medium supplemented with 6% or 8% PEG, or to PEG-free medium as a control, and grown for an additional 7 days. The total root lengths and fresh weights were then measured.

To investigate the role of TaTGA16-2D in ABA-mediated stomatal regulation, a stomatal aperture assay was performed. The stomatal opening solution was prepared with 10 mM MES-Tris (pH 6.15), 10 mM KCl, and 7.5 mM iminodiacetic acid. Mature leaves from the same position in wild-type and TaTGA16-2D-overexpressing Arabidopsis were excised and floated on the stomatal opening solution under strong light for 3 h to induce stomatal opening. Subsequently, the leaves were transferred to stomatal opening solutions containing 0 μM, 5 μM, or 10 μM ABA and incubated under the same light conditions for an additional 2 h. Stomatal apertures were observed under a light microscope, and images were captured. Three biological replicates were performed for each treatment, with at least 30 stomata measured per replicate. The stomatal pore length and width were measured using the ImageJ software version 1.54p [43], and stomatal opening was calculated as

Stomatal opening (%) = (width/length) × 100.

For heat stress treatment, the transformed seeds were grown on MS medium for 5 days and then exposed to 42 °C for 1 day, followed by transfer to 4 °C for 14 h. Plants were subsequently returned to normal conditions at 22 °C for 3 days, after which survival rates were recorded. The experiment was performed in triplicate.

5. Conclusions

This study comprised a comprehensive study of the 48 members of the wheat TGA gene family, which were categorized into four groups. These genes were then subjected to comprehensive analyses, including phylogeny, chromosomal distribution, duplication events, gene structure, and protein motif analyses, transcriptome profiling, and cis-acting element identification. Subsequently, the expression levels of six candidate genes were validated by qRT-PCR. As a result, these TaTGAs underwent several segmental duplication events, which played a dominant role in the expansion of the TaTGA family. In particular, TaTGA16-2D was further investigated and found to be involved in tolerance to drought and heat stress. These results lay a foundation for future studies to characterize wheat TGA genes.