**1. Introduction**

Transcription factors (TFs) play important roles in regulating growth and responding to external environmental stress in plants [1,2], which can regulate gene expression by binding to cis-promoter elements, thereby exerting regulatory roles in morphogenesis and so on [3,4]. TF genes, such as bHLH and Myeloblastosis (MYB), account for a large proportion of almost all plant genomes and are widely involved in plant development, stress response, and other physiological processes by regulating their target gene [5,6].

Basic/helix-loop-helix (bHLH) is a ubiquitous transcription factor family [7], which forms the second largest TF superfamily in plants [8]. The bHLHs have highly conserved alkaline/helix-loop-helix domains with approximately 50–60 amino acid residues [9]. This domain contains two functional regions: the basic region and the helix-loop-helix (HLH) region. The basic region contains approximately 15 amino acids and is located at the Nterminus, which is a critical region for binding to the cis-acting element E-box (5 -CANNTG-3 ) and determining whether the bHLH transcription factor binds to the promoter region of genes [10,11]. The HLH region contains two α-helices connected by a relatively poorly

**Citation:** Xu, Y.; Zhou, W.; Ma, F.; Huang, D.; Xing, W.; Wu, B.; Sun, P.; Chen, D.; Xu, B.; Song, S. Characterization of the Passion Fruit (*Passiflora edulis* Sim) bHLH Family in Fruit Development and Abiotic Stress and Functional Analysis of *PebHLH56* in Cold Stress. *Horticulturae* **2023**, *9*, 272. https://doi.org/10.3390/ horticulturae9020272

Academic Editor: Jia-Long Yao

Received: 6 January 2023 Revised: 11 February 2023 Accepted: 13 February 2023 Published: 17 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

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conserved loop distributed at the C-terminus, and this structure is essential for bHLH transcription factors to form homologous or heterodimers [10,12,13].

With the release of more high-quality genomes, many bHLH families in plants have been identified, such as *Carthamus tinctorius* (41) [14], Chinese jujube (92) [15], pineapple (121) [16], pepper (122) [17], potato (124) [18], peanut (132) [19], Jilin ginseng (137) [20], Brachypodium distachyon (146) [21], common bean (155) [22], tomato (159) [23], rice (167) [24], apple (188) [25], maize (208) [26], Chinese cabbage (230) [27], MOSO bamboo (448) [28], wheat (571) [29].

In current research, bHLHs are involved in responding to light [30], hormone signals [31], regulating anthocyanin biosynthesis [32], epidermal cell fate determination [33], and seed germination [34]. At present, the relationship between the bHLH gene and abiotic stress has attracted more and more attention. The bHLH has been shown to play a crucial role in plant resistance to abiotic stresses, such as abnormal temperature, drought, and high salinity. *FtbHLH3* of Tartary buckwheat participates in abiotic stress in response to changes in the polyethylene glycol (PEG) and the abscisic acid (ABA) [35], *FtbHLH2* can improve cold tolerance in plants [36]. *TabHLH39* augments the tolerance of transgenic *Arabidopsis* seedlings to drought, high salt, and low-temperature stress [37]. Overexpression of *AtbHLH92* can significantly improve salt and drought tolerance [38]. The *AtICE1*/*2* in *Arabidopsis* and their homologs *PtrbHLH* in Poncirus trifoliata can adjust plant cold tolerance and regulate peroxidase to break down hydrogen peroxide [39].

In addition, some studies have reported that *bHLH* genes are related to fruit development. In transgenic tomatoes, *SlbHLH22* is highly induced with the fruit color changed from green to orange, which is achieved by promoting the production of ethylene. Meanwhile, *SlbHLH22* was upregulated by using the exogenous ACC, IAA, ABA, and ethephon [40]. The research from Tan [41] showed that *CmbHLH32* was highly expressed in early developmental fruits. Overexpression of *CmbHLH32* can promote the early ripening of melon fruits, and the transgenic melons ripened earlier than the wild type. Papaya *CpbHLH1* and *CpbHLH2* promote fruit development by regulating genes related to carotenoid biosynthesis [42].

Passion fruit is a rare tropical fruit of the *Passiflora* genus (Passifloraceae), native to South America [43]. *Passiflora* has fresh and ornamental types [44]. *Passiflora* is nutritious and contains more than 100 different fruits in the pulp. At present, the planting area of East Asian countries such as Vietnam and China is growing rapidly, with a growth cycle of 4–6 months [45]. Because of its rich flavor, it has become more and more popular. Similar to other fruit, drought, high salinity, and cold and high-temperature stress seriously affect the development of passion fruit, resulting in a decline in fruit quality and yield. Due to the unpredictability of global climate change in recent years, passion fruit growing areas have been frequently affected by cold injury, which has also caused huge economic losses, resulting in more than 30% yield reduction and fruit stunting. Chilling injury is the most difficult to predict and control among the four abiotic stresses (drought, salt, cold and high temperature). Therefore, the identification of functional genes related to stress resistance and their utilization for variety improvement is of great importance for passion fruit cultivation.

Here, we identified the PebHLH family members in the genome of passion fruit and analyzed the members' biological information. Moreover, the expression patterns of *PebHLH* members were obtained by transcriptome sequencing and qRT-PCR at fruit developmental stages and under typical abiotic stress. More importantly, the *PebHLH* genes that were highly expressed and significantly induced by abiotic stress (drought, high salt, cold, and high temperature) were selected and overexpressed in *Arabidopsis* for functional validation. This provided a good foundation and reserved important information for studying the resistance mechanism in passion fruit and utilizing it for genetic improvement.

#### **2. Materials and Methods**

#### *2.1. Identification of bHLH Members in Passion Fruit*

The genome sequences of passion fruit were obtained from Phytozome V12.1. The HMM file of the NAM domain (PF00011) was downloaded from the PFAM database. In addition, analysis was performed using the bHLH with the highest comparison value to identify credible *PebHLHs*. The identification of PebHLH proteins used two methods described above were integrated and resolved to remove redundancy. AtbHLH protein sequences were obtained from Plant TFDB software (http://planttfdb.gao-lab.org/ accessed on 15 June 2022). The full-length protein sequences of PebHLHs and AtbHLHs were aligned by online ClustalX2. Moreover, the phylogenetic tree was constructed using MEGA (Version 7.0) [46]. A bootstrap test of 1000 repetitions was performed. Finally, PebHLH protein motifs were achieved using the MEME tool to compare the common domain of *PebHLHs*. Through the above procedures, the PebHLH members were finally obtained.

#### *2.2. Gene Structure, and Chromosomal Locations of PebHLHs*

The molecular weight (MW), protein isoelectric point (PI), and molecular formulas of all PebHLH members were reckoned using the online ProtParam. NetPhos (Version 3.1 Server) was used to predict the protein of PebHLH phosphate sites. The WoLF PSORT was used to perform Subcellular localization prediction. The gene structure maps, phylogenetic trees, combinations of motifs and gene structures, visualization of chromosomal localization, and collinearity relationships were obtained using TBtools [47].

## *2.3. Cis-Acting Elements, Protein Interaction Network and Gene Collinearity of PebHLHs*

All *PebHLH* gene transcription start site DNA sequences of the genomes of 2000 bp upstream were imported to PlantCARE and used to analyze the sequence of the promoter region [48]. The Orthovenn2 was used to analyze the orthologous pairs between PebHLHs and AtbHLHs. The regulatory networks of *PebHLHs* and other proteins were identified using the AraNet (Version 2.0). The STRING database and the predicted regulatory network were evinced by Cytoscape software. The PebHLH gene duplication events between different species were analyzed by Multicollinearity Scanning Toolkit (MCScanX) [47].
