Characterization of Dof Transcription Factors and the Heat-Tolerant Function of PeDof-11 in Passion Fruit (Passiflora edulis)

Abiotic stress is the focus of passion fruit research since it harms the industry, in which high temperature is an important influencing factor. Dof transcription factors (TFs) act as essential regulators in stress conditions. TFs can protect against abiotic stress via a variety of biological processes. There is yet to be published a systematic study of the Dof (PeDof) family of passion fruit. This study discovered 13 PeDof family members by using high-quality genomes, and the members of this characterization were identified by bioinformatics. Transcriptome sequencing and qRT-PCR were used to analyze the induced expression of PeDofs under high-temperature stress during three periods, in which PeDof-11 was significantly induced with high expression. PeDof-11 was then chosen and converted into yeast, tobacco, and Arabidopsis, with the findings demonstrating that PeDof-11 could significantly respond to high-temperature stress. This research lays the groundwork for a better understanding of PeDof gene regulation under high-temperature stress.


Introduction
There are about 520 species of passion fruit, which is a tropical and subtropical fruit [1]. The passion fruits grown in East Asia are mainly two varieties of Passiflora edulis (Sims and Degener), of which Sims is the most common and is the dominant variety, including yellow and purple skins [2]. Passion fruit is widely grown throughout tropical areas with less advanced economies and technological infrastructure, has a great therapeutic value, and is nutritious [3]. Abiotic stress is an important limiting factor for the healthy growth of passion fruit, especially heat stress, which negatively affects the flowering and fruiting process, leading to flower abscission and non-growth of fruit, seriously affecting yields, with ambient temperatures above 40 • C leading to over 80% yield reduction [4]. Therefore, it is particularly important to address the impact of high-temperature stress on passion fruit production, and mining resistance genes, especially transcription factors with powerful functions, and studying their mechanisms of resistance action and applying them to biological breeding is an important avenue.
With respect to the Dof gene, a powerful transcription factor, recent research has demonstrated its important role in enhancing plant abiotic stress functions (heat, salt, and drought). For example, overexpression of the tomato SLDOF25/26 genes can improve the tolerance to drought and salt stress in Arabidopsis [5]. TDDF1, which encodes a tomato Dof protein, has been shown to increase plant salt and drought tolerance [6]. With the overexpression of tomato DOF genes SlCDF1/3 in Arabidopsis, transgenic plants showed enhanced drought and salt resistance [5]. The expression of Dof Daily Fluctuations 1 (TDDF1) improves tolerance to drought and salt stress in tomatoes [6]. Furthermore, the Dof gene plays an important role in controlling the development and germination of seeds and the flowering process of plants [7][8][9]. For example, in grapes, several Dof genes play functional roles during flower, berry, and seed development, indicating their importance in grape growth and development [10]. SlDOF10 plays an important role in plant reproductive development, especially during fruit setting [11].
The Dof gene family has a highly conserved Dof domain at the N-terminus, which is composed of the common core sequence (AT)/AAAG and is organized as a C2C2-type zinc-finger-like motif [12]. More Dof family genes have been researched since the first Dof gene (ZmDof1), and with the advent of genome sequencing, Dof genes have been widely identified in higher plants, such as Arabidopsis (36), rice (30) [13], sorghum (18) [14], and potato (35) [15].
In view of the biological functions of Dof genes, we analyzed the members of the PeDof family with a view to obtaining whether the family is amplified/contracted in passion fruit and preliminarily resolving its relevance to environmental adaptation. The members of the PeDof s and their characteristics were obtained by various biological techniques on the basis of a high-quality genome, transcriptome sequencing, and qRT-PCR analysis of PeDof s expression patterns under high-temperature stress and genetic transformation model organisms to verify the heat-tolerant biological functions of PeDof-11. This lays a good foundation for studying the regulatory mechanism of PeDof s and also provides important genes for passion fruit biobreeding.

Identification of Passion Fruit Dof Members and Information Analysis
In total, 13 passion fruit PeDof s were identified, ranging in length from 738 bp (PeDof-9, 245 amino acids, molecular weight 26,871.18 Da) to 1503 bp (PeDof-7, 500 amino acids, molecular weight 54,769.37 Da), with all PeDof proteins containing Ser, Thr and Tyr phosphorylation sites, and PeDof genes located in the Nucleus (Table 1). The phylogenetic tree ( Figure 1A) revealed that PeDof s were divided into three groups, with 3 and 10 members in groups 1 and 3, respectively. Most Dof members were closely homologously clustered in passion fruit with Arabidopsis and rice; e.g., PeDof-13 with AtDof1.7, PeDof-6 with AtDof3.5, PeDof-11 with AtDof4.2, PeDof-9 with AtDof4.7, respectively, showed the closest homology. Chromosomal localization analysis revealed that 13 PeDof genes were scattered on the chromosome ( Figure 1B), with PeDof-1/2/3/4 localized in Chr1. Chr3,4,8 contained two PeDof s, and Chr2,6,7 contained one PeDof s, respectively. Protein interaction analysis revealed that the proteins interacting with PeDof s were mainly HSI2 (high-level expression of sugar-inducible gene 2S), ABA1 (abscisic acid 1), and COL5 (constans-like) and so on, and their functions were mostly involved in plant resistance and development, which provided important clues for further functional studies on the regulation of interactions ( Figure 1C). Most instances of PeDof s 3D-structure modeling were structurally similar in that they contained the extension chain (red part) ( Figure 1D).

Expression of PeDof in Different Tissues of Passion Fruit and under High-Temperature Stress
We have analyzed the expression of some PeDofs in the leaves, roots, stems, and fruits of passion fruit ( Figure 4). Among them, 6 of them were mainly expressed in the fruit (PeDof-1/5/6/10/12/13). PeDof-3/4/7/8/9/11 were mainly expressed in the root. The results showed that each gene was expressed in different parts.
Under high-temperature stress, most PeDof members except PeDof4/5/10/13 could be induced, especially PeDof-11, which is more than 6.7-fold differentially expressed under the stress at 2 h. The qRT-PCR result also verified this finding. Therefore, we also chose this high-expression gene as the research object to further study the relationship between this gene and improving heat tolerance ( Figure 5).  Table S4. Data are means ± SD of n = 3 biological replicates. Means denoted by the same letter are not significantly different at p < 0.05 as determined by Duncan's multiple range test.

Expression of PeDof in Different Tissues of Passion Fruit and under High-Temperature Stress
We have analyzed the expression of some PeDof s in the leaves, roots, stems, and fruits of passion fruit ( Figure 4). Among them, 6 of them were mainly expressed in the fruit (PeDof-1/5/6/10/12/13). PeDof-3/4/7/8/9/11 were mainly expressed in the root. The results showed that each gene was expressed in different parts.

Expression of PeDof in Different Tissues of Passion Fruit and under High-Temperature Stress
We have analyzed the expression of some PeDofs in the leaves, roots, stems, and fruits of passion fruit ( Figure 4). Among them, 6 of them were mainly expressed in the fruit (PeDof-1/5/6/10/12/13). PeDof-3/4/7/8/9/11 were mainly expressed in the root. The results showed that each gene was expressed in different parts.
Under high-temperature stress, most PeDof members except PeDof4/5/10/13 could be induced, especially PeDof-11, which is more than 6.7-fold differentially expressed under the stress at 2 h. The qRT-PCR result also verified this finding. Therefore, we also chose this high-expression gene as the research object to further study the relationship between this gene and improving heat tolerance ( Figure 5).  Table S4. Data are means ± SD of n = 3 biological replicates. Means denoted by the same letter are not significantly different at p < 0.05 as determined by Duncan's multiple range test.  Table S4. Data are means ± SD of n = 3 biological replicates. Means denoted by the same letter are not significantly different at p < 0.05 as determined by Duncan's multiple range test.
Under high-temperature stress, most PeDof members except PeDof4/5/10/13 could be induced, especially PeDof-11, which is more than 6.7-fold differentially expressed under the stress at 2 h. The qRT-PCR result also verified this finding. Therefore, we also chose this high-expression gene as the research object to further study the relationship between this gene and improving heat tolerance ( Figure 5).

Analysis of PeDof-11 in Different Tissues of Passion Fruit and under High-Temperature Stress
For the high-temperature stress experiment, the pYES2-PeDof-11 and pYES2 empty vectors (control) were translated into INVSC1. The modified yeast grew better at 50 °C when exposed to high temperatures. This shows that PeDof-11 may have a function in high-temperature stress ( Figure 6).

Analysis of PeDof-11 in Different Tissues of Passion Fruit and under High-Temperature Stress
For the high-temperature stress experiment, the pYES2-PeDof-11 and pYES2 empty vectors (control) were translated into INVSC1. The modified yeast grew better at 50 • C when exposed to high temperatures. This shows that PeDof-11 may have a function in high-temperature stress ( Figure 6).

Analysis of PeDof-11 in Different Tissues of Passion Fruit and under High-Temperature Stress
For the high-temperature stress experiment, the pYES2-PeDof-11 and pYES2 empty vectors (control) were translated into INVSC1. The modified yeast grew better at 50 °C when exposed to high temperatures. This shows that PeDof-11 may have a function in high-temperature stress ( Figure 6). We then transiently transformed tobacco with the PeDof-11 promoter (Figure 7). The PeDof-11p-transformed tobacco and the control were both treated with a 42 • C high-temperature stress treatment. As can be seen from the figure, under the control of the PeDof-11 promoter, the GUS staining is the deepest under the high-temperature treatment for 4 h, indicating that the PeDof-11 is induced to express by the high-temperature stress. The findings demonstrate that the gene was substantially induced during the four-hour treatments. We then transiently transformed tobacco with the PeDof-11 promoter (Figure 7). The PeDof-11p-transformed tobacco and the control were both treated with a 42 °C high-temperature stress treatment. As can be seen from the figure, under the control of the PeDof-11 promoter, the GUS staining is the deepest under the high-temperature treatment for 4 h, indicating that the PeDof-11 is induced to express by the high-temperature stress. The findings demonstrate that the gene was substantially induced during the four-hour treatments. To determine the role of PeDof-11 in the expression of various tissues of Arabidopsis and in heat stress response, we transformed the PeDof-11 promoter and the gene into Arabidopsis, respectively, and two lines were selected with the experiment, separately (Figure 8). The extent of GUS staining reflects the expression of the PeDof gene driven by the PeDof-11 promoter. As shown in Figure 8A, the leaves of transgenic Arabidopsis had the deepest GUS staining. It shows that the gene is expressed mainly in the leaves. The wild-type and transgenic plants (7 days old) grown in 23 °C MS medium were treated at 42 °C for 36 h, and the GUS staining was greater and concentrated mostly in the leaves. We performed a GUS enzyme activity test and discovered that GUS activities are 3.9-fold higher than the control. The Arabidopsis plants (20 days old) grown in an incubator at 23 °C were treated at 45 °C for 8 h. The results showed that the transgenic plants were phenotypically more heat-tolerant than those of the wild type after high-temperature stress and that the transgenic Arabidopsis grew significantly better than the wild type, with higher survival than that of the wild type, after being restored to the normal conditions of 23 °C for 4 days of incubation. The results show that PeDof-11 was induced by high-temperature stress. To determine the role of PeDof-11 in the expression of various tissues of Arabidopsis and in heat stress response, we transformed the PeDof-11 promoter and the gene into Arabidopsis, respectively, and two lines were selected with the experiment, separately ( Figure 8). The extent of GUS staining reflects the expression of the PeDof gene driven by the PeDof-11 promoter. As shown in Figure 8A, the leaves of transgenic Arabidopsis had the deepest GUS staining. It shows that the gene is expressed mainly in the leaves. The wild-type and transgenic plants (7 days old) grown in 23 • C MS medium were treated at 42 • C for 36 h, and the GUS staining was greater and concentrated mostly in the leaves. We performed a GUS enzyme activity test and discovered that GUS activities are 3.9-fold higher than the control. The Arabidopsis plants (20 days old) grown in an incubator at 23 • C were treated at 45 • C for 8 h. The results showed that the transgenic plants were phenotypically more heat-tolerant than those of the wild type after high-temperature stress and that the transgenic Arabidopsis grew significantly better than the wild type, with higher survival than that of the wild type, after being restored to the normal conditions of 23 • C for 4 days of incubation. The results show that PeDof-11 was induced by high-temperature stress.

Discussion
With more and more gene families of species being mined and analyzed, many Dof genes have been cloned. And some studies have shown that Dofs play a role in regulating gene expression in response to abiotic stress, such as drought, cold, and heat stress. However, the identity and function of passion fruit Dofs have remained unknown. In this research, 13 Dof family members were identified in the passion fruit genome and showed possible roles in PeDofs responses to abiotic stresses.

Discussion
With more and more gene families of species being mined and analyzed, many Dof genes have been cloned. And some studies have shown that Dof s play a role in regulating gene expression in response to abiotic stress, such as drought, cold, and heat stress. However, the identity and function of passion fruit Dof s have remained unknown. In this research, 13 Dof family members were identified in the passion fruit genome and showed possible roles in PeDof s responses to abiotic stresses.

Identification and Characteristics of PeDof Genes
The Dof gene family is a plant-specific transcription factor family. Members of Dof genes have been discovered in different species since the first Dof gene was discovered in maize [16]. In this research, 13 PeDof genes have been identified. This number is similar to the number of Dof reported in spinach (22) and is lower than that of rice (30) [13], wheat (96) [11], and soybean (78) [6]. This indicates a massive shrinkage of the Dof family in passion fruit, and the 13 preserved members appear to be particularly valuable. The theoretical isoelectric points (pI) of Dof proteins ranged from 4.75 to 9.32; this result is similar to that in spinach [17].
In plants, Dof genes typically form polygenic families. The fundamental driving mechanism in the development of Dof genes is assumed to be repetitive events. In poplars, for example, 49% of PTRD genes were discovered to be localized in segmental and tandem repeat areas [18]. In apples, 57 and 18 MDD genes, respectively, are found in segmental and tandem repeat regions [19]. The cucumber genome contains two pairs of tandem repeats and six pairs of segmental repeats [20]. In this study, there are 28, 25, 19, and 2 connections between PeDof family members and poplar, Arabidopsis, grape, and rice Dof members, respectively.
Exon-intron structural arrangement can reveal evolutionary links within gene families [21]. In the current study, the number of introns in members of the PeDof s ranged from 0 to 2, with most members having no introns, a result similar to that of many other plant species, including pigeonpea, cucumber, poplar, and pear [11,18,20,22].

Cis-Elements of PeDof Genes
Cis-acting components, such as plant hormones and stress response, play a vital part in the life cycle of plants [17]. Cis-regulatory elements found in the promoters of co-expressed genes are thought to have a role in gene activity regulation. The majority of cis-elements in the PeDof gene family were associated with responses to light, stress response, and hormones (Table S5). The majority of cis-elements found in the SoDof gene family were connected to light response [23]. Passion fruit growth is strongly light-demanding, and the cis-acting element of PeDof s contains a large number of light components, which also provides a basis for conducting studies on the mechanism of PeDof and light response.

Potential Role of PeDof genes in Different Tissues
The expression profiles of genes under specific conditions are closely related to biological functions [24]. Gene expression in different tissues may be associated with the underlying function of the gene. In general, genes located in fruits, meristems, and other tissues may be involved in the development of organs and tissues. In this study, most of the PeDof genes were distributed in fruits and roots. This predicts that the function of genes may be related to fruit development and stress resistance. In spinach, six SoDof s are expressed at higher levels in flowers than in other tissues, suggesting that they may be involved in reproductive processes such as flowering and fertility through floral organ responses [17].

The Response of Dof Genes in Abiotic Stresses
Dof genes are linked to abiotic stress tolerance in plants, according to a growing body of studies. For instance, in Brassica napus, the Dof gene family is able to respond to different abiotic stresses [25]. Analysis of the cis-acting elements of CcDof s showed a predominance of abiotic stress response elements, suggesting that these genes may be associated with abiotic stresses [22]. Overexpression of the Dof gene TDDF1 in tomato increased blooming time, and transgenic plants were also more drought and salt tolerant [13]. OsDof27 could improve the heat tolerance of transgenic rice [26]. Overexpression of Dof s (SlCDF1/F3) greatly improved Arabidopsis drought and salt tolerance while also activating other stressresponsive genes including COR15, RD29A, and ERD10 [12]. One of the Arabidopsis Dof genes called CDF3 was highly induced by drought, extreme temperatures, and abscisic acid treatment [27].

Identification of Dofs and Information in Passion Fruit
The PeDof members were analyzed by bioinformatics software, including gene sequence, protein sequence, protein iso-electric point (pI) values and molecular weight (MW), prediction of subcellular localization, gene conserved region analysis, promoter structure, collinearity and protein interaction regulation and so on [3,4,28,29]. The relevant software and websites are listed in Table S1.

Plant Materials, Transcriptome, and qRT-PCR Analysis
Healthy and virus-free passion fruit seedlings of the purple fruit varieties were chosen. They were grown in the soil under a growth chamber (Panasonic, MLR-352) (30 • C; 200 µmol·m −2 ·s −1 light intensity; 12-h light/12-h dark cycle; 70% relative humidity) to a height of about 40 cm and with 8-10 functional leaves, which were subjected to hightemperature stress treatments [28]. The seedlings were grown at 45 • C for high-temperature stress treatment, with other conditions unchanged. The plants were subjected to hightemperature stress treatment for transcriptome analysis [3]. The samples from the hightemperature stress treatment were utilized for RNA sequencing. The Biomic Biotechnology company (Beijing, China) was entrusted with sequencing services. In the additional information, the FRKP data of the transcriptome are shown in Table S2. And we have frozen samples of roots, stems, fruits, and leaves for gene expression analysis in passion fruit tissues. The qRT-PCR analysis of PeDof genes was undertaken using suitable equipment (Light 96, Roche, Basel, Switzerland) [29]. Relative expression levels were calculated using the 2 −∆∆Ct method and normalized to the PeDof s. The date of qRT-PCR is shown in Tables S3 and S4.

Cloning and Vector Construction of PeDof-11 and the Promoter
The full-length cDNA and the promoter fragment of PeDof-11 were amplified by PCR from the passion fruit (purple fruit varieties) and then cloned into the pCAM-BIA1304 vector called pCAMBIA1304-PeDof-11 and pCAMBIA1304-PeDof-11p, respectively. pCAMBIA1304-PeDof-11p was used for the tissue specificity and high-temperature stress experiments in the tobacco and Arabidopsis thaliana seedlings (7 days old). pCAMBIA1304-PeDof-11 was used for the high-temperature stress experiments in the Arabidopsis thaliana (30 days old). The pYES2-PeDof-11 vector was used for the yeast experiments.

Functional Complementation of PeDof-11 in Yeast (Saccharomyces cerevisiae)
The INVSc1 strain (Saccharomyces cerevisiae) was transfected with the pYES2-PeDof-11 and the pYES2 vector (control). To perform the yeast complementation assays, the yeast liquid contained pYES2-PeDof-11, and the pYES2 empty vectors were first cultured in SD-Ura liquid medium at 30 • C to an OD600 value of about 1.0. The yeast liquid was diluted with sterile water by factors of 1, 10 −1 , 10 −2 , and 10 −3 , cultivated in SD-Ura liquid medium at 30 • C, and subjected to high-temperature stress (42 • C for 0 h, 12 h, and 24 h) to perform the yeast complementation tests. Three days later, the growth of the yeast was observed. and photos were taken.

Plant Transformation and High-Temperature Treatment
The Agrobacterium transformed with pCAMBIA1304-PeDof-11p was shaken at 28 • C to OD 600 = 0.8-1.0. Transient expression tests were conducted on two-month-old tobacco leaves. After high-temperature stress treatment (30 • C, 40 • C, 50 • C treated for 2 h), for additional staining tests, leaf discs with a diameter of 0.5 cm were cut off. The 7-day-old transgenic Arabidopsis with pCAMBIA1304-PeDof-11p were treated at 23 • C and 42 • C for 36 h. The T3 generation of 20-day-old transgenic Arabidopsis with pCAMBIA1304-PeDof-11 was treated with the high-temperature treatment. The plants were planted in vermiculite and treated in an incubator under 45 • C under the light condition for 8 h, replaced at 23 • C for 4 d to recover cultivation (200 µmol·m −2 ·s −1 light intensity; 8-h light/16-h dark cycle; 70% relative humidity), observed the survival rate of seedlings of different. Two lines of pCAMBIA1304-PeDof-11p (L1-p, L2-p) and pCAMBIA1304-PeDof-11 (L1, L2) transgenic Arabidopsis were taken for correlation studies, respectively.

Detection of GUS Activity
The different tissues of transgenic Arabidopsis with pCAMBIA1304-PeDof-11p, the 7-day-old seedlings, and the transgenic tobacco under normal and high-temperature stress were stained for GUS enzyme activity testing [30].

Conclusions
Dof s are widely reported to be related to the abiotic stress resistance of plants. We have identified 13 Dof members from the genome of the passion fruit. Analyses such as the evolutionary tree, structural domains, promoter cis-acting elements, inter-species, and intraspecies collinearity were completed. In this research, the transcriptome results of PeDof gene members under high-temperature stress were verified by qRT-PCR, and the results showed that most of the members were induced to express by high-temperature stress. One of the Dof genes (PeDof-11) was highly induced by high-temperature stress. Further testing of this gene revealed that it might improve the capacity of transgenic tobacco, Arabidopsis, and yeast to withstand heat stress. The findings provide a solid platform for future research into the ability of passion fruit to withstand abiotic stressors.  Data Availability Statement: The passion fruit genomic data and raw RNA-sequence data have been deposited in (https://ngdc.cncb.ac.cn/search/?dbId=gwh&q=GWHAZTM00000000), (https: //ngdc.cncb.ac.cn/omix/release/OMIX563, accessed on 8 January 2020); the accession number is OMIX563-20-01, and the NCBI SRA number: SRP410034, and also accession number: SRR2240515-20.

Conflicts of Interest:
The authors declare no conflict of interest.