*3.9. Response of Transgenic* Arabidopsis *to Cold Stress*

In A, GUS staining of the pCAMBIA1304-*PebHLH56p* transgenic seedlings was mainly observed in the leaf, and under the cold stress, GUS staining was enhanced and distributed throughout the transgenic plants. With the increase of cold time, GUS staining gradually deepened, and the GUS enzyme activity also increased (Figure 11).

**Figure 7.** Differential expression of *PebHLHs* responding to different levels of drought, high salt, cold, and high temperature (Table S2). CK is normal growth condition, drought 50% and 10% are soil water content indexes, NaCl 3 d and 10 d are the treatment time under 300 mM concentration, cold 20 h, and 48 h are the treatment under 8 ◦C, and high temperature 2 h, 4 h, and 24 h are the treatment time under 42 ◦C, respectively.

**Figure 8.** qRT-PCR analysis of 8 *PebHLHs* under 4 different abiotic stresses (Table S3)**.** Horizontal coordinates indicate different stress treatments (the different processing methods are described above), and vertical coordinates represent relative expression values. The different letters mean significance, which was examined by Duncan's range test (*p* < 0.05).

**Figure 9.** Differential expression of *PebHLHs* during 3 fruit ripening periods (Table S4).

**Figure 10.** qRT-PCR analysis of 8 *PebHLHs* during 3 fruit ripening periods (Table S5). Horizontal coordinates indicate different stress treatments, and vertical coordinates represent relative expression values. Biological replicates, tests, and *p* values are described above. The different letters mean significance, which was examined by Duncan's range test (*p* < 0.05).

**Figure 11.** Induction and expression pattern of *PebHLH56* under cold stress. (**A**) GUS staining of overexpressing *Arabidopsis* strains. (**B**) GUS activity quantitative analysis of overexpressing *Arabidopsis*. Horizontal coordinates indicate different stress treatments, and vertical coordinates represent relative expression values. The different letters mean significance, which was examined by Duncan's range test (*p* < 0.05).

### **4. Discussion and Analysis**

Numerous studies have shown that bHLH transcription factors are involved in diverse biological processes and the whole growth cycle [2]. At present, however, the systematic characterization of the bHLH genes in passion fruit is lacking, although bHLHs have been identified in many plants. The first plant bHLH gene was identified in maize (*Zea mays* L.) [52]. Furthermore, in this study, 117 bHLH genes were identified and characterized in passion fruit. This number is similar to some reported species, for example, the pineapple (121) [53], the pepper (122) [28], the potato (124) [54], the Jilin ginseng (137) [55], and the tomato (159) [56]. The numbers were quite different from some species. For example, the MOSO bamboo was 448 [57], and the wheat was 571 [18]. This shows that the bHLH gene family is diverse in different species. Based on phylogenetic analysis, we classified 117 *PebHLH* genes into 18 subfamilies, which is the same number reported in maize [26]. Previous studies in other species reported six subfamilies in the animal genomes [55], while in plants, the bHLH family genes were divided into 21 in *Arabidopsis*, tomato, and pear [21,23]; 22 in rice [24] and 31 subfamilies in B. napus [53]. Compared with the classification of other plant species, our results show similarities and differences, which also indicate that the classification of bHLH transcription factors in plants is more complex than that in animals, so the classification of plant bHLH families is still needed for more research [58].

The gene structure of the *PebHLH* family was further analyzed. Most family genes contain two or more introns. Among them, *PebHLH12/29/99* have no intron, while seven members (*PebHLH3/45/48/53/65/70/94*) have only one intron. It is generally believed that genes with few or no introns in plants show lower expression levels [59]. The compact gene structure may facilitate the induction of gene expression in response to exogenous stress [60]. For example, intron-less genes, *PebHLH57*, were upregulated under drought, salt, cold, and heat stresses. Genome duplication events have occurred during plant evolution [61]. The evolution of the genome and the expansion of gene families depend primarily on gene duplication events [62]. We have performed the collinearity analysis on passion fruit, rice, and *Populus trichocarpa*, and the results showed that the *PebHLH* gene had the most tandem duplication relationship with the bHLH gene in *Populus trichocarpa*. Therefore, it is speculated that the relationship between passion fruit and *Populus trichocarpa* is the closest. In the same way, *PebHLH31* was found to be an ortholog of *AtPIF4* (AT2G43010.5). The AtPIF gene is a central signaling hub regulating plant growth and development [63].

The functions of bHLH genes in plants are diverse, including plant perception of the growth and development processes such as fruit development [24,64]. *AtCIB1*, together with *AtCRY*, promotes flower opening by stimulating the expression of flowering genes [65,66]. In our results, *PebHLH109* has the highest expression level in fruit, and it is predicted to play a key role in fruit development. It also shows the highest expression level at the T3 stage of fruit ripening. In pepper, *CabHLH33*, a homolog of *AtbHLH31*, was highly expressed in flower buds and petals. Previous studies suggest that *AtbHLH31* regulates petal growth by controlling cell expansion [67].

Recent studies have increasingly focused on the relationship between bHLH genes and abiotic stress. Under stress conditions, certain bHLH TFs are activated, and they combine with the promoters of key genes to regulate the transcription level of the target gene. Several studies have found that *OsbHLH068* of rice and At*bHLH112* of *Arabidopsis* play an active role in response to salt stress [68]. *MfbHLH38* of the *Myrothamnus flabellifolia* was transformed into *Arabidopsis*, and the drought tolerance of transgenic lines was enhanced by the increase of gene expression [69]. *ZmbHLH55* of Maize can increase salt stress tolerance by regulating the expression of ascorbic acid biosynthesis-related genes [70]. Overexpression of *Ntbhlh123* and *IbbHLH79* can improve the cold tolerance of tobacco [71] and sweet potato, respectively [72]. *ZjbHLH076/ZjICE1* of *Zoysia japonica* can enhance the tolerance of transgenic lines to cold stress [73]. The activity of gene expression is quantitatively efficiently regulated by specific or functional promoters that contain multiple cis-acting elements. These acting elements can respond to a variety of stress responses [52,74]. Plant bHLH responds to various stress responses through the activation of long terminal repeat (LTR) reverse transcription transposons [75,76], with activation factors including drought [77], heat [78], and salt [79]. In this work, the cis-element analysis indicated that *PebHLHs* contained elements (such as cold-responsive element, salicylic acid-responsive element, ABRE) that could be responsive to various stresses, which was consistent with previous studies on potato [18], lotus [80], and Pepper [56] bHLHs.

In this experiment, we focused on the function of the bHLH gene under cold stress. Here, we identified one of the *PebHLHs* (*PebHLH56*), which can respond to cold stress in

transgenic *Arabidopsis*. This study provides experimental evidence that bHLH family genes in passion fruit respond to cold stress, and we will further study the resistance mechanism of bHLH genes.
