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Article

DNA-Binding with One Finger (Dof) Transcription Factor Gene Family Study Reveals Differential Stress-Responsive Transcription Factors in Contrasting Drought Tolerance Potato Species

by
Xin Jin
1,2,†,
Zemin Wang
1,2,†,
Qianyi Ai
2,
Xuan Li
2,
Jiangwei Yang
1,2,
Ning Zhang
1,2 and
Huaijun Si
1,2,*
1
State Key Laboratory of Aridland Crop Science, Gansu Agricultural University, Lanzhou 730070, China
2
College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, China
*
Author to whom correspondence should be addressed.
†
These authors have contributed equally to this work.
Int. J. Mol. Sci. 2024, 25(6), 3488; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms25063488
Submission received: 21 February 2024 / Revised: 13 March 2024 / Accepted: 15 March 2024 / Published: 20 March 2024
(This article belongs to the Special Issue Advances in Molecular Plant Sciences)
Browse Figures
Versions Notes

Abstract

:
DNA-binding with one finger (Dof) proteins comprise a large family that play central roles in stress tolerance by regulating the expression of stress-responsive genes via the DOFCORE element or by interacting with other regulatory proteins. Although the Dof TF has been identified in a variety of species, its systemic analysis in potato (Solanum tuberosum L.) is lacking and its potential role in abiotic stress responses remains unclear. A total of 36 potential Dof genes in potato were examined at the genomic and transcriptomic levels in this work. Five phylogenetic groups can be formed from these 36 Dof proteins. An analysis of cis-acting elements revealed the potential roles of Dofs in potato development, including under numerous abiotic stress conditions. The cycling Dof factors (CDFs) might be the initial step in the abiotic stress response signaling cascade. In potato, five CDFs (StCDF1/StDof19, StCDF2/StDof4, StCDF3/StDof11, StCDF4/StDof24, and StCDF5/StDof15) were identified, which are homologs of Arabidopsis CDFs. The results revealed that these genes were engaged in a variety of abiotic reactions. Moreover, an expression analysis of StDof genes in two potato cultivars (‘Long10′ (drought tolerant) and ‘DXY’ (drought susceptible)) of contrasting tolerances under drought stress was carried out. Further, a regulatory network mediated by lncRNA and its target Dofs was established. The present study provides fundamental knowledge for further investigation of the roles of Dofs in the adaptation of potato to drought stress, aiming to provide insights into a viable strategy for crop improvement and stress-resistance breeding.

1. Introduction

Transcription factors (TFs) are essential components of many regulatory and signaling networks in plants [1,2], which have been proven to be pivotal in plants’ responses to abiotic stress, including drought, and low and high temperatures. The survival, growth, and reproduction of plants are impacted by adverse climatic factors such as drought and extreme temperatures. Under the control of a vast array of stress-responsive genes and a sophisticated network of TFs, plants have evolved a variety of biochemical and physiological strategies to maintain growth and cell integrity in adverse environmental conditions. DNA-binding with one finger (Dof) proteins are a group of plant-specific TFs [3]. Following the isolation of the first Dof gene (Zmdof1) from maize, additional Dof genes have been identified in other plants and are the subject of extensive research [4,5,6,7,8]. In N-terminal regions, the Dof proteins contain a highly conserved Dof domain (about 52 amino acids with a Cys2/Cys2 zinc finger structure). T/AAAAG is a common core sequence of the Dof domain recognized in the promoters of target genes. Dof proteins also contain a C-terminal with a transcriptional activation function. Interestingly, the Dof domain can mediate not only protein–DNA interactions, but also protein–protein interactions [9]; for instance, Dof-OBPl (for OBF-binding protein) [10], Dof-bZIP [11], and Dof-MYB [12] have been characterized. At the same time, the conserved motif at the C-terminal of some Dof proteins also plays an important role in specific protein–protein interactions. For example, cycling Dof factor (CDF) proteins often play a regulatory role by interacting with post-translational regulatory proteins (such as the clock gene GIGANTEA (GI) and flavin binding kelch repeat F-box protein 1 (FKF1)) via a special motif in Dof proteins [6,13,14]. In addition, small ubiquitin-like modifier (SUMO) targets ubiquitin ligase (AT-STUbL4) and induces ubiquitination degradation of CDF2 by binding to it, thereby increasing CO mRNA levels and promoting flowering during the photoperiod [15]. These findings indicate that Dof TFs play a regulatory role in multiple dimensions by interacting with TFs and other proteins.
It has been demonstrated that Dofs are involved in the control of gene expression in a variety of physiological processes, including seed germination [16], flowering [17,18], root growth [19], light-mediated regulation [20], and hormone responses [21]. Dof TFs also play essential roles in the reaction to abiotic and biotic stresses, including drought [22,23], salt [24,25], extreme temperature [26,27], and pathogens [28]. The rose genome contains a total of 24 Dof genes, the majority of which have shown elevated expression levels in response to salt (in 21 cases) and drought (19 cases) stress conditions, highlighting the critical role of Dof genes in abiotic stress tolerance [29]. In walnut, JrDof3 directly regulates JrGRAS2, which regulates the expression of HSPs to improve heat resistance [30]. Wang et al. [8] isolated 11 VaDof genes (from a total of 25), which responded to cold stress in grape plants, and VaDof17d, which enhanced the cold tolerance of grape callus by increasing raffinose synthesis. Moreover, the C-repeat Binding Factor/Dehydration-Responsive Element (DRE) Binding Factor (CBF/DREB) transcriptional regulatory network plays a key role in plant abiotic stress. Recent results indicate that CDF plays a role in developmental processes and stress responses by participating in the GI pathway to regulate CBF regulators [31,32,33,34]. In Arabidopsis, AtCDF3 overexpression enhanced the abiotic stress tolerance of transgenic plants and promoted late flowering by direct control of the GI-CDF module through regulating CBFs, DREB2A, and others, also including the GI-independent pathway [32,33]. The Arabidopsis CDF3 T-DNA insertion mutant cdf3-1 was much more sensitive to drought and low temperature stress, whereas CDF3 overexpression enhanced the tolerance to various abiotic stresses (drought, cold, and osmotic stress) of transgenic plants by regulating the CBF/DREB and ZAT10/12 pathways [28]. Dof proteins also play important roles in crop senescence, C/N regulation, photosynthesis regulation, and chloroplast development [6,16,35,36]. Some Dof TFs regulate both abiotic stress resistance and important agronomic traits in crops, such as flowering time and abiotic stress (TDDF1 and SlCDF3) in tomato [32,33,37], biomass and abiotic stress in tomato [33], and GhDof1 improved salt and cold tolerance and seed oil content in Gossypium hirsutum [38]. It is extremely important to harmonize and unify crop yield increases with the development of stress resistance and agronomic improvement.
Potato (Solanum tuberosum L.) is an important non-grain food crop worldwide, which is commonly sensitive to abiotic stress (drought, salinity, and heat, etc.). With increasing global climate change, there are further requirements for the study of potato abiotic resistance, which is a limiting factor affecting potato yield and quality. Finding and researching new abiotic resistance genes is necessary for a better understanding of the control mechanisms of resistance, as well as for future abiotic resistance breeding in potatoes. Despite the importance of Dofs in plant abiotic stress resistance, little is known about the function of Dofs in potato abiotic stress responses. StCDF1, a member of potato Dof TF, acted as a mediator between the circadian clock and the StSP6A mobile tuberization signal to regulate tuberization and potato life cycle length [14]. Recently, a study demonstrated that StCDF1, together with a long non-coding RNA (lncRNA) counterpart named StFLORE, also regulates water loss by affecting stomatal development and diurnal opening [14,39]. This study includes genome-wide identification of the putative StDof gene family in potato, the construction of a phylogenetic tree, and the annotation of the chromosomal position of these genes. We assessed the transcript abundance of the StDof genes in various abiotic conditions using whole-transcriptome data and qRT-PCR. Additionally, the expression profiles of specific Dof genes under drought stress were examined. The structure, chromosomal locations, and evolutionary relationships of the Dof gene family in the potato genome were also carefully examined. The expression profiles of the StDofs in various tissues and stress settings were analyzed to determine whether stress affected the expression levels of the Dof genes in potato. Moreover, a regulatory network mediated by lncRNA and its target Dofs was established. The findings offer baseline knowledge of an important gene family in potato species, which will form the basis for further functional analyses and for breeding stress-tolerant potato.

2. Results

2.1. Identification and Phylogenetic Analysis of Dof Genes in Potato

A total of 36 Dof genes were identified in S. tuberosum by combining HMM (e-value < 0.01) scan, BLAST (e-value < 1 × 10−5 search), and CDD search method. Except for StDof1 and StDof27, the rest of the Dof genes’ accession numbers were obtained from GenBank. The 36 Dof genes in potato showed a variance in gene length, ranging from 498 bp (StDof5) to 1512 bp (StDof15) (Table 1).
Based on the conserved domain, the StDofs can be divided into five groups (Groups A–F, see Figure 1), with Group E being the largest group (containing 13 members) and Group D being the smallest (having only three members). However, Group B was not found in S. tuberosum. These outcomes align with those found in the grapevine.

2.2. Gene Structure, Chromosome Localization, and Duplication Analysis of StDof Genes

The exon–intron organization was previously known to play critical roles in the evolution of several gene families. GSDS 2.0 (http://gsds.gao-lab.org/, accessed on 5 September 2022) was used to generate the exon–intron structure of 36 StDof genes. The gene structure (exon–intron) analysis revealed that StDofs had a maximum of four exons (Figure 2A). Members of Groups A and F had two exons and one intron (except StDof4, five genes in Group A and StDof7 in Group F, which only had one exon), whereas members of Group C had three exons and two introns (Figure 2A). In Group E, there were four StDof genes containing one exon, six StDof genes containing two exons, two genes containing three exons (StDof30 and StDof33), and only one StDof gene containing four exons. Furthermore, only one exon existed in all the members of Group D. Group E member StDof22 contained four exons (Figure 2A). These results indicated that StDofs with a similar number of introns are highly conserved.
The 36 StDof genes were distributed on ten chromosomes except for chromosomes 7 and 12 (Figure 3; Table 1). The largest number of StDof genes were located on chromosome 2, with a total of nine StDof genes. Chromosomes 4, 5, 8, and 10 each included two StDof genes, while chromosome 9 had just one StDof gene. The results of the duplication analysis of StDofs revealed that 28 StDofs were engaged in 20 segmental duplication events, and these genes were found in all five groups (A, C–F). Aside from segmental duplication, no tandem duplication events were discovered, and eight members of the StDof gene were not detected in the duplication events. StDof1 and StDof7 were discovered to be involved in two tandem duplication events. Only one member (StDof1) was involved in both segmental and tandem duplication (Figure 3). These results suggested that segmental duplication may have played the dominant role in the expansion of the potato Dof gene family.

2.3. Protein Structure and Conserved Motifs of the StDofs

Ten conserved motifs were identified by the MEME program with unique phylogenetic distributions (Figure 2B). The StDof proteins exhibited a variable number of motifs, ranging from 1 to 7, with a length spanning between 15 and 50 amino acids. Except for StDof5, all members of Group A contained seven motifs. Motifs 2, 5, and 7–9 were only present in five members of Group A (Figure 1 and Figure 2B; Supplementary Table S1). By contrast, one motif (Motif 1, Dof domain) can be detected in all 36 StDofs. Similar to other plants, the five CDFs belong to Group A and contain the most conserved motifs.

2.4. Profiling of StDof Gene Expression in Different Organs/Tissues

By using RNA-seq data (DM 1-3 516 R44-Gene Expression Matrix (TPM)-v6.1), the expression levels of StDof genes in 14 different organs/tissues (stamen, flower, mature tuber, young tuber, tuber pith, tuber peel, tuber cortex, shoot apex, stem, petiole, and root) were examined. The results displayed diverse response patterns in different tissues. The StDofs can be detected in different tissues and organs, and the expression levels differ in various organs/tissues (Figure 4). For example, the highest expression levels of six genes (StDof15, StDof19, StDof20, StDof23, StDof24, and StDof33) were detected in the leaf, and the other 23 StDof genes were highly expressed in the root. The mRNA levels of StDof17 and StDof33 were particularly high in flowers, while the expression level of StDof4 was found to be higher in mature tubers.

2.5. Analysis of Cis-Acting Elements in Promoter

The cis-acting elements within the region of a 1.0 kb sequence upstream from each gene’s ATG site were analyzed using PlantCARE to get a better understanding of the putative regulation mechanism (stress/hormone-related) of StDofs. Except for the basic gene expression control elements (CAAT and TATA), 12 groups were divided according to their biological function (Figure 5; Table S2). A variety of hormonal response-related cis-acting elements, such as ABA, MeJA, GA, SA and Auxin, have been discovered. ABA and MeJA are frequently involved in plant stress resistance. Drought- or low-temperature-responsive elements, such as the MYB binding site involved in drought-inducibility (MBS) and low temperature-responsive (LTR) elements. The MYB element was found in StDof4/7/9/10/18/19/23. The LTR element was detected in the promoters of StDof2/5/14/22/25. Other abiotic stress-responsive elements, such as the dehydration-responsive element (DRE), MBS, TC-rich repeats, F-box, ARE and MYC, were also detected in StDof gene promoters.

2.6. Expression Analysis of StDof Genes under Abiotic/Biotic Stresses and Hormone Treatment

The functions of the StDof genes in response to abiotic stress remain mostly unclear. To investigate the function of StDof genes, their expression patterns under abiotic conditions (salt stress, 150 mM NaCl; heat stress, 35 °C; mannitol, 24 h of 260 µM), biotic stress (BABA and BTH treated 24 h, respectively; P. infestans infected leaves 24 h), and hormone treatment (ABA, IAA, GA3, and BAP treated 24 h of 50, 10, 50, and 10 μM, respectively) (DM 1-3 516 R44-Gene Expression Matrix TPM-v6.1) were assessed (Figure 6). The results showed that most StDofs genes responded to abiotic stress. Under salt and heat stress, the transcript abundance of eight (StDof7/9/13/17/21/26/28/33) and eleven (StDof2/4/7/11/13/17/19/24/28/29/31) Dof genes increased. In contrast, salt stress decreased the expression levels of StDof6 and StDof12, while under heat stress, the expression levels of StDof15 and StDof34 decreased (Figure 6). After being treated with hormones (24 h of 50 μM ABA, 50 μM GA3, 10 μM IAA, and 10 μM BAP), the genes exhibited distinct patterns of expression in response to each specific hormonal treatment. Both StDof16 and StDof31 exhibited increased expression levels in all hormone treatments, while the expression levels of StDof1 and StDof6 were down-regulated. Some StDof genes also showed variable expression patterns in response to biotic stresses.

2.7. Expression Analysis of StDof Genes in Two Potato Cultivars of Contrasting Tolerance under Drought Stress

Due to differential tolerance to drought stress [39], ‘Long10′ and ‘DXY’ were selected as experimental materials for the expression analysis of Dof genes in potato. The expression of all 36 StDof genes was tracked using RNA-Seq (six-week-old micropropagated plantlets of ‘Long10′ and ‘DXY’ were exposed to drought stress) to acquire insight into their possible roles in drought stress responses (Figure 7A). According to the findings, drought stress caused significant alterations in the expression profiles of StDofs. Drought treatment significantly increased the expression of five StDof genes, while it resulted in a decrease in seven other StDof genes. Six differentially expressed StDofs involved in drought stress response were studied using qRT-PCR to validate the RNA-seq data. The qRT-PCR results agreed with the RNA-seq data (Figure 7; Table S3). Simultaneously, we observed significant differences in the expression levels of StDofs (StCDF1/StDof19, StCDF2/StDof4, StCDF3/StDof11, StCDF4/StDof24, and StCDF5/StDof15) within two cultivars with differing drought tolerances. These findings suggested that StCDFs were involved in potato drought stress response.

2.8. Regulatory Network Mediated by lncRNAs and Their Target Dofs

Long non-coding RNAs (lncRNAs) operate as cis- or trans-acting regulators of protein-coding genes, playing key roles in plant responses to abiotic stressors. Some Dofs were identified as the targets of lncRNAs in potato—StCDF1/StDof19, together with lncRNA counterpart StFLORE; the StCDF1–StFLORE locus is important for vegetative reproduction and water homeostasis [40]. We proceeded to build regulatory networks mediated by lncRNAs and their target Dofs. In potato, 50 lncRNAs, which target 29 StDofs, were predicted and discovered (Figure 8; Table S4). Notably, StDof14 and StDof33 were identified as hub genes in the module, which was cis-regulated by four lncRNAs. Meanwhile, StDof9, StDof25, and StDof26 may be regulated by two lncRNAs, respectively. Furthermore, two specific micRNAs (stu-miR8020 and stu-miR8045) that were targeted by lncRNAs were also identified. These two micRNAs have previously been reported to associate with the drought stress response [41]. We examined the expression patterns of CDF-related lncRNA in various drought-resistant potato cultivars under drought stress. The results indicated significant differential expression of lncRNA under drought stress (Figure 9), suggesting a potential role in regulating CDF and influencing potato’s response to drought stress.

3. Discussion

3.1. Identification and Characterization of StDof in Potato

Dof genes are crucial for various plant physiological processes as well as for abiotic and biotic stress responses. A total of 36 Dof genes were found in potato. These StDofs were categorized into five groups based on the presence of a highly conserved Dof domain in the amino acid sequences (Figure 1 and Figure 3; Table 1) and named in reference to Arabidopsis [42]. The number of Dof genes in potato (36) is comparable to that of Arabidopsis (36) and rice (30) [43,44]. Gene duplication occurrences are a major contributor to the gene family’s expansion. In potato, 28 StDofs were involved in duplication events (Figure 2), which differs from other potato gene families like the WRKY family [45] and the CIPK [46] family. This implies that duplication is one of the most important processes contributing to the expansion of the potato Dof gene family.
Interestingly, the analysis of conserved motifs revealed a series of motifs that were largely confined to a single phylogenetic Group A of Dofs (as the CDF group, Figure 1 and Figure 3). CDF proteins often function as important regulators, along with other proteins, through a special motif from the C-terminal of Dof proteins [6,13,14,47]. Most StDofs contain Motif 1 (Dof domain); however, only the CDF group contains many conserved motifs, with some encompassing up to seven motifs. Indeed, most CDFs are known to work downstream of GI, regulating responses to stress and flowering [6,31]. For example, Motif 2 is a GI- and FKF1-binding domain (Figure 3) [6,47], Motif 9 is a predicted nuclear localization signal, and Motif 5 is an N-myristoylation site [48]; these motifs only exist in the CDFs (Figure 1 and Figure 3). These results suggest that CDF-related Dofs play more extensive regulatory roles in plant growth, development, and stress response than other groups with fewer motifs. This may indicate that these StDof proteins play an equally important role in potato.

3.2. Specific StDofs Involved in Response to Drought Stress in Potato

Previous studies have shown that Dof genes are involved a wide range of stress responses [33,47]. Our findings indicated that the expression of StDofs was extensively responsive to a variety of bio- and abiotic stresses, as well as to hormones, suggesting that StDofs may be involved in multiple abiotic stress responses in potato. As one of the major abiotic stresses affecting plant growth and development, drought stress seriously affects potato yield and quality. When comparing drought-tolerant (Long10) and drought-sensitive (DXY) cultivars, the results showed that Dof TFs were differentially expressed in potato varieties (Figure 7). Due to the important regulatory role of CDF, we also identified potato CDF, StCDF1/StDof19, StCDF2/StDof4, StCDF3/StDof11, StCDF4/StDof24, and StCDF5/StDof15, which are homologs of Arabidopsis CDFs [6,14].
Based on their nature (tolerant or sensitive to drought) and drought treatment, the genotypes were grouped into similar groups, displaying different expression patterns for control and stress (Figure 7). Like StCDF1, its ortholog in tomato SlCDF1 demonstrated increased drought and salt tolerance [47]. In Arabidopsis, overexpression of the orthologs gene AtCDF3 improved the tolerance of the transgenic plant to several abiotic stresses (drought, cold, and osmotic stress) by modulating the CBF/DREB system [32,33]. In potato, StCDF1 was identified as a major-effect quantitative trait locus for plant maturity and the initiation of tuber development. It was also shown that StCDF1, together with the lncRNA StFLORE, regulates water loss by affecting stomatal growth and diurnal opening [14,40]. In our results, StCDF1 was significantly up-regulated in the drought-resistant cultivar ‘Long10’, but not in the drought-sensitive cultivar ‘DXY’. Meanwhile, the expression levels of StCDF2 and StCDF3 were reduced in the drought-sensitive cultivar ‘DXY’, but were dramatically increased in the drought-resistant cultivar ‘Long10’. As a result, research shows that potato StCDFs may play a crucial role in drought and other stress responses.

3.3. The lncRNA-Mediated Regulatory Pathways Involved in Response to Drought Stress

LncRNAs are primarily involved in the epigenetic and transcriptional regulation of gene transcription and expression [49]. LncRNAs have been confirmed to be related to stress in various plants, and several studies have explored the relationship between lncRNAs and mRNAs at a genome-wide level [50,51]. In rice, a total of 191 lncRNAs, 2115 mRNAs, and 32 miRNAs (microRNAs) were found to be differentially expressed under drought stress [52]. The lncRNA Auxin-regulated Promoter Loop (AtAPOLO) interacted with the TF WRKY42 to trigger the cell expansion of root hair in response to cold [53]. In response to cotton salt stress, LncRNA973 regulated reactive oxygen species-scavenging genes, transcription factors, and genes associated with salt stress [54]. In this paper, we predicted that lncRNAs regulated 26 Dofs, and 10 of them were regulated by multiple lncRNAs (Figure 8). Recently, lncRNA StFLORE has been reported to regulate water loss by affecting stomatal growth and diurnal opening by negatively regulating its target gene StCDF1/StDof19 in potato [40]. LncRNAs function as regulators of gene transcription and expression, and they also engage in interactions with miRNAs [55,56]. In this paper, we also identified the target miRNAs of two lncRNAs. like Stu-miR8045-lncRNA-StCDF4. Collectively, we highlight a unique role of Dofs in stress that integrates potato’s response to adverse environmental conditions with different aspects of potato growth and development.

4. Materials and Methods

4.1. Identification of Dof Genes in Potato

Dof proteins were identified by Hidden Markov Models (HMM, e value < 0.01) (PF02701) in the Pfam database (http://pfam.sanger.ac.uk/, accessed on 18 October 2022) and a search against the proteins of the potato reference genome by BLAST (e value < 1 × 10−5), the potato genome database (http://solanaceae.plantbiology.msu.edu/pgsc) based on Dof genes from Arabidopsis and rice [42], Malus domestica [43], sorghum [44], and grape [8]. The candidate members were confirmed by a Conserved Domain Data (CDD) search (http://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/structure/cdd/wrpsb.cgi, accessed on 19 October 2022).

4.2. Phylogenetic and Structural Analysis of StDof Genes

MEGA7.0 (Pennsylvania, USA) and Clustal X (Dublin, Ireland) were used to perform a phylogenetic analysis and amino acid sequences alignment. The phylogenetic tree was built using the neighbor-joining statistical approach and 1000 bootstrap replications. Domain analysis was performed by SMART (http://smart.embl-heidelberg.de/, accessed on 22 October 2022). Gene Structure Display Server 2.0 (GSDS) (http://gsds.cbi.pku.edu.cn, accessed on 22 October 2022) was used to create the gene structure. The annotated potato genome was used to obtain information on the introns and exons of the StDofs.

4.3. Conserved Motifs and Gene Location and Duplication Analysis of StDof Genes

Conserved motifs were identified by MEME (https://meme-suite.org/meme/tools/meme, accessed on 25 October 2022). The Dof genes’ locations were obtained from the potato gene model annotation files v6.1 (http://spuddb.uga.edu, accessed on 25 October 2022). The entire protein sequences were subjected to duplication studies using BLAST (all vs. all), and the duplications were detected using Mcscan X (e-value 1 × 10−5 and on the nearby gene loci).

4.4. Cis-Acting Element of Promoter Analysis

The potato genome database (http://solanaceae.plantbiology.msu.edu/pgsc, accessed on 18 October 2022) was used to obtain the promoter regions of StDofs. Using the PlantCARE databases (http://bioinformatics.psb.ugent.be/webtools/plantcare, accessed on 3 January 2023), we searched the 1.0 kb region upstream of the transcription start site (ATG) to identify cis-acting regulatory elements.

4.5. Microarray Data Analysis

Microarray data were used for further analysis in order to comprehend the geographical and temporal expression patterns of StDof genes. Using publicly available information (DM 1–3 516 R44-Gene Expression Matrix (TPM)-v6.1, http://spuddb.uga.edu/dm_v6_1_download.shtml, accessed on 18 October 2023), the expression profiles of StDofs in various tissues and with different treatments were investigated. A heatmap was constructed using the Pheatmap (https://cran.rproject.org/web/packages/pheatmap/, accessed on 26 October 2023) package of R.

4.6. Plant Materials and Treatments

Potato (Solanum tuberosum L.) cultivars ‘Long10′ (drought tolerant) and ‘DXY’ (drought susceptible) were used in the experiment [39]. The potato tubers, which weighed around 150 g, were collected and grown in vermiculite pots of 4 L in a greenhouse with natural light at a temperature of 25 ± 2 °C. The seedlings were given weekly applications of a complete fertilizer solution and water after sprouting. Drought treatments were carried out on the treatment samples once the plants had grown for 25 days. The soil water content in the pots was monitored two times every day using TDR-300 sensors (Spectrum R, Aurora, IL, USA). The soil water content in the pots was kept at 75–80% in the control group (D0 and L0). When the water content of the soil in the pots had decreased to 35–40%, it was considered the first day of drought treatment. Samples were collected after a 5-day drought treatment, and three replicates (each containing 10 plants) were employed [46,57].

4.7. Quantitative RT-PCR (qRT-PCR) Analysis

IQ SYBR Green Supermix (Bio-Rad, California, America) was used for qRT-PCR. For each sample, three biological replicates, each comprising three technical replicates, were tested. The gene expression levels were normalized against the StEF1a (Soltu.DM.06G005680) gene. The relative expression levels were calculated using the 2−ΔΔCt method.

4.8. Statistical Analysis

SPSS 20.0 (SPSS, Armonk, NY, USA) was used for significant difference analysis. The experiment was performed in three biological replicates; data are mean values ± SE of three biological replicates.

5. Conclusions

In conclusion, the functional characterization of StDofs will benefit future research into the mechanism of abiotic stress tolerance (drought, salt, and cold, etc.). The findings also suggest several StCDFs as potential candidates for enhancing abiotic stress resistance in potatoes. This provides new insights into the Dof gene family’s evolutionary and functional divergence, which can benefit gene functional investigations of prospective Dof genes, providing an essential genetic resource for future studies including multiple gene knockouts and protein–protein interactions. LncRNAs are a class of heterogeneous regulatory transcripts that play a variety of regulatory roles in plant development and stress response. To date, only one report has reported a link between CDFs and lncRNA. Therefore, it is important to determine whether functional roles between Dofs and lncRNA are more widespread than currently known. Therefore, additional studies are required to fully understand the molecular mechanisms by which Dofs orchestrate metabolic homeostasis, stress responses, crop improvement, and plant growth and development. First, the key regulatory genes were mined through the identification and screening of core germplasm resources, and the molecular biological basis of character differences was analyzed. The use of genomics and molecular biology and other modern technological means has the potential to improve the genetic efficiency of crops, improving or innovating crop varieties, through the organic combination of biological breeding and conventional breeding, including genetic engineering.

Supplementary Materials

The following supporting information can be downloaded at: https://0-www-mdpi-com.brum.beds.ac.uk/article/10.3390/ijms25063488/s1.

Author Contributions

X.J. and Z.W. performed bioinformatics analysis of the potato Dof gene family and wrote the manuscript. Q.A., X.L. and N.Z. performed drought treatment and analysis. X.J. and Z.W. performed bioinformatics analysis of lncRNA and miRNA. Z.W., X.J., J.Y., N.Z. and H.S. designed this study and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by The Research Program Sponsored by State Key Laboratory of Aridland Crop Science, Gansu Agricultural University (No. GSCS-2020-07 and GSCS-2022-03); the National Natural Science Foundation of China (32260518); Youth Science and Technology Fund by Science and Technology Department of Gansu Province (21JR7RA844 and 22JR5RA882); The Scientific Research Start-up Fund by Gansu Agricultural University (GAU-KYQD-2019-06 and GAU-KYQD2020-26).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Phylogenetic analysis of the Dof genes in potato and Arabidopsis. The unrooted tree was generated by ClustalW in MEGA7 using the conserved amino acid sequences of the 36 StDof and 36 AtDof proteins. Dof proteins groups are distinguished by different colors.
Figure 1. Phylogenetic analysis of the Dof genes in potato and Arabidopsis. The unrooted tree was generated by ClustalW in MEGA7 using the conserved amino acid sequences of the 36 StDof and 36 AtDof proteins. Dof proteins groups are distinguished by different colors.
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Figure 2. Characterization of potato Dof genes. (A) Potato Dof gene exon/intron structure. Exons are represented as yellow boxes. Introns are shown by black lines, whereas untranslated regions (UTRs) are represented by blue boxes. The size of the exons and introns can be estimated using the scale at the bottom. (B) Conserved motif distribution in the 36 StDof proteins. A number in a colored box represents each motif. For more details on the motifs, see Supplemental Table S1. Different motifs are represented by different colored double-sided wedge boxes.
Figure 2. Characterization of potato Dof genes. (A) Potato Dof gene exon/intron structure. Exons are represented as yellow boxes. Introns are shown by black lines, whereas untranslated regions (UTRs) are represented by blue boxes. The size of the exons and introns can be estimated using the scale at the bottom. (B) Conserved motif distribution in the 36 StDof proteins. A number in a colored box represents each motif. For more details on the motifs, see Supplemental Table S1. Different motifs are represented by different colored double-sided wedge boxes.
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Figure 3. Chromosome distribution and duplication analysis of potato Dof genes. Chromosomes 1–12 are shown in a circular form. Black curves denote the details of duplication between potato Dof genes.
Figure 3. Chromosome distribution and duplication analysis of potato Dof genes. Chromosomes 1–12 are shown in a circular form. Black curves denote the details of duplication between potato Dof genes.
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Figure 4. Expression profiles of the StDof genes at different developmental stages in specific tissues/organs in potato. Dynamic expression profiles of StDof genes for 14 different tissues/organs using publicly available microarray data. Intensity of expression is defined in the colored bar on the right of the chart with red representing increased transcript abundance and blue representing decreased transcript abundance.
Figure 4. Expression profiles of the StDof genes at different developmental stages in specific tissues/organs in potato. Dynamic expression profiles of StDof genes for 14 different tissues/organs using publicly available microarray data. Intensity of expression is defined in the colored bar on the right of the chart with red representing increased transcript abundance and blue representing decreased transcript abundance.
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Figure 5. Schematic representation of cis-elements detected in StDof gene promoters using PlantCARE. All identified cis-acting elements were divided into 12 groups (indicated by different colors).
Figure 5. Schematic representation of cis-elements detected in StDof gene promoters using PlantCARE. All identified cis-acting elements were divided into 12 groups (indicated by different colors).
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Figure 6. Expression profiles of the StDof family genes with multiple treatments in potato. Gene expression was hierarchically clustered. Salt stress, 150mM NaCl; Heat stress, 35 °C; Mannitol, 24 h of 260 µM; BABA and BTH treated 24 h, respectively; P. infestans infected leaves 24 h. Up-regulation is indicated by red colors, whereas down-regulation is indicated by blue colors.
Figure 6. Expression profiles of the StDof family genes with multiple treatments in potato. Gene expression was hierarchically clustered. Salt stress, 150mM NaCl; Heat stress, 35 °C; Mannitol, 24 h of 260 µM; BABA and BTH treated 24 h, respectively; P. infestans infected leaves 24 h. Up-regulation is indicated by red colors, whereas down-regulation is indicated by blue colors.
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Figure 7. Expression patterns of differentially expressed Dof genes in two cultivars. (A) RNA-Seq data of differentially expressed Dof genes in two cultivars; (B) The qRT-PCR validation of differentially expressed Dof genes. D0 is the control of DXY and L0 is the control of Long10, D1 and L1 represents DXY and Long10 five days after drought treatment. * p < 0.05 and ** p < 0.01 (Student’s t-test).
Figure 7. Expression patterns of differentially expressed Dof genes in two cultivars. (A) RNA-Seq data of differentially expressed Dof genes in two cultivars; (B) The qRT-PCR validation of differentially expressed Dof genes. D0 is the control of DXY and L0 is the control of Long10, D1 and L1 represents DXY and Long10 five days after drought treatment. * p < 0.05 and ** p < 0.01 (Student’s t-test).
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Figure 8. Network diagram of interaction between LncRNA and target Dof genes. LncRNA is shown in light red, mRNA is shown in blue, mircroRNA is shown in fuchsia, and the line represents the targeting relationship.
Figure 8. Network diagram of interaction between LncRNA and target Dof genes. LncRNA is shown in light red, mRNA is shown in blue, mircroRNA is shown in fuchsia, and the line represents the targeting relationship.
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Figure 9. Expression patterns of differentially expressed lncRNAs in two cultivars. D0 is the control of DXY and L0 is the control of Long10. D1 and L1 represent DXY and Long10 five days after drought treatment.
Figure 9. Expression patterns of differentially expressed lncRNAs in two cultivars. D0 is the control of DXY and L0 is the control of Long10. D1 and L1 represent DXY and Long10 five days after drought treatment.
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Table 1. Features of Dof gene family in S. tuberosum.
Table 1. Features of Dof gene family in S. tuberosum.
Gene IDGene SymbolGroupChromosomeStartEndGene Length (bp)Protein Length (aa)NCBI Accession
Soltu.DM.01G002740StDof1C012,885,9502,886,891819272-
Soltu.DM.01G035240StDof2C0174,752,80874,755,079852283XP_006339731.1
Soltu.DM.02G008570StDof3D0223,280,58323,279,690585194XP_006367424.1
Soltu.DM.02G009620StDof4A0224,499,78924,496,0551398465XP_006363404.1
Soltu.DM.02G016040StDof5A0230,575,387 30,576,594498165XP_006362415.1
Soltu.DM.02G017380StDof6E0231,859,70031,861,6851197398XP_006347015.1
Soltu.DM.02G017390StDof7F0231,876,79831,874,957858285XP_006347016.1
Soltu.DM.02G018030StDof8E0232,428,49332,429,942849282XP_006347078.1
Soltu.DM.02G029480StDof9E0242,036,36742,037,640843280XP_006338231.1
Soltu.DM.02G029580StDof10D0242,088,87742,087,455723240XP_006338240.1
Soltu.DM.02G031630StDof11A0243,678,53843,675,0141410469XP_006338444.1
Soltu.DM.03G001980StDof12E031,907,5511,908,434777258XP_015162392.
Soltu.DM.03G022110StDof13C0346,929,01946,927,517786261XP_006350511.1
Soltu.DM.03G026810StDof14F0351,573,01351,570,712993330XP_006344507.1
Soltu.DM.03G030070StDof15A0354,420,29254,417,5251512503XP_006364590.1
Soltu.DM.03G036130StDof16E0359,179,73359,179,107627208XP_006343007.1
Soltu.DM.04G026220StDof17C0456,550,62256,549,822801266XP_015158674.1
Soltu.DM.04G034500StDof18D0465,958,71165,957,509717238XP_015167546.1
Soltu.DM.05G005140StDof19A054,485,5314,488,4951329442XP_006355111.1
Soltu.DM.05G025040StDof20E0553,480,94553,479,248909302XP_006346013.1
Soltu.DM.06G005290StDof21F068,124,9678,122,936942313XP_006366600.2
Soltu.DM.06G022770StDof22E0649,163,82149,167,8351131376KAH0749417.1
Soltu.DM.06G025370StDof23E0651,314,59951,316,145960319XP_015167508.1
Soltu.DM.06G025850StDof24A0651,760,52651,757,7141479 492XP_006354775.1
Soltu.DM.06G026620StDof25F0652,433,44152,435,513843280XP_006347454.1
Soltu.DM.06G030750StDof26F0655,730,22855,727,997909302XP_006365745.1
Soltu.DM.06G031420StDof27E0656,312,49656,309,962963320-
Soltu.DM.08G004890StDof28C086,182,8486,184,359756251XP_006352301.2
Soltu.DM.08G029850StDof29C0858,857,29258,860,058999332XP_006344006.1
Soltu.DM.09G002280StDof30E091,781,0871,778,7031212403XP_006341037.1
Soltu.DM.10G005360StDof31C104,885,7614,883,678816271XP_006362132.1
Soltu.DM.10G020610StDof32E1052,237,95552,239,7891125374XP_006349511.1
Soltu.DM.11G002690StDof33E112,808,209 2,810,6101140379XP_006353273.1
Soltu.DM.11G020390StDof34E1140,267,21840,265,223936311KAH0712769.1
Soltu.DM.11G025780StDof35F1145,731,87745,733,968909302XP_006366395.1
Soltu.DM.01G002730StDof36C012,882,119 2,881,166954317KAH0724332.1
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Jin, X.; Wang, Z.; Ai, Q.; Li, X.; Yang, J.; Zhang, N.; Si, H. DNA-Binding with One Finger (Dof) Transcription Factor Gene Family Study Reveals Differential Stress-Responsive Transcription Factors in Contrasting Drought Tolerance Potato Species. Int. J. Mol. Sci. 2024, 25, 3488. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms25063488

AMA Style

Jin X, Wang Z, Ai Q, Li X, Yang J, Zhang N, Si H. DNA-Binding with One Finger (Dof) Transcription Factor Gene Family Study Reveals Differential Stress-Responsive Transcription Factors in Contrasting Drought Tolerance Potato Species. International Journal of Molecular Sciences. 2024; 25(6):3488. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms25063488

Chicago/Turabian Style

Jin, Xin, Zemin Wang, Qianyi Ai, Xuan Li, Jiangwei Yang, Ning Zhang, and Huaijun Si. 2024. "DNA-Binding with One Finger (Dof) Transcription Factor Gene Family Study Reveals Differential Stress-Responsive Transcription Factors in Contrasting Drought Tolerance Potato Species" International Journal of Molecular Sciences 25, no. 6: 3488. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms25063488

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