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Article

Comparative Analysis of TALE Gene Family in Gramineae

by
Zicong Liang
1,†,
Shuai Shi
2,†,
Baoping Xue
3,
Dongyang Li
1,
Yue Liu
4 and
Chang Liu
1,*
1
College of Agronomy, Shenyang Agricultural University, Shenyang 110866, China
2
Department of Plant Sciences, College of Life Sciences, Wuhan University, Wuhan 430072, China
3
Department of Gastroenterology, Xinqiao Hospital, Third Military Medical University (Army Medical University), Chongqing 400037, China
4
Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110866, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2025, 15(6), 1460; https://doi.org/10.3390/agronomy15061460
Submission received: 30 April 2025 / Revised: 12 June 2025 / Accepted: 13 June 2025 / Published: 16 June 2025
(This article belongs to the Section Crop Breeding and Genetics)
Browse Figures
Versions Notes

Abstract

:
The transcription factor known as TALE (three-amino acid loop extension) is essential for plant growth, cell differentiation and responses to environmental stresses. Although the TALE gene family has been identified in various plants, there has been a lack of comprehensive whole-genome identification and analysis in Gramineae species. In this study, 123 TALE family genes were identified in five Gramineae species, which can be categorized into two main subgroups: KONX and BELL. Most of the TALE genes in the same subgroup displayed analogous gene structures and conserved motifs. Furthermore, whole genome duplication (WGD) significantly contributes to the expansion of the TALE gene family in Gramineae. The promoter region of TALE genes in Gramineae contains a large number of cis-elements associated with abiotic stress and hormone response. Tissue-specific expression analysis indicated that most OsTALE, ZmTALE and AtTALE genes were highly expressed in stems and leaves. Additionally, RNA-seq data revealed that OsTALE, ZmTALE and AtTALE genes were found to respond to abiotic stress treatments. Furthermore, we found that the expression levels of SbTALE11/19 were up-regulated in response to PEG and NaCl treatment, respectively. This study provides a significant reference for further research on the biological function of TALE transcription factors in Gramineae plants.

1. Introduction

The TALE (three-amino acid loop extension) protein was first identified in the genome of fruit flies [1]. Subsequently, 70, 68, 24, 35, 17 and 46 TALEs have been identified in T. aestivum (Triticum aestivum L.) [2], G. max (Glycine max L.) [3], S. lycopersicum (Solanum lycopersicum L.) [4], P. tomentosa (Populus tomentosa) [5], P. granatum (Punica granatum L.) [6] and G. hirsutum (Gossypium hirsutum L.) [7]. The conserved domain of the TALE consists 60 amino acids and forms three helical regions. The first and second helices form a ring structure, while the second and third helices form a helix–angle–helix configuration [8]. TALE transcription factors can be divided into two subfamilies: BELL and KNOX. The BELL subfamily contains two conserved domains, POX and homeodomain, which play important roles in plant growth and development, hormone regulation, signal transduction, meristem formation and stress response [9,10,11,12]. Conversely, in the KONX subfamily, most members contain the domains KNOX2, KNOX1, ELK and ELK-Superfamily, which play important roles in gene regulation [9,10,11,12].
TALE typically exists as heterodimers, such as OSH15-SH5, which enhances seed shedding by inhibiting lignin biosynthesis [13]. Additionally, TALE interacts with other family proteins, including BEL1-STM complexes, which are essential for maintaining indeterminate inflorescence meristem development in A. thaliana (Arabidopsis thaliana L.) [14], and GhKNL1 interacts with GhOFP4 [15]. Moreover, AtBLE1 is crucial for ovule development [16]. In Z. mays (Zea mays L.), BLH12/BLH1 regulate stem and vascular development through interaction with KN1, with double mutants exhibiting reduced plant height and fewer vascular bundles [17]. Furthermore, BELL family members are involved in secondary cell wall formation and fruit ripening. For example, tomato SIBL4 plays a role in cell wall metabolism and carotenoid accumulation [12], while OsBLH6 is implicated in cell wall formation [18]. Additionally, TALE genes also regulate flower development [19]. In A. thaliana, ATH1 controls floral competency through positive regulation of FLC, which leads to late flowering [20]. The over-expression of MdKNOX19 increases ABA sensitivity, up-regulates MdABI5 gene expression and affects fruit size and seed yield [21], revealing a complex KNOX regulatory network in plant growth and stress responses. It is worth noting that GmSBH1 not only participates in growth and development processes but also increases tolerance to high temperatures and high humidity stresses [22]. In P. tomentosa, the KNOX gene PagKNAT2/6b enhances drought resistance by inhibiting gibberellin (GA) synthesis [23], and another TALE gene responds to salt stress [5]. PvKN1 is involved in regulating the lignification and cell wall development of switchgrass [24]. During tomato fruit set, auxin regulates GA synthesis through the up-regulation of GA20ox1 and the down-regulation of KONX, maintaining a delicate balance between auxin and GA [25,26].
Gramineous crops, such as O. sativa (Oryza sativa L.), Z. mays and S. italica (Setaria italica L.), not only hold substantial economic importance but also function as crucial model organisms for investigating gene functionality [27,28,29,30]. TALE transcription factors are widespread in both plants and animals, playing a role in numerous physiological processes. Recent research has identified TALE family genes across numerous plant species [2,3,4,5,6,7]. However, a systematic bioinformatics analysis of TALE genes in gramineous plants, like Z. mays, S. italica, S. bicolor (Sorghum bicolor L.) and B. distachyon (Brachypodium distachyon L.), remain absent. Consequently, we conducted a whole-genome-level identification and analysis of the TALE gene family in five gramineous species, discovering a total of 123 TALE genes. Our research included chromosomal distribution, phylogenetic relationships, gene duplication and collinearity, evolutionary selection pressures, gene structure and cis-acting elements. Additionally, we investigated the expression variations of OsTALE, ZmTALE and AtTALE genes under abiotic stress to understand the functional differentiation of the TALE gene family in various plants. This study will provide valuable references for the further utilization of TALE genes for the development of drought-tolerant plants in the context of global land loss, and will facilitate the functional characterization of TALE gene responses to stress and developmental signals.

2. Materials and Methods

2.1. Sources of Genomic Data for Different Species

The species selected for this study include O. sativa, Z. mays, S. italica, B. distachyon, S. bicolor and A. thaliana (Arabidopsis thaliana L.). The genome files were downloaded from the Phytozome v13 database (https://phytozome-next.jgi.doe.gov/, accessed on 5 February 2025) [31]. Additionally, the sequences of the TALE family protein in A. thaliana were obtained from TAIR (https://www.Arabidopsis.org/, accessed on 5 February 2025) [32].

2.2. Identification of TALE Family Proteins in Different Species

The reference sequences for blast alignment (E-value < 1 × 10−5) to identify potential candidate TALE proteins in various species were from the protein sequences of AtTALE. The specific domain files (PF05920) and (PF07526) associated with the TALE protein family were obtained from the PFAM database (http://pfam-legacy.xfam.org/, accessed on 15 February 2025) [33]). HMMER 3.0 software was utilized to screen the potential members of the TALE family among the five species (E-value < 1 × 10−5). To validate the presence of the conserved homeobox domain in the candidate proteins, NCBI-CDD (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi, accessed on 5 February 2025) was employed. Based on this analysis, proteins that did not possess the TALE domain were excluded from the candidate list.

2.3. Evolution Analysis of TALE Proteins

The sequences of the TALE proteins identified in O. sativa, Z. mays, S. italica, B. distachyon and S. bicolor were utilized to create an evolutionary tree using MEGA7.0 software and the neighbor-joining (NJ) method [34]. The parameter settings included the Poisson model, pairwise deletion and 1000 bootstrap replicates.

2.4. Analysis of TALE Gene Structures, Conserved Domains and Protein Motifs

The gene structures, conserved domains and protein conserved motifs of Gramineous and A. thaliana TALE were identified using genome annotation files, NCBI-CDD database (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi, accessed on 15 February 2025) and MEME (with a motif length using built-in parameters of 6–60 amino acids and a motif number of 10) [35]. The visualization, which combines evolutionary trees, conserved motifs, conserved domains and gene structures, was created by Tbtools v2.121 software [36].

2.5. Gene Duplication, Collinearity and Ka/Ks Ratio Analysis of TALE Gene

The TALE gene locations within each species were derived from the corresponding gene structure annotation files (GFF3), named based on their chromosomal order, and they were visualized using TBtools [36]. Gene duplication events were investigated with the Multiple Collinearity Scan Toolkit (MCScan, McScan is an application in the jcvi toolkit) [37], with all parameters left at their defaults. With the O. sativa genome as a reference, collinearity analyses were performed against Z. mays, S. italica, S. bicolor, B. distachyon and A. thaliana. The figure was visualized by TBtools software. Additionally, the Ka/Ks ratios for both TD (tandem duplication) and WGD (whole genome duplication) gene pairs were calculated via TBtools v2.121 software.

2.6. Cis-Element Analysis of TALE Genes in Gramineous and A. thaliana

The 2000 bp sequences upstream of the TALE gene in O. sativa, Z. mays, S. italica, S. bicolor, B. distachyon and A. thaliana were extracted as the promoter sequence. Subsequently, these sequences were submitted to the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 20 February 2025) to predict the cis-acting elements [38].

2.7. Cluster Heat Map Analysis

The expression data of the TALE gene in different tissues (leaves, embryo, anther, endosperm, inflorescence, root and stem) and under different abiotic stresses (drought, salt, cold and heat) for O. sativa, Z. mays and A. thaliana were obtained from the Plant Public RNA-seq Database (PPRD) (http://plantrnadb.com, accessed on 20 February 2025) [39]. The relative expression levels of the TALE gene were calculated using 1 + FPKM (treatment group)/1 + FPKM (control group) values under abiotic stresses. Red indicates up-regulated gene expression and blue denotes down-regulated gene expression. Heat maps were generated using Tbtools, scaled by row with Euclidean clustering. The “house-keeping” gene of O. sativa is LOC_Os03g61970 (Actin); the “house-keeping” gene of Z. mays is Zm00001d027254 (Actin) and the “house-keeping” gene of A. thaliana is AT3G18780 (Actin).

2.8. Protein–Protein Interaction

A protein–protein interaction network was constructed using STRING databases (https://string-db.org/, accessed on 25 February 2025).

2.9. RT-PCR

S. bicolor was cultured in a growth chamber at Shenyang Agricultural University with 16.0 h of light, temperature maintained at 25 °C and 70% humidity. To determine the expression level of the TALE gene after NaCl and PEG treatments, S. bicolor seedlings at the three-leaf–one-heart stage were selected and treated with 150 mM NaCl and 15% PEG, respectively, followed by the collection of S. bicolor root samples. All experiments were performed in three biological replicates, and three different sets of samples were used in each experiment. Total RNA from S. bicolor roots was isolated using the Plant Total RNA Kit (Beijing Chuangmeng International BioGenetics Co. Ltd, Beijing, China) from Beijing Chuangmeng International BioGenetics Co. Ltd. and reverse transcription was performed using HiScript IV All-in-One Ultra RT SuperMix (Nanjing Vazyme Biotech Co., Ltd, Nanjing, China) for qPCR from Nanjing Vazyme Biotech Co., Ltd. The RT-PCR amplification reaction system consisted of 5 μL 2 × SYBR, 3 μL ddH2O, 1 μL cDNA template and 0.5 μM forward and reverse primers in a total volume of 10 μL. The expression level of the SbTALE gene was analyzed by the 2−ΔΔCT method in response to different stress treatments, and SbACTIN was used as an internal reference gene to analyze the expression level of the SbTALE gene under different stress treatments.

3. Results

3.1. Identification of TALE Genes in Gramineae

In this research, we discovered a total of 123 TALE genes among five Gramineae species: O. sativa (26), Z. mays (28), B. distachyon (23), S. bicolor (23) and S. italica (23) (Figure 1 and Supplemental Table S1). Importantly, both O. sativa and Z. mays displayed a greater quantity of TALE genes relative to B. distachyon, S. bicolor and S. italica, indicating a possible expansion of the TALE gene family in these species. The identified TALE genes were named based on their chromosomal locations, designated as OsTALE1-OsTALE26 for O. sativa, ZmTALE1-ZmTALE28 for Z. mays, BdTALE1-BdTALE23 for B. distachyon, SbTALE1-SbTALE23 for S. bicolor and SiTALE1-SiTALE23 for S. italica (Figure 1). The 26 OsTALE genes showed a wide distribution across nearly all chromosomes, with the exception of chromosomes 4, 7 and 9. Conversely, the 23 BdTALE genes were found on almost all chromosomes, except chromosome 5. The distribution of 28 ZmTALE genes were uneven across the chromosomes. Twenty-three SbTALE genes were distributed across all chromosomes except for chromosomes 6, 7 and 8. 23 SiTALE genes were present on all chromosomes except chromosome 7. Moreover, AtTALE genes were present on all chromosomes, apart from chromosome 3.

3.2. Phylogenetic Analysis of TALE Proteins in the Gramineae

To explore the evolutionary relationship for the TALE gene family within the Gramineae, an evolutionary tree was generated utilizing the protein sequences of five Gramineae species and A. thaliana. As illustrated in Figure 2, these TALE proteins can be categorized into two primary subgroups: BELL and KONX. The KONX subgroup is further divided into KONX-I, KONX-II, KONX-III and KONX-IV, while the BELL subgroup consists of BELL-I, BELL-II, BELL-III, BELL-IV and BELL-V. Notably, KONX-III is uniquely present in the Gramineae. Furthermore, most proteins are classified under the KONX-IV classification, in contrast to KONX-I, which has the smallest number of TALE members.

3.3. Gene Structure and Protein Conserved Motifs of TALE Members in Gramineae

To investigate the variations in gene structure and conserved motifs among members in the Gramineous TALE family, we employed MEME for the analysis of their amino acid sequences. As shown in Figure 3, the motif count varies between two and seven. The majority of the KONX subgroup members feature Motif 1, Motif 3, Motif 4, Motif 7, Motif 8 and Motif 10, while the TELL subgroup include Motifs 1, 2, 5, 6 and 9. Notably, OsTALE8 is found to contain only Motif 1 and Motif 2, while another instance of OsTALE8 is characterized by the presence of only Motif 1 and Motif 10. Interestingly, Motif 3 is specifically associated with the KONX-I subgroup. In addition, we found that homologous genes have similar conserved motifs in different plants.
Additionally, we analyzed the conserved domains present within TALE family members in Gramineous species. Members of the TELL subgroup generally exhibit POX and Homeobox-KN domains. In contrast, BdTALE5, OsTALE10, OsTALE20 SbTALE6, SiTALE18, SbTALE16, ZmTALE19 and ZmTALE26 possess POX-superfamily and Homeobox-KN domains. On the other hand, a majority of KONX subgroup members display KNOX2, KNOX1, ELK, and ELK-superfamily domains, which play crucial roles in gene regulation. Uniquely, ZmTALE9, SiTAL15 and OsTALE13 are found to carry KNOX1-superfamily, KNOX1, ELK and ELk-Superfamily domains, suggesting they may offer unique regulatory functions or expression patterns. Other KNOX members, including ZmTALE1, ZmTALE28, SiTAL123, OsTALE5, BdTALE12 and SbTALE11, demonstrate a unique combination of domains, consisting of KNOX1, KNOX2-superfamily and ELK-superfamily domains, which may indicate their specific biological roles. On the other hand, homologous genes have similar conserved domains in different plants.
Moreover, we examined the gene structure of the TALE family in both Gramineous and A. thaliana. Figure 3 illustrates that the gene structure of the TALE family reveals significant variations; the quantity of introns shows patterns specific to each subfamily: members of the BELL subfamily generally have between three and six introns, whereas those in the KNOX subfamily usually have around four to five introns. This pattern could suggest a relationship between gene structure and functionality. Notably, conserved motif analysis revealed that OsTALE9 contains only Motifs 3 and 4, as well as the Homeobox-KN structural domain. In Gramineae and A. thaliana, homologous genes have similar intron–exon structures. In addition, we found considerable disparities in the untranslated region (UTR) present among various members of the Gramineae and A. thaliana TALE genes. For instance, AtKNAT6, AtBEL5, OsTALE9 and ZmTALE24 do not possess both 5’UTR and 3’UTR, while ZmTALE7 and ZmTALE11 are missing 5’ UTR, AtBEL11 is lacking 3’UTR and others show full UTR coverage. Considering the critical role that UTR plays in mRNA stability and translation, the lack of UTR in these particular genes necessitates further inquiry.

3.4. Homologous Gene Pair Analysis of TALE in Gramineae and A. thaliana

Gene duplication is crucial for plant gene family expansion [40,41]. To evaluate its influence on the TALE gene family in Gramineae and A. thaliana, we investigated potential duplication events in these species. Figure 4 illustrates that in O. sativa, B. distachyon, S. bicolor, Z. mays, S. italica and A. thaliana, we found 12, 10, 14, 26, 14 and 8 genome-wide duplication (WGD) genes, as well as 2, 2, 2, 0, 2 and 0 tandem duplication (TD) genes, respectively. Noteworthy is that Z. mays contains the highest number of WGD TALE genes when compared to the other Gramineae and A. thaliana species, with these genes distributed all chromosomes. Furthermore, our investigation uncovered variations in duplication patterns among species: Z. mays and A. thaliana showed an absence of TD genes, while O. sativa, B. distachyon, S. bicolor and S. italica each demonstrated an identical count of TD genes. These results suggest that the expansion of the TALE gene family in Gramineae and A. thaliana is primarily driven by WGD, especially in Z. mays.
To evaluate the pressure of natural selection on the TALE gene family in these species, we computed the Ka/Ks ratio, which represents the ratio of non-synonymous substitutions per non-synonymous site (Ka) to synonymous substitutions per synonymous site (Ks). The outcomes indicated that the Ka/Ks ratio for all TALE duplication genes in Gramineae and A. thaliana were less than 1, ranging from 0.07 to 0.7 (Supplemental Table S2), indicating that TALE genes in these species have generally experienced purifying selection during evolution throughout their evolutionary history.

3.5. Collinearity Analysis of TALE Genes

To further elucidate the evolutionary relationship among the TALE gene family in Gramineae, we conducted a collinearity analysis comparing O. sativa with four Gramineae (B. distachyon, S. bicolor, Z. mays and S. italica) species and the dicotyledonous plant A. thaliana. As shown in Figure 5, we discovered 39, 20, 33 and 34 homologous gene pairs between O. sativa and Z. mays, S. italica, S. bicolor and B. distachyon, respectively. In contrast, only six homologous gene pairs were identified between O. sativa and A. thaliana. This notable disparity indicates that the functionality of the TALE gene family in Gramineae might be more conserved, reflecting a greater differentiation in comparison to A. thaliana. Furthermore, the collinearity events predominantly occurred on chromosomes 1, 2, 3, 5, 6, 10, 11 and 12. These genomic regions may represent hot-spots in the evolution of the TALE gene family within Gramineae, containing significant genetic variation and functional conservation. Importantly, the number of collinear gene pairs between O. sativa and Z. mays, S. italica, S. bicolor and B. distachyon is relatively high, which reflects their close evolutionary relationship and suggests that the TALE genes in these Gramineae crops may exhibit functional similarities. Specifically, we identified four genes, OsTALE2, OsTALE18, OsTALE21 and OsTALE25 that share homologous gene pairs with other Gramineae species and A. thaliana, implying that these genes existed prior to the divergence of monocotyledons and dicotyledons.

3.6. Cis-Element Analysis of TALE Genes in Gramineae

To gain deeper insights into the potential role of the TALE gene in Gramineae, we conducted an analysis of cis-acting elements located in the 2 kb upstream region of the translation initiation site of the TALE gene. We categorized these cis-acting elements into five separate categories: growth and development, response to plant hormone, stress response, light regulation, and binding sites for transcription factors. Our results related to growth and development revealed a strong connection between the TALE gene and various processes, such as endosperm formation, meristem expression and circadian rhythm, as well as root-specific and seed-specific regulatory elements. Additionally, the TALE gene displayed a wide array of responsiveness to plant hormones, indicating its involvement in a complex hormone signaling network that finely regulates plant growth, development and response to stress. The most prevalent observed elements included abscisic acid response elements (ABREs) followed by jasmonic acid response elements (CGTCA-motif and TGACG-motif), as well as ethylene response elements (EREs), implying the significant role of the TALE gene in coping with abiotic stress (Supplemental Table S3).
The examination of elements related to stress response provided further evidence for the possible function of the TALE gene in the adaptation of plants to stress. The occurrence of low-temperature and drought response elements in both Gramineae and A. thaliana indicates that the TALE gene may be crucial in managing these two stresses. Moreover, additional stress response elements, including those associated with anaerobic induction and mechanical injury, were also identified in some TALE genes, which enhances our comprehension of their functional roles.
The examination of light elements and binding site transcription factors has uncovered the possible involvement of the TALE gene in the transduction of light signals and the regulation of gene expression. The existence of light-responsive elements such as G-box and I-box implies that TALE genes might play a role in photosynthesis in plants. In addition, the discovery of transcription factor binding sites, including W-box, MYB and MYC, indicates that multiple transcription factors may regulate the TALE gene.
Additionally, we observed differences in the cis-acting elements of the TALE gene across various grasses and A. thaliana. For example, seed-specific regulatory elements are identified in Gramineae but are absent in A. thaliana, and circadian-related elements are observed in O. sativa, Z. mays, S. bicolor, B. distachyon and A. thaliana, yet they are missing in S. italica. In summary, our findings suggest that the TALE gene has diverse and intricate functions in Gramineae and A. thaliana, involving growth and development, hormone signaling and stress adaptation. Additionally, we have recognized both similarities and differences in the function of TALE gene among different species, providing a new perspective for understanding the evolution and functional differentiation of the TALE gene family.

3.7. The Expression Patterns of OsTALE, ZmTALE and AtTALE Genes Across Various Tissues

Based on the published RNA-seq in the PPRD database, we mapped the TALE gene expression heat map across the organs of A. thaliana, O. sativa and Z. mays to investigate the differences in TALE gene expression patterns between monocotyledonous plants and dicotyledonous plants. The organs included roots, stems, leaves, anthers, embryos, endosperm and inflorescence (Supplemental Table S4). The expression levels of the TALE gene in A. thaliana, O. sativa and Z. mays were relatively high in stems, leaves and inflorescence, while the expression levels in endosperm and embryos were comparative low. Notably, the expression level of the A. thaliana TALE gene was significantly higher in the embryo than in O. sativa and Z. mays, highlighting its unique expression pattern. Furthermore, we observed that OsTALE1, OsTALE10, ZmTALE20, ZmTALE25 and AtBEL11 were specifically highly expressed in the roots, while OsTALE4, OsTALE15, OsTALE16, OsTALE20, ZmTALE13, ZmTALE22 and ZmTALE23 exhibited high expression in inflorescence. These findings suggest a differentiated regulatory role of TALE gene family members across various tissues. Additionally, we found that OsTALE2, ZmTALE14, ZmTALE22, ZmTALE24 and AtBLH9 were orthologous genes. Importantly, OsTALE2, ZmTALE14 and ZmTALE24 were highly expressed in stems, while ZmTALE22 and AtBLH9 showed high expression in inflorescence. This phenomenon not only reveals the functional differentiation of monocotyledonous and dicotyledonous plants throughout the evolutionary process but also indicates functional conservation and differentiation among grasses. Similar patterns were observed with OsTALE21, ZmTALE16, ZmTALE18 and AtKNAT5, as well as with OsTALE25, ZmTALE15 and AtBLH1, which further confirmed the complexity and diversity of TALE gene functions in plant evolution.

3.8. The Expression Profiles of the OsTALE, ZmTALE and AtTALE Genes Under Abiotic Stresses

The TALE gene has been identified as playing a crucial role in plant responses to abiotic stress. In this study, we used RNA-seq data to conduct a comprehensive analysis of the expression profile changes of ZmTALE, OsTALE and AtTALE genes under drought, cold, heat and salt stress (Supplemental Table S5). The results showed that 3, 10, 6 and 16 OsTALE genes were up-regulated under drought, heat, salt and cold stress, respectively, while 14, 7, 7 and 5 OsTALE genes exhibited down-regulated under the same conditions. For the ZmTALE gene family, the number of up-regulated genes was 2, 4, 2 and 9, under the corresponding stress conditions, with the number of down-regulated genes being 0, 7, 10 and 9. In contrast, the AtTALE gene family showed 3 up-regulated genes under drought, heat, salt and cold stress, but the number of down-regulated genes varied, recorded as 3, 12, 8 and 4, respectively. Notably, we identified several homologous genes exhibiting distinct expression patterns in response to stress. OsTALE18, ZmTALE16, ZmTALE18 and AtKNAT4 are homologous genes; OsTALE18 and ZmTALE18 were up-regulated under cold stress, while AtKNAT4 was down-regulated. In addition, OsTALE2, ZmTALE14, ZmTALE22, ZmTALE24 and AtBLH9 also belong to homologous genes, each displaying different expression patterns. Specifically, OsTALE2 was down-regulated under all stress conditions. ZmTALE14 and ZmTALE24 were up-regulated under cold stress but down-regulated under salt stress. AtBLH9 was up-regulated specifically under drought and salt stress, showing no significant response to cold and heat stress. These findings not only enhance our understanding of the functional diversity within the TALE gene family but also suggest that TALE genes exhibit both functional conservation and significant differentiation among various species throughout the course of evolution. This has considerable implications for how plants adapt to complex and dynamic environments.

3.9. Analysis of Protein–Protein Interaction (PPI) Networks

Previous studies have shown that TALE proteins can form homologous or heterodimers. Therefore, we used the STRING database to analyze the interaction network of OsTALE proteins and found intricate interactions among family members. Notably, the (OsTALE1) OSH1, OSH15 (OsTALE22) and OSH71 (OsTALE17) genes play an important role in the formation and maintenance of undifferentiated cell meristem states. As shown in Figure 6, OsTALE17 interacts with OsTALE2, OsTALE19, OsTALE6, OsTALE15 and OsTALE24, suggesting that they may participate in the formation and maintenance of meristem states. Additionally, OsTALE3 interacts with OsTALE5, OsTALE7, OsTALE10, OsTALE11, OsTALE12 and OsTALE21, while OItsTALE2 interacts with OsTALE8, OsTALE9, OsTALE17, OsTALE18 and OsTALE22. Previous studies have revealed that OSH15 (OsTALE22) forms heterodimers with SH5 (OsTALE18) and qSH1 (OsTALE2), repressing lignin biosynthesis genes expression and facilitating seed shedding. This suggests that OsTALE8, OsTALE9 and OsTALE17 proteins may also play a significant role in this biological process. These studies laid a solid foundation for further exploring the mechanism of TALE proteins in plant growth and development and stress response (Figure 7 and Figure 8).

3.10. The Expression Patterns of SbTALE Genes in NaCl and PEG Stress

To further explore the function of the TALE gene in the Gramineae family, we utilized S. bicolor as model organism and subjected it to NaCl and PEG treatments. A random selection of 10 S. bicolor TALE genes was verified. The results are shown in Figure 9. The expression levels of several SbTALE genes significantly altered under 150 mM NaCl and 15% PEG6000 treatment. Under NaCl treatment, the expressions of genes such as SbTALE11, SbTALE18 and SbTALE19 were up-regulated, while SbTALE2, SbTALE5 and SbTALE16 were down-regulated. Conversely, during PEG treatment, the expressions levels of SbTALE18 and SbTALE46 were up-regulated, whereas SbTALE2, SbTALE5, SbTALE8, SbTALE16 and SbTALE17 exhibited down-regulation. Notably, the SbTALE2, SbTALE5 and SbTALE16 genes demonstrated down-regulation following both drought and salt stress treatment. Importantly, under the combined stress of NaCl and PEG6000, the expression levels of TALE8, TALE15, TALE16, TALE17 and TALE19 were significantly up-regulated.

4. Discussion

TALE transcription factors are crucial in the regulation of plant growth and development, especially in meristem formation and the maintenance of organ morphogenesis. In this study, 26, 28, 23, 23 and 23 TALE genes were identified in the genomes of O. sativa, Z. mays, S. italica, S. bicolor and B. distachyon, respectively. Given that the differentiation of crops dates back to 50 to 70 million years ago [42], these results imply that the TALE gene family in Gramineae might have expanded at a relatively uniform rate. Furthermore, we observed that the number of TALE genes in Gramineae is comparable to A. thaliana [43], more than in P. granatum [6] and less than in G. max [3], T. aestivum [2], P. tomentosa [5] and G. hirsutum [7]. This comparison highlights the significant diversity of the TALE gene across various plant species. This study not only revealed the distribution of TALE genes in Gramineae but also offered insights into the evolutionary and functional differentiation of TALE gene family in plants through cross-species analysis.
Conserved domains or motifs are crucial for protein interactions. In this study, we discovered that the TALE gene family, which consists of five Gramineae and A. thaliana, features a conserved domain known as Homeobox-KN. Additionally, both Gramineae and A. thaliana TALE family members contain motif1, likely representing the most conserved protein motif. Further investigation revealed that the BELL subfamily also encompasses POX and POX-superfamily domains. The KNOX subfamily comprises KNOX1, KNOX2 and ELK domains. In A. thaliana, BELL subfamily proteins interact with KNAT2 and KNAT5, influencing plant growth and development [44]. Conversely, KNOX1 plays a crucial role in repressing the expression of downstream target genes, while KNOX2 is essential for the formation of homodimers [45].
The TALE family members of five Gramineae species and A. thaliana were divided into two subgroups, KNOX and Bell, and the KNOX subgroup was further divided into four subgroups, while the BELL subgroup was further divided into five subgroups, which was consistent with the previous classification in A. thaliana, cotton and poplar [5,7,43]. In A. thaliana, O. sativa, Z. mays, S. italica, S. bicolor and B. distachyon, the BELL and KNOX subfamilies have distinct branches, which reflects the significant differences in amino acid sequences, and it is possible that these two subfamilies diverged early in the evolutionary process. In contrast, Gramineae plants show consistency in differentiation, which may be due to the fact that the five plants belong to the same family of Gramineaes and are closely related to each other. However, Gramineae and A. thaliana show great differences in differentiation, which may be attributed to the difference in evolutionary paths between monocotyledonous and dicotyledonous plants.
Previous studies have revealed that gene replication events play a key role in the evolution of the TALE gene family in plants [2]. We identified 12, 10, 14, 26, 14 and 8 WGD genes and 4, 4, 4, 0, 2 and 0 TD genes in O. sativa, B. distachyon, S. bicolor, Z. mays, S. italica and A. thaliana, respectively. These results suggest that WGD is a major factor driving the expansion of the TALE gene family in these species. In addition, the Ka/Ks values of all homologous gene pairs were less than one, revealing that these gene pairs underwent different degrees of purification selection. The results of Huang et al. (2020) in G. raimondii and G. arboretum further support our conclusion [46]. Additionally, collinearity analysis can be used to study the evolution of gene family in different species. The number of homologous gene pairs between O. sativa and Z. mays, S. italica, S. bicolor and B. distachyon were significantly more than that between O. sativa and A. thaliana, and the number of TALE homologous gene pairs with S. italica, S. bicolor and B. distachyon was the most, indicating that O. sativa and Z. mays, S. bicolor and B. distachyon were closely related in evolution. It is worth noting that collinear gene pairs of OsTALE2, OsTALE18, OsTALE21 and OsTALE25 exist in dicotyledonous and monocotyledonous plants, which not only reflects gene conservation but also suggests that these homologous genes may have originated before ancestral differentiation. In addition, most of the TALE genes of O. sativa had a one-to-many collinearity relationship with Z. mays, S. italica, S. bicolor and B. distachyon and A. thaliana, which further confirmed that after the differentiation of O. sativa and these species, gene family duplication events occurred, promoting the diversity and complexity of their respective genomes.
The TALE gene family plays a crucial role in regulating plant growth and development. For example, Wang et al. (2020) found that the TALE gene in P. granatum may be involved in regulating shoot apical meristem (SAM), flower and ovule development [6]. Similarly, TALE genes showed different expression levels at different stages of walnut flower bud development, revealing its dynamic regulatory role in this process [47]. The expression levels of GhSTM3 were down-regulated and significantly affected flowering time [48]. In poplar, most TALE genes are highly expressed in stem tissue suggests that they may play a key regulatory role in wood formation [5]. Consistent with this study, our results also showed that most TALE genes in O. sativa, Z. mays and A. thaliana were specifically highly expressed in stems, while some TALE genes were highly expressed in leaves, suggesting that these TALE genes may play an important role in different developmental stages of O. sativa, Z. mays and A. thaliana. It is worth noting that TALE proteins usually form heterodimers to function [5]. In our study, there may be interactions between OsTALE17 and OsTALE1 proteins, in addition to OsTALE3 and OsTALE5 as well as OsTALE7, OsTALE10, OsTALE11, OsTALE12 and OsTALE21. It further revealed the complex regulatory mechanism of the TALE gene family in plant growth and development.
Previous studies have found that the function of TALE genes is significantly related to the complex hormone network in plants. For example, the KNOX gene promoter region in Orchidaceae contains MeJA and ABA-responsive elements [49], while the TALE gene promoter of pomegranates contains auxin and gibberellin-responsive elements [6]. In this study, we identified a variety of hormone response elements, including auxin, gibberellin, methyl jasmonate, abscisic acid and salicylic acid in Gramineae and A. thaliana. These findings suggest that hormone signaling pathways may finely regulate TALE gene expression in Gramineae and A. thaliana. Recent studies have shown that the TALE gene plays an important role in plants response to environmental stress. For example, the PagKNAT2/6b gene in poplar mediates the response to drought stress by down-regulating the expression of PagGA20ox1 [25]. Similarly, the expression of the TALE gene is not only finely regulated by plant hormones but also influenced by external abiotic stress [50,51]. In soybean, the TALE gene can finely regulate its expression level to adapt to adverse environments under salt and drought stress [3]. Similarly, 11 TALE genes in poplars were found to have significant responses to salt stress [5]. As shown in Figure 7, the promoter region of the TALE gene in Gramineae and A. thaliana is rich in a variety of abiotic stress response elements, including low temperature and drought. A further study found that the expression of OsTALE6/8/14/17/18/19/24/26 and ZmTALE1/4/14/15/18/24/28 exhibits an increasing trend under cold stress, while OsTALE7 and ZmTALE2/11 show significant increases under drought stress. Moreover, OsTALE2, ZmTALE14, ZmTALE22, ZmTALE24 and AtBLH9 are orthologous genes. However, in response to different stresses, their expression patterns showed significant differences: OsTALE2 was down-regulated under drought and salt stress, while ZmTALE14 and ZmTALE24 were down-regulated under salt stress. In contrast, AtBLH9 is up-regulated under drought and salt stress. These results suggest that TALE genes are functionally differentiated and functionally different in different species during evolution. Different SbTALE genes showed differential expression patterns under drought and salt stress conditions (Figure 10). Under salt stress (NaCl), SbTALE11, SbTALE18 and SbTALE19 were up-regulated, while SbTALE2, SbTALE5 and SbTALE16 were down-regulated. During drought stress (PEG), SbTALE18 and SbTALE46 were up-regulated, whereas SbTALE2, SbTALE5, SbTALE8, SbTALE16 and SbTALE17 were down-regulated. Notably, SbTALE2, SbTALE5 and SbTALE16 were consistently down-regulated under both stresses. Under combined NaCl+PEG6000 stress, TALE8, TALE15, TALE16, TALE17 and TALE19 showed significant up-regulation. Analysis of the cis-acting elements of the SbTALE promoters revealed that SbTALE2, SbTALE5, SbTALE8, SbTALE17, SbTALE18 and SbTALE19 contain regulatory elements related to drought response. These results suggest that the SbTALE gene family plays an important role in the S. bicolor response to drought and salt stress. This finding is consistent with previous research indicating that TALE genes play important roles in drought and salt tolerance in other species. These findings not only reveal the functional differentiation of TALE genes across species but also highlight their functional differences in response to complex environmental stresses, providing valuable insights into the molecular mechanisms of plant stress resistance.

5. Conclusions

A total of 123 TALE genes were identified across five Gramineous plants. Phylogenetic analysis revealed that that TALE can be divided into the KONX and BELL subfamilies. Gene duplication events indicated that WGD was the primary driver of TALE gene family expansion. Collinearity analysis demonstrated a higher number of collinear gene pairs between O. sativa and other Gramineous plants, whereas relatively few collinear gene pairs were observed between O. sativa and A. thaliana. The prediction of protein interaction showed that O. sativa TALE proteins could form heterodimers. Tissue expression patterns revealed that OsTALE, ZmTALE and AtTALE genes were highly expressed in stems and leaves, while expression levels were lower in the endosperm and embryo. The RNA-seq data showed that the TALE genes in O. sativa, Z. mays and A. thaliana exhibited not only functional differentiation but also some functional discrepancies. Furthermore, the expression levels of SbTALE11/19 were up-regulated in response to PEG and NaCl treatment, respectively. These findings provide important insights into the diversity and specificity of TALE gene functions across different species.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15061460/s1, Table S1: Physicochemical properties of TALE family genes. Table S2: Ka/Ks values of duplicate gene pairs. Table S3: The Cis-elements was analyzed of TALE genes by plantcare. Table S4: The expression patterns of TALE genes. Table S5: The expression patterns of TALE genes.

Author Contributions

Z.L.: writing—original draft, validation and data curation. S.S.: data curation and writing—review and editing. B.X.: writing—review and editing, visualization and investigation. D.L.: visualization, investigation and data curation. Y.L.: writing—review and editing. C.L.: writing—review and editing, visualization and supervision. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Phd Starting Foundation of Shenyang Agricultural University (X2021022).

Data Availability Statement

Rest assured, I have ensured that all data, materials, software applications, and custom code supporting the claims made in this article are in full compliance with field standards. Data are contained within the article or Supplementary Material. The datasets about the Public RNA-seq data during the current study are available in the PPRD (http://plantrnadb.com, accessed on 20 February 2025).

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The chromosomal positions of the TALE genes have been identified in five Gramineae, such as (A) O. sativa, (B) S. italica (C) S. bicolor, (D) Z. mays, (E) B. distachyon and (F) A. thaliana.
Figure 1. The chromosomal positions of the TALE genes have been identified in five Gramineae, such as (A) O. sativa, (B) S. italica (C) S. bicolor, (D) Z. mays, (E) B. distachyon and (F) A. thaliana.
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Figure 2. The evolutionary tree of the 123 identified TALE proteins sequences from O. sativa, Z. mays, S. italica, S. bicolor, B.distachyon and A. thaliana was constructed. These 123 TALE proteins were divided into two primary subgroups: BELL and KONX. The KONX subgroup is further divided into four categories KONX-I, KONX-II, KONX-III and KONX-IV, while the BELL subgroup comprises BELL-I, BELL-II, BELL-III, BELL-IV and BELL-V. The tree was constructed by the neighbor-joining method using MEGA 7.0 software with bootstrap values calculated for 1000 replicates. Different colors represent distinct subgroups.
Figure 2. The evolutionary tree of the 123 identified TALE proteins sequences from O. sativa, Z. mays, S. italica, S. bicolor, B.distachyon and A. thaliana was constructed. These 123 TALE proteins were divided into two primary subgroups: BELL and KONX. The KONX subgroup is further divided into four categories KONX-I, KONX-II, KONX-III and KONX-IV, while the BELL subgroup comprises BELL-I, BELL-II, BELL-III, BELL-IV and BELL-V. The tree was constructed by the neighbor-joining method using MEGA 7.0 software with bootstrap values calculated for 1000 replicates. Different colors represent distinct subgroups.
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Figure 3. Analysis of the phylogenetic relationships, conserved motifs, domains and gene structures of TALE in Gramineae and A. thaliana. (A) A phylogenetic tree representing TALE proteins in Gramineae and A. thaliana was created using MEGA 7, employing the amino acid sequences of TALE proteins from both plant groups, with the bootstrap test set to 1000 replicates. (B) The distribution of conserved motifs across TALE proteins in Gramineae and A. thaliana is displayed, with ten potential motifs highlighted in various colored boxes. (C) The conserved domain analyses of full-length TALE amino acid sequences in both Gramineae and A. thaliana. (D) The distribution of exons and introns for TALE genes in Gramineae and A. thaliana is illustrated.
Figure 3. Analysis of the phylogenetic relationships, conserved motifs, domains and gene structures of TALE in Gramineae and A. thaliana. (A) A phylogenetic tree representing TALE proteins in Gramineae and A. thaliana was created using MEGA 7, employing the amino acid sequences of TALE proteins from both plant groups, with the bootstrap test set to 1000 replicates. (B) The distribution of conserved motifs across TALE proteins in Gramineae and A. thaliana is displayed, with ten potential motifs highlighted in various colored boxes. (C) The conserved domain analyses of full-length TALE amino acid sequences in both Gramineae and A. thaliana. (D) The distribution of exons and introns for TALE genes in Gramineae and A. thaliana is illustrated.
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Figure 4. The analysis of duplication events concerning TALE genes in Gramineae and A. thaliana encompasses (A) O. sativa, (B) B. distachyon, (C) S. bicolor, (D) Z. mays, (E) S. italica and (F) A. thaliana. The TD gene names are highlighted in red, while WGD genes are represented by purple lines.
Figure 4. The analysis of duplication events concerning TALE genes in Gramineae and A. thaliana encompasses (A) O. sativa, (B) B. distachyon, (C) S. bicolor, (D) Z. mays, (E) S. italica and (F) A. thaliana. The TD gene names are highlighted in red, while WGD genes are represented by purple lines.
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Figure 5. Collinearity analysis was conducted for O. sativa in relation to Z. mays, S. italica, S. bicolor, B. distachyon and A. thaliana. The collinear regions observed between O. sativa and the other species are depicted with gray lines. Meanwhile, the syntenic TALE gene pairs corresponding to O. sativa and the other plants are emphasized with red lines.
Figure 5. Collinearity analysis was conducted for O. sativa in relation to Z. mays, S. italica, S. bicolor, B. distachyon and A. thaliana. The collinear regions observed between O. sativa and the other species are depicted with gray lines. Meanwhile, the syntenic TALE gene pairs corresponding to O. sativa and the other plants are emphasized with red lines.
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Figure 6. The distribution of cis-acting elements within the TALE gene family among Gramineae and A. thaliana species. These cis-acting elements were located in the 2 kb promoter region of TALE genes found in both Gramineae and A. thaliana.
Figure 6. The distribution of cis-acting elements within the TALE gene family among Gramineae and A. thaliana species. These cis-acting elements were located in the 2 kb promoter region of TALE genes found in both Gramineae and A. thaliana.
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Figure 7. Analysis of the expression patterns of OsTALE, ZmTALE and AtTALE genes across various tissues. (A) A heat map illustrating the expression levels of OsTALE genes in four different tissues (root, stem, leaves, inflorescence, anther, endosperm and embryo). (B) A heat map displaying the expression of ZmTALE genes in four tissues (root, stem, leaves, inflorescence, anther, endosperm and embryo). (C) A heat map displaying the expression of AtTALE genes in four tissues (root, stem, leaves, inflorescence, anther, endosperm and embryo). The heatmaps were generated using TBtools software. The colors red and blue denote elevated and reduced expression levels of the OsTALE, ZmTALE and AtTALE genes, respectively.
Figure 7. Analysis of the expression patterns of OsTALE, ZmTALE and AtTALE genes across various tissues. (A) A heat map illustrating the expression levels of OsTALE genes in four different tissues (root, stem, leaves, inflorescence, anther, endosperm and embryo). (B) A heat map displaying the expression of ZmTALE genes in four tissues (root, stem, leaves, inflorescence, anther, endosperm and embryo). (C) A heat map displaying the expression of AtTALE genes in four tissues (root, stem, leaves, inflorescence, anther, endosperm and embryo). The heatmaps were generated using TBtools software. The colors red and blue denote elevated and reduced expression levels of the OsTALE, ZmTALE and AtTALE genes, respectively.
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Figure 8. Expression profiles of the OsTALE, ZmTALE and AtTALE genes under abiotic stresses. (A) The heatmap displays the relative expression levels for OsTALE genes subjected to abiotic stress treatments, including drought, heat, cold and salt. (B) The heatmap illustrates the relative expression levels for ZmTALE genes under cold, drought, heat and salt. (C) The heatmap illustrates the relative expression levels for AtTALE genes under cold, drought, heat and salt. The construction of the heatmap utilized TBtools software. High and low expression levels of OsTALE, ZmTALE and AtTALE genes are represented by red and blue boxes, respectively.
Figure 8. Expression profiles of the OsTALE, ZmTALE and AtTALE genes under abiotic stresses. (A) The heatmap displays the relative expression levels for OsTALE genes subjected to abiotic stress treatments, including drought, heat, cold and salt. (B) The heatmap illustrates the relative expression levels for ZmTALE genes under cold, drought, heat and salt. (C) The heatmap illustrates the relative expression levels for AtTALE genes under cold, drought, heat and salt. The construction of the heatmap utilized TBtools software. High and low expression levels of OsTALE, ZmTALE and AtTALE genes are represented by red and blue boxes, respectively.
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Figure 9. Protein–protein interaction network was constructed using STRING databases (https://string-db.org/, accessed on 27 February 2025).
Figure 9. Protein–protein interaction network was constructed using STRING databases (https://string-db.org/, accessed on 27 February 2025).
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Figure 10. The relative expression levels of SbTALE genes under 150 mM NaCl and 15% PEG6000 treatment. The data are expressed as the standard error (SE) based on three replicates. Different letters indicate significant differences determined by one-way analysis of variance (ANOVA). RT-qPCR analysis utilized the Sobic.001G112600.1 gene as an internal control.
Figure 10. The relative expression levels of SbTALE genes under 150 mM NaCl and 15% PEG6000 treatment. The data are expressed as the standard error (SE) based on three replicates. Different letters indicate significant differences determined by one-way analysis of variance (ANOVA). RT-qPCR analysis utilized the Sobic.001G112600.1 gene as an internal control.
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Liang, Z.; Shi, S.; Xue, B.; Li, D.; Liu, Y.; Liu, C. Comparative Analysis of TALE Gene Family in Gramineae. Agronomy 2025, 15, 1460. https://doi.org/10.3390/agronomy15061460

AMA Style

Liang Z, Shi S, Xue B, Li D, Liu Y, Liu C. Comparative Analysis of TALE Gene Family in Gramineae. Agronomy. 2025; 15(6):1460. https://doi.org/10.3390/agronomy15061460

Chicago/Turabian Style

Liang, Zicong, Shuai Shi, Baoping Xue, Dongyang Li, Yue Liu, and Chang Liu. 2025. "Comparative Analysis of TALE Gene Family in Gramineae" Agronomy 15, no. 6: 1460. https://doi.org/10.3390/agronomy15061460

APA Style

Liang, Z., Shi, S., Xue, B., Li, D., Liu, Y., & Liu, C. (2025). Comparative Analysis of TALE Gene Family in Gramineae. Agronomy, 15(6), 1460. https://doi.org/10.3390/agronomy15061460

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