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Article

Genome-Wide Identification of the Trihelix Transcription Factor Family and Functional Analysis of ZmTHX15 in Maize

by
Yanyong Cao
1,†,
Zeqiang Cheng
1,†,
Xinyan Sun
1,
Meichen Zhu
1,
Ling Yue
2,
Hui Liu
2,
Xiaolin Wu
2,
Jinghua Zhang
2,* and
Canxing Duan
3,*
1
Institute of Cereal Crops, Henan Academy of Agricultural Sciences, The Shennong Laboratory, Zhengzhou 450002, China
2
National Key Laboratory of Wheat and Maize Crop Science, College of Life Sciences, Henan Agricultural University, Zhengzhou 450046, China
3
Key Laboratory of Grain Crop Genetic Resources Evaluation and Utilization, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2024, 25(24), 13257; https://doi.org/10.3390/ijms252413257
Submission received: 5 November 2024 / Revised: 5 December 2024 / Accepted: 8 December 2024 / Published: 10 December 2024
(This article belongs to the Special Issue Molecular Research in Plant Adaptation to Abiotic Stress)
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Abstract

The trihelix transcription factor, which is a plant-specific family, play a critical role in plant growth and development and stress responses. Drought is the main limiting factor affecting yield of maize (Zea mays). However, the identification and characterization of this gene family in maize and its biological functions in response to drought stress have not been reported. Here, 46 Zea mays trihelix genes (ZmTHXs) were identified in the genome. Phylogenetic analysis of the ZmTHXs revealed that the genes were clustered into five subfamilies: GT-1, GT-2, GTγ, SH4, and SIP1. Chromosomal localization analysis showed that the 46 ZmTHXs were unevenly distributed across 10 chromosomes in maize. Cis-acting elements related to abiotic stress in ZmTHXs were found. Most ZmTHXs genes showed significant changes in expression levels under drought treatment. In addition, ZmTHX15-overexpressing Arabidopsis exhibited stronger drought tolerance with less secondary oxidative damage and higher photosynthetic rate. These findings could serve as a basis for future studies on the roles of ZmTHXs and the potential genetic markers for breeding stress-resistant and high-yielding maize varieties.

1. Introduction

The growth and development of plants and their adaptation to the environment are regulated by multiple genes. Transcription factors, the most important regulatory elements, are widely involved. Also known as trans-acting factors, transcription factors can bind to specific cis-acting elements of eukaryotic genes to regulate the transcription of related genes, thereby enabling the target genes to express in a specific temporal and spatial manner [1]. To date, more than 64 types of transcription factors have been identified in higher plants, playing crucial roles in plant growth, development, and responses to abiotic stresses [2].
Trihelix (THXs) are a unique class of transcription factors specific to plants, first discovered and isolated in peas [3], and play important roles in developmental processes. THXs generally bind to the core sequence 5′-G-Pu-(T/A)-A-(T/A)-3′ in the promoter region of the ribulose-1,5-biphosphate carboxylase small subunit 3A (rbcS-3A) gene, regulating light response reactions [4].
THXs contain three tandem α-helix domains (helix-loop-helix-loop-helix), which is why members of this family are called trihelix transcription factors. THXs can specifically bind to GT elements related to light response to regulate the expression of related genes; thus, they are also known as GT factors [5]. GT elements are highly degenerate cis-acting elements, with the core sequence 5′-GTGTGGTTAATATG-3′ [4].
Research has found that the conserved domains of trihelix transcription factors overlap and share similarities with the helix-loop-helix structure of MYB transcription factors [6]. Therefore, the domains of trihelix transcription factors include the main features of MYB transcription factors [6]. Based on their domains and other sequence characteristics, this family is divided into five subfamilies: GT-1, GT-2, GTγ, SH4, and SIP1 [7]. The GT-2 subfamily has two DNA-binding domains, while the other four subfamilies have only one DNA-binding domain [7]. Each subfamily is named after the first member discovered.
Additionally, based on the recognized elements, members of this family can be divided into three categories: GT-1, GT-2, and GT-3. GT-1 and GT-3 can specifically bind to the Box II (5′-GTGTGGTTAATATG-3′) and 5′-GTTAC-3′ motifs. However, GT-2 is different; its N-terminal can bind to the GT-3 box (5′-GAGGTAAATCCGCGA-3′), while its C-terminal can bind to the GT-2 box (5′-GCGGTAATTAA-3′) [8].
Recent studies have shown that THXs not only respond to light induction but also play important roles in flower organ morphogenesis, trichome formation, embryo development, and grain development [9,10,11,12,13]. For example, the trihelix transcription factor GT-2-LIKE1 (GTL1) and its homolog DF1 inhibit root hair growth by directly binding to the promoter of ROOT HAIR DEFECTIVE 6-LIKE4 (RSL4) in Arabidopsis [14]. In Arabidopsis, the trihelix transcription factor PETAL LOSS (PTL) is involved in the morphogenesis of various floral organs, including the perianth, petal, and stamen [15]. The GT-1 subfamily gene At5g63420, which encodes a metal-β-lactamase-trihelix complex, is highly expressed in seeds and is essential for early embryogenesis [16]. Through map-based cloning, a trihelix gene named Shattering 1 (SHA1) was identified in rice that played an important role in regulating seed shattering [17].
In addition to their involvement in plant growth, development, and light responses, THXs also play a crucial role in responding to biotic and abiotic stresses. In Arabidopsis, the trihelix transcription factor ARABIDOPSIS SH4-RELATED3 (ASR3) played a negative role in regulating pattern-triggered immunity (PTI) since the ASR3 mutant showed enhanced disease resistance to virulent bacterial pathogen infection [18,19]. Another trihelix family member, ARS3-Interacting Transcriptional Factor 1 (AITF1), regulates plant immune responses and resistance to phytobacteria [20]. Overexpression of AITF1 causes autoimmunity in plants dependently on ASR3 [20]. A cotton (Gossypium hirsutum L.) trihelix family gene, GhGT-3b_A04, was strongly induced by the fungal pathogen Verticillium dahlia [21]. Overexpression of GhGT-3b_A04 in Arabidopsis enhanced the plant’s resistance to Verticillium wilt [21]. Arabidopsis thaliana GT-2-LIKE 1 (AtGTL1) acts as a positive regulator of PTI since Atgtl1 mutants are compromised in basal resistance to Pst DC3000 infection, whereas the overexpression of AtGTL1 leads to a reduced susceptibility [22]. In kiwifruit, the trihelix transcription factor GT1 interacts with E3 ubiquitin ligase PUB23 to negatively regulate immune responses against Pseudomonas syringae pv. actinidiae [23]. ZmGT-3b, a GT-1 subfamily trihelix transcription factor, negatively regulates plant defense responses as knocking down of ZmGT-3b in seedlings led to enhanced resistance to Fusarium graminearum infection [24].
A study has found that AtGT-4 interacted with AP2/ERF domain-containing protein (TEM2) to positively co-regulate the expression of the salt responsive gene Cor15A, thereby enhancing salt stress tolerance in Arabidopsis [25]. The transcript level of OsGTγ-1 is strongly induced by salt stress, and mildly induced by drought, low temperature, and ABA treatment [5]. Overexpression of OsGTγ-1 in rice enhanced the salt tolerance of transgenic plants, whereas the homozygous mutant osgtγ-1 (T-DNA insertion in the promoter region of OsGTγ-1) exhibited reduced salt tolerance and increased sensitivity to salt stress [5]. Moreover, a study has found that OsGTγ-2 directly bound to the promoter of ion transporting genes to regulate salt response [26]. Overexpression of OsGTγ-2 in rice improved the seed-germination rate, seedling growth, and survival rate under salt stress. However, CRISPR/Cas9-mediated OsGTγ-2 knockout lines showed salt-hypersensitive phenotypes [26]. AtGT2L was involved in plant responses to low temperature and salt stress, as in plants overexpressing AtGT2L, both basal and chilling/NaCl-induced expression levels of cold- and salt-inducible marker genes RD29A and ERD10 were significantly higher than those in wild-type (WT) plants [27]. A member of the cotton trihelix transcription factor (GhGT26) positively regulates salt stress as overexpression of GhGT26 in Arabidopsis improved the tolerance to salt stress [28].
Arabidopsis SIP1 clade Trihelix 1 (AtAST1) could regulate the expression of peroxidase (POD), superoxide dismutase (SOD), and late embryogenesis abundant (LEA) genes by binding to the AGAG-box, thus improving plant tolerance to salt and osmotic stress [29]. Transgenic Arabidopsis plants overexpressing AtAST1 increased the tolerance to drought, salt, and osmotic stress, whereas the AST1 mutants displayed sensitive phenotype [29]. Chromatin immunoprecipitation (ChIP) analysis indicated that AtGTL1 interacted with the GT-3 box in the STOMATAL DENSITY AND DISTRIBUTION1 (AtSDD1) promoter, negatively regulating SDD1 to modulate stomatal density [30]. The expression level of AtGTL1 was downregulated under water stress, and GTL1 loss-of-function mutants improved drought tolerance by reducing stomatal density, decreasing water loss through transpiration, and increasing water use efficiency [30]. TaGT2L1D, a transcriptional repressor, has similar effects to AtGTL1 in regulating drought resistance and stomatal aperture [31]. It could suppress the expression of AtSDD1 by directly binding to the GT-3 box in its promoter that negatively regulated drought resistance [31]. Overexpression of TaGT2L1D significantly increased stomatal density and reduced drought tolerance in GTL1-3 plants [31]. Conversely, plants overexpressing AtSDD1 gene exhibited reduced stomatal density, decreased transpiration, and improved drought tolerance [31]. Similarly, the PtaGTL1 gene in poplar (Populus L.) enhanced the drought resistance of transgenic plants by regulating leaf stomatal density [32].
A member of the cotton trihelix transcription factors family (GhGT23) positively regulates salt and drought stress responses, since overexpression of GhGT23 in Arabidopsis improved drought and salt tolerance [33]. Overexpression of Gh_A05G2067 (GT-2) showed enhanced drought tolerance with lower malondialdehyde (MDA), hydrogen peroxide contents and higher oxygen-scavenging enzyme activities in cotton [34]. Knocking down of ZmGT-3b led to enhanced drought tolerance in maize [24]. ShCIGT, a GT-1 subfamily trihelix transcription factor, mediates cold and drought tolerance in tomato [35]. SlGT-26 negatively regulates drought and salt stress resistance in tomato since SlGT-26-RNAi transgenic plants significantly enhanced drought resistance and salt tolerance [36]. SlGT-30 functions as a negative regulator of drought resistance as CRISPR-mediated loss of function mutant of SlGT-30 exhibited improved plant performance under drought stress in tomato [37].
Overall, trihelix transcription factors play a crucial role in regulating plant development and abiotic/biotic stress responses. Although many trihelix genes have been reported, their functions still require further investigation. In this study, we used bioinformatics to identify the members, physicochemical properties, classification, chromosomal localization, cis-acting elements, and differential expression analysis under drought stress of the trihelix family in maize. Moreover, ZmTHX15-overexpressing Arabidopsis exhibited greater drought tolerance compared to WT plants, with reduced secondary oxidative damage and enhanced photosynthetic rate. These findings provide a foundation for future research on the roles of ZmTHXs, which may be potential genetic markers for breeding stress-resistant and high-yielding maize varieties.

2. Results

2.1. Identification and Physicochemical Properties of ZmTHXs

To determine the composition and physicochemical properties of the ZmTHXs gene family, we identified and analyzed their members and properties. First, searches were conducted in the PlantTFDB and Uniprot databases, using the presence of the MYB binding 4 domain as the criterion for sequence alignment and selection. After removing redundant proteins or those lacking the typical domain, a total of 46 ZmTHXs family members were identified. Based on their chromosomal locations, these genes were sequentially named ZmTHX01 to ZmTHX46 (Table 1).
Physicochemical property analysis indicated that the 46 members of the ZmTHXs family ranged in size from 187 to 863 amino acids (aa), with an average of 421 aa. The molecular weight (MW) of these proteins ranged from 8,855.39 to 96,306.71 kDa. The theoretical isoelectric point (pI) ranged from 4.55 to 11.19, with 22 proteins having a pI above 7, categorizing them as basic proteins, while the remaining proteins had a pI below 7, making them acidic proteins. The instability index, an important indicator for measuring protein stability, ranges from 32.16 to 96.72, with proteins theoretically considered unstable if the index exceeds 40; all family proteins, except for ZmTHX15 and ZmTHX35, are unstable. The average hydropathicity index ranges from −1.259 to −0.296, and proteins with an index below 0 are generally hydrophilic, indicating that all family members are hydrophilic. Subcellular localization predictions showed that, except for ZmTHX30, which is localized to the chloroplast, all other members were localized to the nucleus. Although ZmTHXs vary greatly in the number of aa and pI values, they exhibit good similarity in hydrophilicity, instability index, and subcellular localization, indicating a high degree of sequence conservation within this protein family (Table 1).

2.2. Phylogenetic Analysis and Subfamily Classification of ZmTHXs

To better understand the subfamily classification and evolutionary relationships of THX family members, we performed sequence alignment on 27 Arabidopsis THX sequences, 31 rice THX sequences, and 46 maize THX sequences. We used MEGA X and iTOL for the construction and visualization of the phylogenetic tree. The analysis results showed that this family was divided into five subfamilies: GT-1, GT-2, GTγ, SH4, and SIP1 (Figure 1). The SIP1 subfamily had the most members, with 17, accounting for 36.9% of the total family members, while the GT-1 subfamily had the fewest members, with only three, accounting for 6.5% of the family members. The phylogenetic tree showed that the GT-1 and GTγ subfamily members were located on the same branch, indicating that these two subfamilies diverged later and have a closer evolutionary relationship. Each subfamily branch contained THX family proteins from both Arabidopsis and rice. Compared to dicotyledonous Arabidopsis, the THXs of maize and rice generally occupy lower nodes in the branches and converge with Arabidopsis sequences at higher nodes, suggesting a higher degree of homology between maize and rice as they are monocot.

2.3. Chromosomal Localization and Synteny Analysis of ZmTHXs

To determine the distribution of ZmTHXs on maize chromosomes, we conducted a chromosomal localization analysis of the family members. The results showed that the 46 ZmTHXs are unevenly distributed across 10 chromosomes in maize (Figure 2A). Among them, chromosome 2 had the highest number of ZmTHXs, with nine genes, accounting for 19.6% of the total family genes. Chromosomes 6 and 7 had the fewest, with only one gene each. The number of ZmTHXs on the other chromosomes ranged from two to eight. The ZmTHXs on chromosomes 6 and 7 were located downstream, while the ZmTHXs on chromosome 10 were distributed at both ends. The remaining ZmTHXs were mostly randomly distributed on various chromosomes (Figure 2B).
Using TBtools, combined with the multiple collinearity scanning toolkit (MCScanX), we analyzed tandem repeat events among ZmTHXs genes. The results showed that there are no tandem repeat events within this gene family in maize. By using TBtools in conjunction with BLASTP and MCScanX to analyze segmental duplication events, we identified a total of 23 gene pairs with segmental duplication events. The results indicated that segmental duplication events were a major driving force behind the diversity of ZmTHXs genes (Figure 3).

2.4. Analysis of Cis-Acting Elements in ZmTHXs

To explore the stress-responsive elements in this gene family, we performed a cis-acting element analysis on the 46 ZmTHXs of the family. As shown in the Figure 4, in addition to core enhancer elements, transcription initiation elements, and WRKY binding site elements, 16 types of other elements were identified (Figure 4). All ZmTHXs genes contained light-responsive elements and also included five types of phytohormone-related elements: Gibberellins (GA), Auxin (IAA), abscisic acid (ABA), salicylic acid (SA), and Methyl Jasmonate (MeJA) response elements. These cis-acting elements suggested that the expression of ZmTHXs was regulated by various phytohormones. Additionally, we found cis-acting elements related to abiotic stress in ZmTHXs, including anaerobic response elements (ARE), hypoxia-specific inducible elements, drought stress response elements (DRE and MBS), and low-temperature response elements (LTR). The results indicated that ZmTHXs may play an important role in stress response as well as in plant growth, development, and metabolic regulation.

2.5. Gene Expression Profile Analysis

To investigate the expression patterns of the ZmTHXs under drought stress, we constructed an expression heat map of ZmTHXs under drought conditions. The heat map results showed that most genes in this family responded to drought stress. Genes such as ZmTHX24, ZmTHX41, ZmTHX42, ZmTHX46, ZmTHX17, ZmTHX26, ZmTHX7, ZmTHX34, ZmTHX19, ZmTHX14, and ZmTHX43 had low expression levels under normal conditions, showed no significant change in expression level under mild drought (the soil moisture content is between 12% and 16%), but exhibited a significant increase in expression level under severe drought (the soil moisture content is between 6% and 8%) (Figure 5). Genes such as ZmTHX30, ZmTHX29, ZmTHX22, ZmTHX35, ZmTHX33, ZmTHX9, and ZmTHX28 had increasing expression levels with increasing drought severity (Figure 5). Genes such as ZmTHX39, ZmTHX3, ZmTHX6, ZmTHX31, ZmTHX2, ZmTHX37, ZmTHX12, and ZmTHX15 were relatively sensitive to drought stress and could be highly induced even under mild stress (Figure 5). Genes such as ZmTHX32, ZmTHX5, ZmTHX8, ZmTHX45, ZmTHX10, ZmTHX18, ZmTHX23, ZmTHX36, ZmTHX20, ZmTHX25, and ZmTHX01 had high expression levels under normal conditions but showed a significant decrease in expression level after drought treatment (Figure 5), possibly acting as negative regulators of stress signaling. In summary, most genes in this family showed significant changes in expression levels under drought treatment, suggesting that these genes were widely involved in drought stress responses.

2.6. ZmTHX15 Involved in Osmotic Stress Regulation

To verify the role of ZmTHXs under drought stress, we constructed ZmTHX15-overexpressing transgenic Arabidopsis. To investigate the growth of ZmTHX15-overexpressing transgenic plants under osmotic stress, we treated them with mannitol. The results showed that, whether grown on regular 1/2 MS medium or medium containing 100 mM mannitol, longer root lengths were exhibited in the ZmTHX15-overexpressing plants compared to the WT (Figure 6A). Root length statistics of seedlings grown for 6 days revealed that, regardless of the control or mannitol treatment, the root length of ZmTHX15-overexpressing plants was significantly longer than that of the WT (Figure 6B). The root length of WT under mannitol treatment was 66% of the control, while the root length of transgenic plants under mannitol treatment was 82% of the control, indicating that ZmTHX15-overexpressing plants were insensitive to mannitol treatment (Figure 6B). The results suggest that ZmTHX15 may play an important role in the regulation of osmotic stress.

2.7. ZmTHX15-Overexpressing Arabidopsis Is More Drought Tolerant than WT

To determine whether the ZmTHX15-overexpressing Arabidopsis are more drought-tolerant than WT, we subjected both WT and ZmTHX15-overexpressing Arabidopsis plants to soil drought treatment. After 10 days of drought treatment, the WT plants showed obvious wilting, while most of the ZmTHX15-overexpressing plants were still able to grow normally (Figure 7A,B). Survival rate statistics indicated that after 10 days of drought treatment, the survival rate of WT plants was only about 22%, while the survival rate of ZmTHX15-overexpressing plants was as high as 87%, significantly higher than that of the WT (Figure 7C).
Aseorbate peroxidase (APX) and glutathione reductase (GR) are key enzymes in plants responsible for scavenging excess H2O2 and mitigating stress-induced damage. Under drought stress, plants with strong drought tolerance exhibit higher APX and GR activity compared to drought-sensitive plants. Ascorbic acid (AsA) is a non-enzymatic free radical scavenger in plants that effectively eliminates ROS generated by stress, thereby alleviating oxidative damage in plants. Based on this, we measured the activity of APX and GR, as well as the AsA content, in both WT and ZmTHX15-overexpressing plants before and after drought treatment. The results showed that before drought treatment, there were no significant differences in APX, GR, and AsA levels between WT and ZmTHX15-overexpressing plants. However, after drought treatment, the ZmTHX15-overexpressing plants exhibited higher APX and GR enzyme activities and accumulated more AsA (Figure 7D–F).
To investigate the oxidative damage experienced by ZmTHX15-overexpressing plants under drought stress, we measured the MDA content. The results indicated that the MDA content accumulated in the ZmTHX15-overexpressing Arabidopsis was lower than that in the WT, suggesting that the ZmTHX15-overexpressing plants experienced less oxidative damage under drought stress compared to the WT (Figure 7G).
To explore whether there are differences in photosynthetic efficiency between the ZmTHX15-overexpressing and WT plants before and after drought, we measured the photosynthetic capacity of both groups. Chlorophyll fluorescence measurements showed that the fluorescence index decreased in both WT and ZmTHX15-overexpressing plants after drought treatment, but the decrease was less pronounced in the ZmTHX15-overexpressing plants, and the fluorescence index was significantly higher than that of the WT (Figure 7H). Chlorophyll content measurements indicated that the chlorophyll content in the leaves of ZmTHX15-overexpressing plants was higher than that of WT after drought treatment (Figure 7I), consistent with the chlorophyll fluorescence index results.
When measuring photosynthetic rate and intercellular CO2 concentration, we found that before drought treatment, the CO2 concentration in the ZmTHX15-overexpressing plants was slightly higher than in the WT. After drought treatment, the situation was reversed: the intercellular CO2 concentration in the ZmTHX15-overexpressing plants decreased, while it significantly increased in the WT (Figure 7J), indicating excessive CO2 accumulation and a significant reduction in photosynthetic efficiency in WT. The results suggest that under drought stress, the ZmTHX15-overexpressing plants can continue to utilize intercellular CO2 for photosynthesis.
The net photosynthetic rate measurements showed that both WT and ZmTHX15-overexpressing plants experienced a decline in photosynthetic rate after drought treatment, but the decline was less severe in the ZmTHX15-overexpressing plants, and the photosynthetic rate remained significantly higher than that of WT (Figure 7K). These results indicate that ZmTHX15-overexpressing plants can better utilize intercellular CO2 under drought conditions, maintaining higher photosynthetic efficiency, which supports the normal growth under drought stress.
In summary, through experimental analysis, we found that ZmTHX15-overexpressing Arabidopsis exhibit stronger drought tolerance. Under drought conditions, the ZmTHX15-overexpressing plants suffered less secondary oxidative damage compared to the WT. The osmotic substances and antioxidants in the ZmTHX15-overexpressing plants worked together under drought stress to mitigate the damage caused by the stress, thereby maintaining a higher photosynthetic rate.

3. Discussion

In recent years, the frequent occurrence of high temperatures, low rainfall, and drought has led to significant crop yield reductions worldwide. Therefore, understanding the mechanisms of plant drought stress responses has become a major focus in the field of stress research [38]. Studies have shown that osmotic regulation, antioxidant defense systems, transcription factor regulation, and signaling pathways such as ABA play crucial roles in drought stress responses [39,40,41].
Maize, as one of the three major cereal crops, is increasingly in demand due to the growing focus on maize production and development. Therefore, research aimed at identifying key transcription factors based on maize genomic information databases is of significant scientific and practical value for understanding maize genomic information and improving maize quality.
Transcription factors are abundantly present in plants, with 56 transcription factor families and a total of 2298 members identified in maize [42]. Research has shown that the Arabidopsis genome contains more than 1500 transcription factors, accounting for over 5% of the genome [43]. Since transcription factors exert their regulatory functions by interacting with specific DNA sequences, understanding the sequences they recognize is crucial for comprehending the functions and regulatory mechanisms.
There are various transcription factor families in higher plants, most of which have been proven to be associated with plant stress responses and quality trait improvement. Transcription factors that regulate responses to drought, cold, and salinity stress have also been cloned [44,45,46]. Research on the trihelix transcription factor family in plants is an emerging field. This family has gained significant attention due to its extensive involvement in plant growth, development, and responses to various biotic and abiotic stresses. Previous studies have analyzed the trihelix transcription factor families in many plants, such as rice, wheat, maize, alfalfa, tomato, sesame, quinoa, buckwheat, and sorghum [47,48,49,50,51,52,53,54,55]. However, systematic studies on the evolution and protein structure of the maize trihelix family have not yet been fully conducted. Therefore, this study utilized the latest databases to perform a preliminary bioinformatics analysis of the maize trihelix transcription factor family. Compared to previous analyses of the trihelix transcription factor families in plants like Arabidopsis and rice, this study identified a larger number of gene family members and included additional data analysis at the gene structure and protein levels. This work contributes to the further understanding of the transcription factor family information in maize.
In this study, we conducted a phylogenetic analysis of maize trihelix transcription factor family members using bioinformatics methods. The results revealed that 46 trihelix transcription factors were identified in maize, and the family was divided into five subfamilies: GT-1, GT-2, GTγ, SIP1, and SH4, with each subfamily containing a different number of members. The GTγ subfamily has the fewest members, while the SIP1 subfamily has the most. The family members are unevenly distributed across the 10 chromosomes, and some genes exhibit gene clustering on the chromosomes. The family contains a variety of conserved motifs, and the differences in these motifs suggest the diversity of gene functions. Members of the same subfamily have similar structural characteristics. These results indicate that the maize genome may have undergone changes during evolutionary development. By predicting the biological functions of the trihelix family, the basic structure and functions of the family members were predicted, providing an important theoretical basis for future research on the role of monocot gene families in quality improvement and stress response.

4. Materials and Methods

4.1. Identification and Physicochemical Properties of ZmTHXs

The maize genome sequence, CDS sequence, genome annotation files, and protein sequences were downloaded from the EnsemblPlants database. Using the MYB binding 4 domain as a basis for searches in PlantTFDB and UniProt, the obtained sequences for domains in the Pfam, SMART, and NCBI-CDD databases were validated. After removing sequences lacking relevant domains and redundancies, 46 sequences were ultimately identified. The ExPASy (https://www.expasy.org, accessed on 7 December 2024) [56] tool was used to calculate properties such as pI, MW, instability index, and average hydrophobicity of the trihelix family genes. Subcellular localization was predicted using the Plant-mPLoc 2.0 tool (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/, accessed on 7 December 2024) [57].

4.2. Phylogenetic Analysis of the ZmTHXs Gene Family

Multiple sequence alignments were performed using the ClustalW tool (https://www.genome.jp/tools-bin/clustalw, accessed on 7 December 2024) [58]. The phylogenetic tree of THX protein sequences of maize, rice, and Arabidopsis was constructed using MEGAX [59] software with the Neighbor-Joining (NJ) method and 1000 bootstrap replicates. The constructed tree was imported into the iTOL v7 online software (http://itol.embl.de/, accessed on 7 December 2024) for visualization.

4.3. Cis-Acting Element Analysis of ZmTHXs

The 2,000 bp sequence upstream of THX genes start codon was extracted using TBtools [60,61]. Cis-element analysis of the promoter regions was performed using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 7 December 2024) [62]. After organizing and adjusting the analysis results, they were visualized using TBtools.

4.4. Chromosomal Localization and Synteny Analysis of ZmTHXs

The chromosomal location information of ZmTHXs genes was obtained from the maize genome annotation file, and TBtools 1.120 was used for the chromosomal localization visualization. Synteny analysis was performed using the MCScanX tool (https://github.com/wyp1125/MCScanX, accessed on 7 December 2024) [63], and the results were visualized with TBtools.

4.5. Differential Expression Analysis of ZmTHXs Under Drought Stress

The specific expression data of relevant genes was downloaded from the MaizeGDB-qteller database, and the gene-specific expression heat map was created using TBtools.

4.6. Gene Cloning

Target gene sequences were searched on the NCBI (http://www.ncbi.nlm.nih.gov, accessed on 7 December 2024) website. Primers were designed to obtain its complete cDNA using Polymerase Chain Reaction (PCR) and then cloned into the vector pcambimGFP-1302 for sequencing. Primers are listed in Table S1.

4.7. Generation of Transgenic Plants

The genetic transformation of Arabidopsis thaliana was based on the Agrobacterium-mediated floral dip method [64]. After the T1 transgenic plants were obtained, we identified the positive plants by PCR using primers: a 35S sequence primer as the forward primer and a gene-specific primer as the reverse primer. A minimum of 12 positive transgenic lines were identified [65]. Primers are listed in Table S1.

4.8. Mannitol Treatment of Transgenic Arabidopsis

Sow the sterilized WT and transgenic Arabidopsis seeds separately on 1/2 MS medium containing 100 mmoL mannitol and 1/2 MS medium without mannitol. Vernalize them at 4 °C for 3 days, then transfer them to an incubator set at 22 °C with a 16/8 h light/dark cycle. Observe their phenotype daily and measure their root length using Image J (https://imagej.net/ij/, accessed on 7 December 2024) during this period [66].

4.9. Measurement of Chlorophyll Content

Take 0.2 g of fresh leaves and place them in a mortar, add 95% ethanol, and grind thoroughly. Let it stand for 5 min and then transfer the grinding solution to a 25 mL centrifuge tube. Centrifuge at 4000 rpm for 10 min and take the supernatant as the extract. Using ethanol as the control, measure the optical density (OD) values at wavelengths of 649 nm and 665 nm. Calculate the chlorophyll content using the following formulas:
Ca = 13.95A665 − 6.88A649, Cb = 24.96A649 − 7.32A665 (Ca, Cb: mg/L)
Chlorophyll content (mg/g) = (C × V)/Wf
C = Ca + Cb,
V is the volume of the extract, and Wf is the fresh weight of the tissue.

4.10. Measurement of Photosynthetic Rate (Pn) and Intercellular CO2 Concentration

On a clear morning, use the LI-6400XT photosynthesis system with a red/blue light source to measure the net photosynthetic rate (Pn, μmol·m−2·s−1) and intercellular CO2 concentration (Ci, μmol/mol) of the leaves.

4.11. Chlorophyll Fluorescence Measurement

Use the FluorCam 800MF chlorophyll fluorescence imaging system from Czech PSI to detect the Fv/Fm of the leaves. Open the FluorCam 800MF software on the computer, adjust the focus in the live window, select “Flashes” for the “light sources” parameter, set “act1” and “act2” to 0, and “super” to 24. Adjust the EI. Shutter to “10” and the “sensitivity” to “40”. Click “use”. After selecting the machine’s preset program, you can start the measurement. Record and save the results after the measurement is completed.

4.12. APX and GR Enzyme Activity

Weigh 0.1 g of Arabidopsis leaves for each sample. Use the APX Activity Assay Kit (Catalog No.: BC0220) and the GR Activity Assay Kit (Catalog No.: BC1160) from Solarbio to measure APX and GR activities, respectively. Follow the instructions in the manual for the detection steps. Perform three biological replicates for each group. Enzyme activity is expressed in units (U).

4.13. Determination of AsA and MDA Content

Weigh 0.1 g of Arabidopsis leaves for each sample. Use the AsA Content Assay Kit (Catalog No.: BC1230) and the MDA Content Assay Kit (Catalog No.: BC0020) from Solarbio for detection, following the steps in the manual. Perform three biological replicates for each group. AsA content is expressed in μmol·g-1, and MDA content is expressed in mmol·g−1.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms252413257/s1.

Author Contributions

Conceptualization, Y.C. and Z.C.; Methodology, X.W.; Validation, X.S., M.Z., and L.Y.; Data curation, L.Y.; Writing—original draft preparation, X.W and J.Z.; Writing—review and editing, H.L. and J.Z.; Supervision, Y.C.; Project administration, Y.C. and C.D.; Funding acquisition, Y.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the key Research Projects of Higher Education Institutions of Henan Province (25A180015 and 24A180011), the key Research and Development Program of Henan Province (241111112300), and the funding of Hainan Seed Industry Laboratory.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Analysis of the evolutionary relationship of THXs in maize, rice, and Arabidopsis.
Figure 1. Analysis of the evolutionary relationship of THXs in maize, rice, and Arabidopsis.
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Figure 2. The location of ZmTHXs on chromosomes. (A) Distribution of ZmTHXs on chromosomes; (B) The number of ZmTHXs on each chromosome.
Figure 2. The location of ZmTHXs on chromosomes. (A) Distribution of ZmTHXs on chromosomes; (B) The number of ZmTHXs on each chromosome.
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Figure 3. ZmTHXs collinearity analysis. Heat maps and histograms along the rectangles represent gene densities on chromosomes. Gray lines indicate syntenic blocks in the poplar genome, and red lines between chromosomes indicate gene pairs with segmental duplications.
Figure 3. ZmTHXs collinearity analysis. Heat maps and histograms along the rectangles represent gene densities on chromosomes. Gray lines indicate syntenic blocks in the poplar genome, and red lines between chromosomes indicate gene pairs with segmental duplications.
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Figure 4. Analysis of cis-acting regulatory elements in ZmTHXs. The key cis-acting regulatory elements are distributed in the 2000 bp region upstream of the ZmTHXs, and different elements are shown in different colors.
Figure 4. Analysis of cis-acting regulatory elements in ZmTHXs. The key cis-acting regulatory elements are distributed in the 2000 bp region upstream of the ZmTHXs, and different elements are shown in different colors.
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Figure 5. Heat map of differential expression of ZmTHXs. Drought treatment time is 6 h. Different-colored rectangles on the right side of the evolutionary tree represent genes of different subfamilies.
Figure 5. Heat map of differential expression of ZmTHXs. Drought treatment time is 6 h. Different-colored rectangles on the right side of the evolutionary tree represent genes of different subfamilies.
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Figure 6. Phenotype of transgenic Arabidopsis. (A) The phenotypes of WT and ZmTHX15-overexpressing transgenic Arabidopsis line 1 and line 2 (OE-1, OE-2) treated with/without 100 mmol/L mannitol after 6 days of germination; (B) The root-length statistics of WT, OE-1, and OE-2 treated with/without 100 mmol/L mannitol after 6 days of germination. Data represent the mean ± SD of three biological repeats, and asterisks indicate significant differences among groups (p < 0.05). Bar, 1 cm.
Figure 6. Phenotype of transgenic Arabidopsis. (A) The phenotypes of WT and ZmTHX15-overexpressing transgenic Arabidopsis line 1 and line 2 (OE-1, OE-2) treated with/without 100 mmol/L mannitol after 6 days of germination; (B) The root-length statistics of WT, OE-1, and OE-2 treated with/without 100 mmol/L mannitol after 6 days of germination. Data represent the mean ± SD of three biological repeats, and asterisks indicate significant differences among groups (p < 0.05). Bar, 1 cm.
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Figure 7. ZmTHX15-overexpressing Arabidopsis is more drought tolerant than WT. (A) Phenotype of WT, OE-1, and OE-2 plants transplanted for 10 days under normal watering; (B) Phenotype of WT, OE-1, and OE-2 plants after 10 days of drought treatment; (C) Plant survival rate after drought treatment; (D) Ascorbate peroxidase activity; (E) glutathione reductase activity; (F) ascorbic acid content; (G) malondialdehyde content; (H) chlorophyll fluorescence index; (I) chlorophyll content; (J) intercellular CO2 concentration; (K) net photosynthetic rate. The data represent the mean ± SD of three biological repeats, and the letters represent significant differences among groups (p < 0.05). Bar, 1 cm.
Figure 7. ZmTHX15-overexpressing Arabidopsis is more drought tolerant than WT. (A) Phenotype of WT, OE-1, and OE-2 plants transplanted for 10 days under normal watering; (B) Phenotype of WT, OE-1, and OE-2 plants after 10 days of drought treatment; (C) Plant survival rate after drought treatment; (D) Ascorbate peroxidase activity; (E) glutathione reductase activity; (F) ascorbic acid content; (G) malondialdehyde content; (H) chlorophyll fluorescence index; (I) chlorophyll content; (J) intercellular CO2 concentration; (K) net photosynthetic rate. The data represent the mean ± SD of three biological repeats, and the letters represent significant differences among groups (p < 0.05). Bar, 1 cm.
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Table 1. Physicochemical properties of ZmTHXs identified in maize.
Table 1. Physicochemical properties of ZmTHXs identified in maize.
GenesGene IDAmino AcidsMW (KDa)pIInstability IndexGRAVYSubcellular Localization
ZmTHX01Zm00001eb00088080984,901.286.5569.6−0.736Nucleus
ZmTHX02Zm00001eb01359037740,843.310.1257.94−0.649Nucleus
ZmTHX03Zm00001eb02387032134,106.449.4174.38−0.567Nucleus
ZmTHX04Zm00001eb03357031935,873.995.675.37−0.966Nucleus
ZmTHX05Zm00001eb04358066970,672.049.2263.48−0.734Nucleus
ZmTHX06Zm00001eb06550020422,859.119.3841.64−0.412Nucleus
ZmTHX07Zm00001eb06677038140,870.939.3880.33−0.64Nucleus
ZmTHX08Zm00001eb07178027332,311.318.3762.18−1.259Nucleus
ZmTHX09Zm00001eb07508020822,949.1611.0996.5−0.682Nucleus
ZmTHX10Zm00001eb07525077483,274.25.973.29−0.882Nucleus
ZmTHX11Zm00001eb08172038540,978.966.6864.43−0.783Nucleus
ZmTHX12Zm00001eb08430035437,623.598.0841.35−0.716Nucleus
ZmTHX13Zm00001eb08486034837,680.898.6952.69−0.773Nucleus
ZmTHX14Zm00001eb09204043649,833.696.3253.26−1.023Nucleus
ZmTHX15Zm00001eb10464035738,923.088.2332.16−0.642Nucleus
ZmTHX16Zm00001eb1291907888,55.3910.0153.05−0.553Nucleus
ZmTHX17Zm00001eb13074053757,507.767.163.71−0.731Nucleus
ZmTHX18Zm00001eb14260029532,148.048.643.47−0.617Nucleus
ZmTHX19Zm00001eb15708031834,724.079.5970.81−0.75Nucleus
ZmTHX20Zm00001eb18080034136,829.895.2761.81−0.743Nucleus
ZmTHX21Zm00001eb18176039845,440.355.9749.73−0.814Nucleus
ZmTHX22Zm00001eb18620066871,709.65.7855.45−0.758Nucleus
ZmTHX23Zm00001eb19911026228,997.619.4964.97−0.834Nucleus
ZmTHX24Zm00001eb20241042648,757.536.6455.07−1.055Nucleus
ZmTHX25Zm00001eb20988071476,269.475.9356.8−0.862Nucleus
ZmTHX26Zm00001eb21778036039,267.536.264.99−0.882Nucleus
ZmTHX27Zm00001eb22025077782,583.326.1157.98−0.827Nucleus
ZmTHX28Zm00001eb22940047651,138.974.9859.64−0.848Nucleus
ZmTHX29Zm00001eb23335038740,507.554.5566.65−0.522Nucleus
ZmTHX30Zm00001eb23462087496,306.718.9640.31−0.296Chloroplast
ZmTHX31Zm00001eb24203034937,637.715.0564.55−0.662Nucleus
ZmTHX32Zm00001eb24326040546,187.925.9147.34−0.896Nucleus
ZmTHX33Zm00001eb24890067272,812.615.8751.7−0.865Nucleus
ZmTHX34Zm00001eb29570034336,918.889.9162.4−0.57Nucleus
ZmTHX35Zm00001eb32047033736,717.56.9437.51−0.631Nucleus
ZmTHX36Zm00001eb34055053357,191.26.0957.65−0.727Nucleus
ZmTHX37Zm00001eb36012031534,672.999.8770.51−0.824Nucleus
ZmTHX38Zm00001eb36159035038,799.789.774.3−0.847Nucleus
ZmTHX39Zm00001eb39091031833,599.959.1770.04−0.458Nucleus
ZmTHX40Zm00001eb39691025127,105.296.2761.82−0.65Nucleus
ZmTHX41Zm00001eb40769044650,509.256.1146.7−1.011Nucleus
ZmTHX42Zm00001eb42138033436,440.245.8951.63−0.794Nucleus
ZmTHX43Zm00001eb42333039242,106.226.6166.72−0.846Nucleus
ZmTHX44Zm00001eb42523037941,935.716.2848.46−0.727Nucleus
ZmTHX45Zm00001eb42763061666,455.629.3471.11−0.931Nucleus
ZmTHX46Zm00001eb42779020622,628.811.1996.72−0.657Nucleus
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MDPI and ACS Style

Cao, Y.; Cheng, Z.; Sun, X.; Zhu, M.; Yue, L.; Liu, H.; Wu, X.; Zhang, J.; Duan, C. Genome-Wide Identification of the Trihelix Transcription Factor Family and Functional Analysis of ZmTHX15 in Maize. Int. J. Mol. Sci. 2024, 25, 13257. https://doi.org/10.3390/ijms252413257

AMA Style

Cao Y, Cheng Z, Sun X, Zhu M, Yue L, Liu H, Wu X, Zhang J, Duan C. Genome-Wide Identification of the Trihelix Transcription Factor Family and Functional Analysis of ZmTHX15 in Maize. International Journal of Molecular Sciences. 2024; 25(24):13257. https://doi.org/10.3390/ijms252413257

Chicago/Turabian Style

Cao, Yanyong, Zeqiang Cheng, Xinyan Sun, Meichen Zhu, Ling Yue, Hui Liu, Xiaolin Wu, Jinghua Zhang, and Canxing Duan. 2024. "Genome-Wide Identification of the Trihelix Transcription Factor Family and Functional Analysis of ZmTHX15 in Maize" International Journal of Molecular Sciences 25, no. 24: 13257. https://doi.org/10.3390/ijms252413257

APA Style

Cao, Y., Cheng, Z., Sun, X., Zhu, M., Yue, L., Liu, H., Wu, X., Zhang, J., & Duan, C. (2024). Genome-Wide Identification of the Trihelix Transcription Factor Family and Functional Analysis of ZmTHX15 in Maize. International Journal of Molecular Sciences, 25(24), 13257. https://doi.org/10.3390/ijms252413257

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