Background

Tumorous stem mustard (Brassica juncea var. tumida) is a dicotyledonous plant belonging to Brassicaceae family of cruciferous crops [1]. The genus Brassica mainly includes three diploid species (Brassica rapa [AA]), Brassica nigra [BB], and Brassica oleracea [CC]) and three allopolyploid species (Brassica napus L. [AACC], Brassica juncea [AABB], and Brassica carinata [BBCC]). Brassica juncea is produced through hybridization between the diploid ancestors of Brassica rapa and Brassica nigra [2]. The evolutionary relationships among these Brassica species can be described using the well-known “triangle of U” model. Tumorous stem mustard is a major vegetable crop that has high economic value because of its primary use as a fresh vegetable or a raw material for Fuling mustard [3,4,5]. Tumorous stem mustard crops are majorly distributed in Chongqing, Zhejiang, Sichuan, Hunan, and Hubei in the Yangtze River basin, East China. The growth of Brassica juncea var. tumida involves four stages: germination, seedling, stem swelling, and flowering. However, owing to the influence of variety, photoperiod, and cultivation conditions, this crop may transit early from vegetative to reproductive growth. These factors often lead to early flowering and bolting, which reduces crop yield.

Phosphatidylethanolamine-binding protein (PEBP) is a class of evolutionarily conserved proteins that are widely present in plants, animals, microorganisms [6,7,8]. It plays an important role in regulating floral transition and seed germination [9,10,11]. Six PEBP genes have been reported in the model plant Arabidopsis thaliana: FLOWERING LOCUS T (FT), TWIN SISTER OF FT (TSF), TERMINAL FLOWER 1 (TFL1), BROTHER OF FT AND TFL1 (BFT), MOTHER OF FT AND TFL1 (MFT), and Arabidopsis thaliana CENTRORADIALIS (ATC) [12, 13]. They were classified into three subfamilies: FT-like, TFL1-like, and MFT-like subfamilies [10]. Recently, a new member of this gene family, AT5G01300 (PYBHB), was detected in Arabidopsis thaliana by Sheng et al. [14]. They classified it into the fourth subfamily called the ybhB-like subfamily [15]. Thus far, a total of seven Arabidopsis PEBP genes have been identified. Arabidopsis FT, TSF, and MFT promote flowering and TFL1, ATC, and BFT repress it [16,17,18,19]. FT belongs to the FT-like subfamily; it is a florigen encoding gene [18, 19]. Recent studies have identified several regulatory pathways associated with flowering: photoperiod, temperature-sensitive, vernalization, autonomous, hormone, and age pathways [20,21,22]. By integrating signals sensed by the photoperiodic, vernalization, and autonomous pathways, FT protein plays a major role in the photoperiodic pathway as a flowering regulation integrator [23], downstreaming flowering development CONSTANS (CO). Under prolonged daylight conditions, CO proteins induce the expression of FT genes [24]. FT protein is transferred from the leaves to the shoot apical meristem, and it then binds to FD protein [24]. These complexes induce the expression of the following genes: SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1, FRUITFUL, and APETALA1 (AP1) [25, 26]. TFL1 belongs to the TFL1-like subfamily. Unlike FT, TFL1 inhibits the plant’s transition from inflorescence meristem to floral meristem, thus delaying flowering time [27]. TFL1 functions in infinite inflorescence branching species by maintaining infinite inflorescence growth and in limited inflorescence branching species by flowering transition and inflorescence structure maintenance. In Arabidopsis sp., TFL1 regulates the meristem genes LEAFY (LFY) and AP1 to control the plant’s morphological structure [28, 29]. MFT belongs to the MFT-like subfamily and is the ancestor of FT and TFL1. Overexpressed AtMFT leads to early flowering, but this exhibits a weak activity in the promotion of flowering. MFT is expressed in seed in Arabidopsis thaliana, and regular seed germination through the abscisic acid (ABA) and gibberellic acid (GA) signaling pathways [30].

The PEBP family has been identified in various plants such as Moso Bamboo (gene number [n] = 6) [31], Oryza sativa (n = 19) [11], Gossypium hirsutum (n = 8) [32], common wheat (n = 76, 38, 16, and 22) [33], Glycine max (n = 27) [34], Vitis cinifera (n = 5) [35], Rosaceae tree species (n = 56) [36], rice (n = 19) [11], and corn (n = 25) [37].

Because the entire Brassica juncea var. tumida genome has been sequenced [2], a genome-wide analysis of PEBP genes was performed for the first time in this study. The phylogenetic relationship, gene structure, protein motif, chromosome location, and expression profile of a total of 21 identified BjPEBP genes in different tissues were analyzed. The results may provide valuable information for classifying BjPEBP genes and lay the foundation for exploring the molecular mechanisms underlying stem swelling and flowering orchestrated by PEBP genes in Brassica juncea var. tumida.

Results

Identification of the PEBP family members of Brassica juncea var. tumida

In this study, a total of 21 genes were identified in Brassica juncea var. tumida using the protein families database (Pfam), National Center for Biotechnology Information (NCBI), Conserved Domains Database (CDD), and Simple Modular Architecture Research Tool (SMART) database. These 21 BjPEBP genes were found to possess the typical PEBP domain (PF01161) and were named in reference to AtPEBPs (Table 1). These BjPEBP genes possess only one PEBP domain, except BjATC1 that possesses two PEBP domains. The number of coding amino acids ranges from 135 to 281; BjMFT1 and BjATC1-1 are 135-aa and 281-aa long, whereas the others are approximately 175-aa long. The isoelectric point ranged from 5.34 to 9.69. These BjPEBP proteins were mainly subcellularly located on the cytoplasm (Table 1).

Table 1 The PEBP genes family members in Brassica juncea var. tumida
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Of the 21 genes, 20 were located on 11 chromosomes, except BjFT2, which was anchored in contig429. There was one BjPEBP gene each on chromosomes A03, A09, and B05; two BjPEBP genes each on chromosomes A06, A07, A10, B02, B03, B04, and B08; and three BjPEBP genes on chromosome B06 (Fig. 1).

Fig. 1
figure 1

The gene locations of BjPEBPgene family. The chromosome name is at the top of each bar. The scale of the chromosome is in millions of bases (Mb)

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Construction of a molecular evolutionary tree of PEBP genes

To further elucidate the evolutionary relationship among the members of the PEBP gene family, an unrooted molecular evolutionary tree was constructed using the neighbor-joining (NJ) method; the 21 identified BjPEBPs of Brassica juncea var. tumida and 7 AtPEBPs of Arabidopsis were analyzed. PEBP proteins were subjected to multiple sequence alignment via ClustalW; the results showed that most proteins possess an interaction site for 14-3-3 protein (RXF motif), and all proteins possess an anion-binding site (GIHR and DPDxP motif) (Fig. 2). The evolutionary tree constructed using the NJ method with Arabidopsis, Brassica juncea var. tumida, Brassica napus L., and Brassica nigra indicated that the genes could be divided into four subfamilies (Fig. 3): FT-like, TFL1-like, MFT-like, and ybhB-like subfamilies. In Brassica juncea var. tumida, the MFT-like subfamily comprises five members: BjMFT1, BjMFT2, BjMFT3, BjMFT4, and BjMFT5. The TFL1-like subfamily comprises eight members: BjTFL1, BjTFL2, BjTFL3, BjTFL4, BjTFL5, BjATC1, BjATC2, and BjATC3. Furthermore, the FT-like subfamily comprises six members: BjBFT1, BjBFT2, BjFT1, BjFT2, BjFT3, and BjTSF1. Finally, the ybhB-like subfamily comprises two members: BjPYBHB1 and BjPYBHB2.

Fig. 2
figure 2

Sequence alignment of 28 PEBP proteins of Brassica juncea var. tumida and Arabidopsis. The sequences were aligned using ClustalW. The conserved protein motif 14-3-3 interaction interface and anion-binding site are rectangle with the color of red and pink, respectively. The rest is not shown

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Fig. 3
figure 3

Molecular evolutionary analysis of PEBP proteins from Arabidopsis, Brassica juncea var. tumida, Brassica napus L. and Brassica nigra. Star: the PEBP genes of Brassica napus L. Triangle: the PEBP genes of Arabidopsis. Check: the PEBP genes of Brassica nigra. The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. This analysis involved 58 amino acid sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 314 positions in the final dataset. Evolutionary analyses were conducted in MEGA X

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Analysis of the gene structure and conserved motifs of BjPEBPs

In this study, BjPEBP genes could be divided into four categories (Fig. 4 A). The gene structure indicated that most BjPEBP genes have four exons, except BjATC1, which contains seven exons, and BjPYBHB1 and BjMFT1, which contain three exons each. The sizes of exons and introns in the same cluster genes showed high similarity (Fig. 4B). The conserved motifs present in the 21 BjPEBP proteins were identified (Fig. 4 C). In total, 10 motifs were identified: motifs 1–10. The BjPEBP genes contain motif1, motif2, motif3, motif4, and motif5, except BjMFT1, BjPYBHB1, and BjPYBHB2. The character sequence of the BjPEBPs motif helps identify the motif that is conserved and can bind an anion. (Fig. 4D).

Fig. 4
figure 4

Genomic structure and motif composition of BjPEBPs. (A) The phylogenetic tree of BjPEBP proteins. (B) Genomic structure of BjPEBP genes family members in Brassica juncea var. tumida. Exons and introns are indicated with green boxes and black lines. (C) The conserved motifs in Brassica juncea var. tumida PEBP proteins identified using MEME online website. Each motif is indicated with a special color. (D) Two major motif logo of BjPEBPs. The character sequence of BjPEBPs motif

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Analysis of promoter cis-acting elements of BjPEBP genes

The promoter cis-acting elements of a gene are associated with its expression and function. In this study, multiple promoter cis-acting elements in were observed in BjPEBP promoters. There are four primary types of cis-acting elements (Table 2; Fig. 5): light-responsive, hormone-responsive, biotic or abiotic stress response, and growth and development–related elements.

Table 2 The information of BjPEBP genes promotor cis-acting element
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Fig. 5
figure 5

Cis-acting elements on promoters of BjPEBP genes. Orange mean Light response elements. Green showed involved in biotic or abiotic stress response components. Cyan showed elements related to growth and development. Blue showed the element of ABRE. Black showed the element of GARE-motif. Red showed the element of P-box. Purple showed the element of TATC-box. Other hormome response elements were showed yellow

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The following hormone-responsive cis-acting elements were identified: ABRE, MeJA response elements (CGTCA-motif and TGACG-motif), GARE-motif, p-box, and growth-hormone response element (AuxRR-core); they are mostly present in the members of the MFT-like and TFL1-like subfamilies. The following 11 light-responsive cis-acting elements were also identified: AE-box, G-box, GA-motif, GT1-motif, Gap-box, I-box, LAMP-element, MRE, sp1, TCT-motif, and chs-CMA2a. Furthermore, the following six growth and development–related elements were detected: HD-zip, MBSI, MSA-like, circadian, GCN4-motif, and CAT-box. The biotic or abiotic stress response elements identified were as follows: MBS, ARE, LTR, GC-motif, TC-rich, MYB, and MYC. Nearly all genes contain the aforementioned regulatory elements, except for BjATC1, BjATC3, BjFT1, BjMFT1, BjMFT2, BjMFT3, BjMFT4, BjMFT5, BjPYBHB1, and BjTSF1. These genes do not contain any growth and development–related elements. In BjTFL1-like genes, except BjTFL2, BjATC1–3 contain hormone-responsive cis-acting element, but the other genes do not contain this regulatory cis-acting element in their promoters. Among these four types of elements, the light-responsive and biotic or abiotic stress response elements were the most diverse and numerous. The abundant information on cis-acting elements suggest that this gene family is be involved in various regulatory mechanisms and play an important role in the stress response as well as growth and development of Brassica juncea var. tumida.

Expression of BjPEBPgenes

Based on RNA-Seq data collected in a previous study, the expression patterns of BjPEBP genes in different tissues were analyzed [38]. The expression of BjTFL1, BjTFL2, BjATC2, BjATC1, BjATC3, BjMFT4, BjBFT2, BjBFT1, BjTFL4, BjTSF1, and BjTFL5 genes were detected in at least one type of tissue (Fig. 6). BjTFL1 and BjTFL3 belong to the TFL1-like subfamily, and their expression pattern was similar. They were expressed in almost all tissues, except for BjTFL3, which was not detected in the root. The expression of BjTFL1 and BjTFL3 was increased and then decreased from YA1 to YA4. BjATC2 expression was detected in YA3, YA4, and YAr, whereas BjATC1 expression was detected in only YAr. BjMFT4 expression was similar to that of BjATC1. The expression of BjBFT1, BjBFT2, and BjATC3 was detected in YA1 and YA3. BjATC3 expression was also detected in YAr. BjTFL2 expression was noted to be weak in YA3 and YA4. BjTFL4, BjTFL5, and BjTSF1 showed low expression in Dayejie (DY), YA1, and YA2. The expression of other genes in these tissues remained undetected.

Fig. 6
figure 6

Expression patterns of BjPEBP genes in different development stages. DY, Dayejie stems were collected 22 weeks after seeding (daye3bianzhong); YA1-4, The stems of Yongan were collected 18, 20, 22, and 25 weeks after seeding; YAr, The mix roots samples of 18 and 22 weeks after seeding. The expression levels are represented by the color bar

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The expression of BjATC1, BjTSF1, BjBFT1, BjMFT4, BjTFL1, BjPYBHB1, and BjFT1 in plant tissues was further detected via qRT–PCR. BjATC1 exhibited weak expression in the tissues except the root (Fig. 7 A). BjTSF1 showed high expression in the leaf, flower, and fruit pod, with the highest expression detected in the fruit pod (Fig. 7B). BjBFT1 showed a higher expression in the stem, followed by that in the root and leaf; the lowest expression was detected in the flower and fruit pod (Fig. 7 C). BjMFT4 and BjBFT1 exhibited a similar expression pattern (Fig. 7D). Furthermore, BjTFL1 and BjPYBHB1 showed a similar expression pattern in the tissues. These genes exhibited high expression in the flower and leaf and weak expression in the fruit pod, root, and stem (Fig. 7E and F). BjFT1 showed a higher expression in the root, flower, and fruit pod than in the stem and leaf (Fig. 7G).

Fig. 7
figure 7

Expression levels of seven PEBP genes in Brassica juncea var. tumida different tissues by qRT-PCR. Statistically significant differences between tissues are indicated using asterisks (*p < 0.05, **p < 0.001; independent t-test)

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Subcellular localization of BjFT1

The subcellular localization of a protein helps predict its functions. The BjFT1–GFP fusion protein was transiently expressed in tobacco leaves. The results of fluorescence analysis revealed that BjFT1–GFP is accumulated in the plasma membrane (Fig. 8 A).

Fig. 8
figure 8

Subcellular localization and the interaction of BjFT1 protein. (A) Cells with only GFP reporter gene and BjFT1 gene under fluorescence and white light. The scale bar is 50 μm. (B) Yeast two-hybrid assay of BjFT1-Bj14-3-3 interaction. The interaction of BjFT1 and Bj14-3-3 in yeast cells. BD-53 + AD-T and BD + AD as the positive and negative controls, respectively. The yeast co-transformed BD-BjFT1 + AD-Bj14-3-3, BD-BjFT1 + AD, BD + AD-Bj14-3-3 and the control groups grown on the SD-Leu-Trp medium, and then grown on the SD-Leu-Trp-His-Ade medium

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BjFT1 interacts with Bj14-3-3

Most members of the PEBP family possess interaction sites for the members of the 14-3-3 family proteins. 14-3-3 may interacts with FT/Hd3a in cytoplasm and then the FT/Hd3a-14-3-3 complex interacts with FD, which is called the florigen activation complex (FAC) [39]. In this study, one PEBP gene, BjFT1, and one Bj14-3-3 gene were selected for to assess the interaction. The result showed that the experimental (pGBKT7::BjFT1 and pGADT7::Bj14-3-3) and positive (pGBKT7-53 + pGADT7-T) groups grew well on the SD-Leu-Trp and SD- Leu-Trp-His-Ade media. Thus, BjFT1 and Bj14-3-3 appear to interact with each other (Fig. 8B).

Discussion

Plant PEBP genes are associated with flowering and growth development. These were conserved in many plants. In B. juncea var. tumida, B. napus L., and B. nigra, a total of 21, 19, and 11 BjPEBP genes were identified, respectively. A previous study identified PEBP genes in Arabidopsis sp. [14]. Therefore, as tetraploid plants, Brassica juncea var. tumida and Brassica napus L. possess nearly three times more PEBP family genes than Arabidopsis sp. The BjPEBP gene family comprises three ATC, five TFL, five MFT, two BFT, three FT, one TSF, and two PYBHB genes. A total of 11 BjPEBP genes were identified in B. nigra (BB), a number higher than that noted for Arabidopsis sp. B. juncea is a tetraploid derived from the hybridization of B. rapa (AA) and B. nigra (BB); the increase in the number of BjPEBP genes might have resulted before the formation of the tetraploid. TFL-like genes play an important role in nutritional growth and inflorescence meristem-specific growth maintenance [10]. Five TFL-like subfamily genes are present in Brassica juncea var. tumida, which may originate through a multifunctional differentiation of TFL genes during growth and development. Different BjTFL genes regulate specific pathways. In Arabidopsis, the expression of BFT gene was upregulated under ABA, drought, and osmotic stress conditions. BFT genes may play a regulatory role in flowering time and inflorescence structure under drought conditions [40]. In Brassica juncea var. tumida, two BjBFT genes that may be closely associated with flowering and stress response function and the domestication of this species were identified.

Regarding the structural composition of PEBP, all PEBP genes were found to have four exons and three introns, except BjMFT1 and BjPYBHB1; this finding is consistent with that of Zhang et al. [36] who identified the PEBP gene family in nine Rosaceae trees species. The second and third exons of BjPEBP were noted to be short and the first and fourth exons were noted to be long; this finding is similar to that observed in Jatropha curcas [41]. The short motifs DPDxP (Asp-Pro-Asp-X-Pro) and GIHR (Gly-Ile-His-Arg) are highly conserved and represent the characteristic motifs of the PEBP protein family [36]. The conserved protein motif identifies motif1 and motif5 as the characteristic motifs of Brassica juncea var. tumida. This finding suggests that these genes have been relatively conserved during the evolution of this species.

The results of cis-acting elements present in the promoter of the members of the Brassica juncea var. tumida PEBP gene family showed that each gene contains various promoter cis-acting elements such as GARE-motif, p-box, and AuxRR-core for hormone regulation; AE-box and LAMP-element for light response; MBS and TC-rich for stress response; and HD-zip and CAT-box for growth and development. MFT-like genes integrate ABA and GA signaling pathways to control seed germination [42]. ABRE elements respond to GA; GARE-motif, TATC-box, and p-box respond to ABA. The results of cis-acting element analysis revealed that all BjMFT genes contain ABRE and p-box, which is consistent with the result of a previous study [30]. The light-responsive elements were mainly present in the MFT-like subfamily, with a higher distribution in the FT-like and ybhB-like subfamilies. The FT-like subfamily regulates plant flowering mainly under photoperiodic conditions. The growth and development–related elements are mainly present in the TFL1-like subfamily, which also reflects the primary function of this family in maintaining the nutritional growth of plants and the infinite growth state of inflorescences. All these elements have their specific functions and are involved in the regulation of gene expression. These elements are involved in the transcriptional regulation of genes via their binding with regulatory proteins and are thus important for the analysis of possible signaling pathways as well as functions. Therefore, the members of the PEBP gene family may play diverse functions during the growth and development of Brassica juncea var. tumida.

The specific expression patterns of genes in tissues usually reflect their biological functions. The RNA-Seq data obtained from different tissues of Brassica juncea var. tumida showed that the expression of both BjTFL1 and BjTFL3 was detected in YA1–YA4; these genes were highly expressed at the stage of stem inflation and thereafter, implying that these two genes are involved in the inflation or growth and development of Brassica juncea var. tumida. BjATC2, BjATC3, BjBFT1, BjBFT2, and BjTFL2 genes showed a weak increase in expression in the YA3 period. YA3 is the period of stem inflation and the transition from nutritional to reproductive growth in Brassica juncea var. tumida. In Arabidopsis sp., the AtATC, AtBFT, and AtTFL genes repress flowering [17]. Whether the BjATC2, BjATC3, BjBFT1, BjBFT2, and BjTFL2 genes in Brassica juncea var. tumida have similar functions warrant further studies. Owing to the high similarity of homologous gene sequences on the same branch in molecular evolutionary tree, primers do not distinguish between BjPEBP homologs. In Arabidopsis sp., TSF overexpression results in significantly early flowering [43]. In Brassica juncea var. tumida, BjTSF was expressed in the leaf, flower, and fruit pod; the expression was particularly high in the fruit pod. Therefore, TSF regulates plant flowering and probably seed development. BjBFT1 was detected in all tissues but showed relatively high expression in the root, stem, and leaf. This finding is consistent with that of a previous study by Zhang et al. [36] who stated that BFT expression is relatively high in the stem and leaf of Prunus yedoensis and Rosaceae occidentalis. In Arabidopsis sp., MFT4 plays a redundant role in flowering [9]. BjMFT4 expression was noted to be higher in the root, stem, and leaf than in the flower and fruit pod, suggesting that BjMFT4 is involved in nutritional growth, but not reproductive growth, in Brassica juncea var. tumida. BjTFL1 belongs to the TFL1-like subfamily. Members of the TFL1-like subfamily aid in flower-forming transformation and inhibit flowering [41]. The expression of BjTFL1 was higher in the leaf and flower than in other tissues. BjPYBHB1, a homolog of PYBHB, was highly expressed in the flowers of Brassica juncea var. tumida. However, to the best of our knowledge, no function of PYBHB has been reported yet. BjFT and BjTSF belong to the FT-like subfamily. They promote the flowering of plants. This result suggests that PEBP genes play an important role in different stages of the growth and development of Brassica juncea var. tumida.

Through subcellular localization prediction, BjFT1 was observed to localize at multiple sites; experimental validation revealed that it localizes primarily on the plasma membranes. Plant 14-3-3 proteins are involved in the flowering, growth, and developmental processes [44]. Most proteins that interact with 14-3-3 proteins contain the following motifs; RSXpSXP [45], RXSXpSXP [46], RXF/YpSXP [47], and YpTV [48]. The multiple sequence alignment result showed that BjPEBP proteins contain the RXF motif. Yeast two-hybrid experiment showed that Bj14-3-3 protein interacted with BjFT1 protein, suggesting that BjFT1 protein has a similar function with FT in Arabidopsis that regulate the flowering and seeding process of Brassica juncea var. tumida.

To the best of our knowledge, this study was the first to identify 21 BjPEBP genes in Brassica juncea var. tumida and reveal the roles of these genes in plant growth and development. This study speculated that these genes are involved in various processes such as hormone response, flowering transition of plants from nutritional to reproductive growth, and morphological structural changes. Our results may provide a reference for further studies on the molecular mechanism of the BjPEBP gene family of Brassica juncea var. tumida as well as a theoretical basis for molecular breeding.

Conclusions

A genome-wide analysis was performed in this study, which resulted in the identification of a total of 21 BjPEBP genes of Brassica juncea var. tumida. Based on the classification of PEBP genes in Arabidopsis sp., these 21 genes were categorized into four subfamilies: FT-like, MFT-like, TFAL1-like, and ybhB-like. Of these 21 BjPEBP genes, 20 were located on 11 chromosomes and the remaining one was anchored in a contig. Based on the results of motif analysis, it appears that the BjPEBP genes are highly conserved. Although some genes show high expression during the growth and development of Brassica juncea var. tumida, the expression of some other genes is low.

Materials and methods

Plant materials and growth conditions

Brassica juncea var. tumida cultivar Yonganxiaoye was provided by Dr. Jinjuan Shen of the Institute of Chongqing Fuling Agricultural Sciences and used to analyze gene expression patterns. Seeds were sowed into nutrient soil and cultured at a constant temperature of 22 °C in long-day photoperiod (16 h of light, eight hours of dark) in the culture room.

Identification of PEBP proteins in Brassica juncea var. tumida

The genome data of Brassica juncea var. tumida, Brassica napus L. (Bna_zs11) and Brassica nigra (Bnigra_N100.v2) were downloaded from the Brassica Database (BRAD; http://brassicadb.cn/) [2, 49]. Arabidopsis PEBP gene data were obtained from the TAIR database (https://www.arabidopsis.org/download/index-auto.jsp?dir=/download_files/Proteins). The Hidden Markov Model of the PEBP gene (PF01161) was downloaded from the Pfam website (http://pfam.xfam.org/family/PF01161). The PF01161 was searched in all protein sequences of Brassica juncea var. tumida, Brassica napus L. and Brassica nigra using the Hmmer software with an E-value of < 1.2e-12, and the screened out results were submitted to Pfam, NCBI CDD, and SMART for further verification [50,51,52].

Sequence and molecular evolutionary analysis

The ClustalW program was used to perform multiple alignments of PEBP protein sequences from Brassica juncea var. tumida, Arabidopsis. A phylogenetic tree was constructed using MEGA 10.2.6 software [53] and the NJ method based on the passion correction model and bootstrap test replication 1000 times [54]. A gene structure diagram was drawn using the online software of the GSDS 2.0 server [55]. The physical location data of BjPEBP genes were retrieved from Brassica juncea var. tumida. Conserved protein motifs were identified using default parameters for the Multiple Em for Motif Elicitation (MEME) website (https://memesuite.org/meme/doc/meme.html?man_type=web), and a maximum of ten motifs were sat. The subcellular location of BjPEBPs was PSORT website (https://wolfpsort.hgc.jp). Using Expasy analysis, the physicochemical properties of BjPEBP gene family proteins. Finally, 1500-bp the 5’ sequence was used as each PEBP gene’s promotor region to analyze the cis-acting elements using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [56].

Expression profile of PEBP genes

RNA-sequencing (RNA-seq) data were downloaded from the NCBI Sequence Read Archive database. The accession numbers are, SRX108496 (Dayejie [DY] stems, a mutant variety without inflated stems, were collected 22 weeks after seeding), SRX108498 (YA1; Yonganxiaoye [YA] stems were collected 18 weeks after seeding), SRX108499 (YA2; YA stems were collected 20 weeks after seeding), SRX108500 (YA3; YA stems were collected 22 weeks after seeding), SRX108501 (YA4; YA stems were collected 25 weeks after seeding), and SRX108502 (YAr; YA mix roots were collected 20 and 22 weeks after seeding). The computed reads per kilobase of transcript per million (RPKM) value was referred to in our previous report [57]. Screening of PEBP family genes data from raw data and using TBtools with selecting log scale, horizontal clustering, and the rest of the parameters are default to analyze the gene expression level.

RNA extraction and real-time quantitative PCR analysis

Root, stem, leaf, flower, and fruit pod’s tissues were collected. Then, total RNA was extracted from different plant materials using RNA Plant Kit (Takara, Qingdao, China), and then reverse transcription was conducted using the PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara, Qingdao, China) to get genome DNA. Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed with 20-µL volume using SYBR qPCR Master Mix (Vazyme, Nanjin, China). The internal reference gene for qRT–PCR was Bj18s; Table S1 lists gene-specific primers.

Three replicate samples of each period were subjected to three biological replicates using the BioRad IQ5 Real-Time PCR instrument (BioRad Laboratories, Hercules, CA, USA). Amplification parameters were as follows: activation at 50 °C for two minutes, predenaturation at 95 °C for two minutes, denaturation at 95 °C for 15 s, and annealing at 60 °C for one minute for 40 cycles. Finally, the relative gene expression level was calculated using the 2−ΔΔCt method [58].

Subcellular localization of BjFT1 protein

The BjFT1 gene was cloned into PCAMBIA1300-35 S-GFP vector and transformed into Agrobacterium tumefaciens strain LBA4404. Primers were designed according to the sequences of the BjFT1 gene (Table S1). Agrobacterium containing only the GFP reporter gene and Agrobacterium containing the BjFT1 gene was injected into Nicotiana benthamiana leaves, respectively. The transient transgenic Nicotiana benthamiana were darkened for 24 h and incubated under normal conditions for three days, and protein localization was observed under fluorescence microscopy.

Yeast two-hybrid experiment

Total leaf RNA was extracted from Brassica juncea var. tumida and reverse transcribed to obtain cDNA. Primers were designed according to the sequences of BjFT1 and Bj14-3-3 genes (Table S1), and PCR amplified the target genes. Restriction endonucleases, EcoRI and BamHI, cut the pGADT7 and pGBKT7 vectors, ligating target genes to construct a recombinant vector. The Plasmids of pGBKT7-BjFT1 + pGADT7-Bj14-3-3, pGADT7 + pGBKT7-BjFT1, pGBKT7 + pGADT7-Bj14-3-3, pGADT7-T + pGBKT7-53, and pGADT7 + pGBKT7 combinations of transformed yeast receptor cells were coated onto two-deficiency SD medium and incubated upside down at 30 °C for 2–3 days. Colonies larger than 2 mm in diameter were transferred to a four-deficiency SD medium and incubated upside down at 30 °C for 4–5 days.