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Genome-wide identification and expression analysis of calmodulin and calmodulin-like genes in passion fruit (Passiflora edulis) and their involvement in flower and fruit development

BMC Plant Biology volume 24, Article number: 626 (2024) Cite this article

Abstract

Background

The calmodulin (CaM) and calmodulin-like (CML) proteins play regulatory roles in plant growth and development, responses to biotic and abiotic stresses, and other biological processes. As a popular fruit and ornamental crop, it is important to explore the regulatory mechanism of flower and fruit development of passion fruit.

Results

In this study, 32 PeCaM/PeCML genes were identified from passion fruit genome and were divided into 9 groups based on phylogenetic analysis. The structural analysis, including conserved motifs, gene structure and homologous modeling, illustrates that the PeCaM/PeCML in the same subgroup have relative conserved structural features. Collinearity analysis suggested that the expansion of the CaM/CML gene family likely took place mainly by segmental duplication, and the whole genome replication events were closely related with the rapid expansion of the gene group. PeCaM/PeCMLs were potentially required for different floral tissues development. Significantly, PeCML26 had extremely high expression levels during ovule and fruit development compared with other PeCML genes, suggesting that PeCML26 had potential functions involved in the development of passion fruit flowers and fruits. The co-presence of various cis-elements associated with growth and development, hormone responsiveness, and stress responsiveness in the promoter regions of these PeCaM/PeCMLs might contribute to their diverse regulatory roles. Furthermore, PeCaM/PeCMLs were also induced by various abiotic stresses. This work provides a comprehensive understanding of the CaM/CML gene family and valuable clues for future studies on the function and evolution of CaM/CML genes in passion fruit.

Conclusion

A total of 32 PeCaM/PeCML genes were divided into 9 groups. The PeCaM/PeCML genes showed differential expression patterns in floral tissues at different development stages. It is worth noting that PeCML26, which is highly homologous to AtCaM2, not only interacts with multiple BBR-BPC TFs, but also has high expression levels during ovule and fruit development, suggesting that PeCML26 had potential functions involved in the development of passion fruit flowers and fruits. This research lays the foundation for future investigations and validation of the potential function of PeCaM/PeCML genes in the growth and development of passion fruit.

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Background

As the main second messenger in plant cells, calcium plays an important role in regulating plant signal transduction [1]. It is involved in regulating plant stress response, growth and development, programmed cell death and other cell processes [2]. A large number of environmental and developmental signals can cause rapid and transient changes in intracellular free Ca2+ concentration [3]. Calcium-binding protein acts as a Ca2+ sensor to detect and decode calcium signals, thereby activating downstream reactions [4]. In plants, there are four main types of Ca2+ sensors: calmodulins (CaMs) and calmodulin-like proteins (CMLs), calcineurin B-like proteins (CBLs) and calcium-dependent protein kinases (CDPKs) [5,6,7]. Most Ca2+ sensors have been found to have a conserved EF-hand (helix-loop-helix structure), which is a Ca2+ binding site [8]. Ca2+ binding leads to changes in protein conformation, which in turn interacts with downstream target proteins and affects their activity, forming a complex calcium signal transduction network system, eventually leading to changes in specific cellular physiological processes [9]. Previous studies have shown that CaM is a highly conserved calcium-binding protein that is widely present in all eukaryotes and contains four EF-hand; CML only exists in plants and some protists, containing 1–6 EF-hand [10, 11]. Each EF-hand contains two α-helices connected by a 12-amino acid loop region [11]. CaM and CML have no catalytic activity, but act as sensor relays to regulate downstream targets [11].

As important Ca2+ sensors, CaMs and CMLs are widely involved in plant growth and development at various stages [12]. For example, AtCML23 and AtCML24 can inhibit the expression of FLOWERING LOCUS C (FLC) gene, thereby affecting the autonomous regulation pathway of the transition to flowering [13]. Moreover, AtCML24 and AtCML25 play a crucial role in pollen germination and pollen tube elongation [14, 15]. AtCML39 plays a vital role in promoting the transduction of light signals during early seedling establishment in Arabidopsis. Besides, overexpression of TaCML20 enhanced the accumulation and yield of water-soluble carbohydrates in wheat [16]. Cotton GhCaM7 promotes cotton fiber elongation by regulating the production of reactive oxygen species (ROS) [17]. In addition, CaMs and CMLs also play an important role in plant response to biotic and abiotic stresses [12, 18]. Overexpression of AtCML43 in Arabidopsis enhances sensitivity and can play a role in the plant’s immune response to pathogens [19]. AtCML20 negatively regulates ABA and drought stress responses in Arabidopsis guard cells [20], while AtCML37 positively regulates ABA accumulation induced by drought stress [21]. Overexpression of OsMSR2 (rice CML gene) can enhance the salinity tolerance, drought tolerance, and ABA sensitivity in Arabidopsis [22]. Overexpression of GmCaM4 in soybean can enhance resistance to plant pathogens and improved tolerance to salt stress [23].

Passion fruit (Passiflora edulis) is a woody vine of the Passifloraceae family which is widely distributed in tropical and subtropical regions [24]. It enjoys the reputation of “the king of juice” due to its attractive aroma of juice that has more than ten kinds of fruits, including mango, pomegranate, banana, strawberry, lemon, pineapple, and litchi [25]. Previous studies have shown that passion fruit is rich in essential vitamins, amino acids, flavonoids, alkaloids, and other bioactive components [26]. Due to the good taste and great nutritional benefits, passion fruit is highly appreciated for fresh consumption and industrial purposes [25, 27]. Additionally, many varieties of passion fruit also have important ornamental value with colorful flowers which characterized with large floral tissues and bright coronal filaments. Moreover, passion fruit is susceptible to environmental factors such as temperature, light, and water, which will seriously affect the flowering, yield and fruit quality of passion fruit [28]. Primary and secondary metabolism processes are closely related to the formation of flower appearance, fruit flavor and quality, as well as stress responses [29]. Thus, it is of great significance to explore the regulatory mechanism of growth and development, and stress responses for maintaining the economic benefits of passion fruit.

Systematic identification and investigations of the CaM/CML genes in various species would further increase our understanding of their evolution and functions, which has been performed in many model plants and crops, such as Arabidopsis [30], rice (Oryza Sativa L.) [31], grapevine (Vitis amurensis) [32], tomato (Solanum lycopersicum) [33], apple (Malus × domestica) [34], Cucumber (Cucumis sativus L.) [35]. However, the CaM/CML gene has not been systematically identified and analyzed in passion fruit, an important tropical fruit crop. Recently, the genome sequence of passion fruit has been published [36, 37], which provides a good opportunity to identify the CaM/CML gene family members and explore their potential regulatory roles in flower and fruit development and stress responses of passion fruit. Here, a total of 32 PeCaM/PeCML genes were identified, and a series of systemic analysis were also performed including the phylogenetic relationships, gene structure, conserved motifs, protein-protein interaction network and expression pattern analysis. Interestingly, we found that although most PeCML genes were expressed at different developmental stages of flowers and fruits, PeCML26 had extremely high expression level during ovule and fruit development compared with other PeCML genes, indicating that PeCML26 may be involved in the flower and fruit development of passion fruit. Furthermore, we also analyzed the response of PeCaM/PeCMLs under different abiotic stresses. Our work could contribute to lay a foundation for further functional investigation on the CaM/CML gene family in passion fruit.

Materials and methods

Identification and characterization of the PeCaM/PeCML genes in passion fruit.

The passion fruit (Passiflora edulis) genomic data was retrieved from the National Genomics Data Center (NGDC) (https://ngdc.cncb.ac.cn/gwh/Assembly/17982/show, accession number: GWHAZTM00000000) [38]. The hidden Markov Model profile (HMM) of the EF-hand_7 (PF13499) was obtained from the Pfam server (https://www.ebi.ac.uk/interpro/entry/pfam), which was used as the seed model for the HMMER search of candidate PeCaM/PeCML genes from the passion fruit genome in TBtools (v1.120) [39], with a cut off E-value of 1 × 10− 5. The redundant sequences were removed manually. The Simple Modular Architecture Research Tool (SMART) (http://smart.embl-heidelberg.de/) [40] and the NCBI-Conserved Domains Database (CDD) (https://www.ncbi.nlm.nih.gov/cdd) [41] database were used to cross-check the presence of the EF-hand domains in candidate sequences. Finally, the sequence containing only the EF-hand domain was retained as the potential PeCaM/PeCML member.

The physicochemical feature of the potential PeCaM/PeCML protein sequences, including molecular weight, theoretical isoelectric point (pI), and the number of amino acids was further analyzed using ExPASy (https://www.expasy.org/) [42]. In addition, the subcellular localization of deduced PeCaM/PeCMLs was predicted through the online website Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/) [43].

Phylogenetic Relationships of CML Proteins.

Then the Arabidopsis CaM and CML protein sequences were downloaded from the Arabidopsis Information Resource (TAIR) (http://www.Arabidopsis.org/). The CML protein sequences of Oryza sativa L., and Populus trichocarpa were obtained according to relevant literatures [31, 34, 44]. A neighbor-joining (NJ) phylogenetic tree was constructed by MEGA (version 11.0) [45] based on the alignment of the CaM/CML protein sequences of passion fruit and Arabidopsis. The parameters were set as follows: bootstrap analysis with 1,000 replicates, poisson correction, and pairwise deletion. In addition, we also constructed a phylogenetic tree of CaM/CML proteins from passion fruit, Arabidopsis, rice, and Populus trichocarpa using the neighbor-joining method and the same parameters. The phylogenetic tree was visualized using iTOL (https://itol.embl.de/).

Gene structure, conserved motif, and cis-elements analysis.

The exon-intron distributions of the PeCaM/PeCML genes were obtained from the gene feature file (GFF) of the passion fruit genome. The domain distributions of PeCaM/PeCMLs were analyzed using the NCBI-CDD (https://www.ncbi.nlm.nih.gov/cdd) [41] database. The conserved motifs of the PeCaM/PeCMLs were analyzed using the MEME online program (http://memesuite.org/tools/meme) [46], with the parameters as follows: the maximum number of motifs, 10; other default parameters. The gene structures, domain, and conserved motifs of PeCaM/PeCMLs were displayed using TBtools (v1.120) software [39].

The upstream 2000 bp sequences of the PeCaM/PeCML were retrieved by TBtools (v1.120) software [39]. The cis-regulatory elements in the 2000 bp upstream putative promoter region of PeCaM/PeCML genes were speculated by the PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [47]. And the results were visualized by TBtools (v1.120) software [39].

Chromosome distribution, gene duplication, and synteny of PeCaM/PeCML genes.

The genome information of Arabidopsis was downloaded from the TAIR (http://www.Arabidopsis.org/). The genomic data of four representative species (Populus trichocarpa, Vitis vinifera, Oryza sativa L., and Zea mays) were downloaded from the Ensembl database (http://plants.ensembl.org/index.html).

The chromosome distribution information of PeCaM/PeCML genes was visualized using Circos [48] in TBtools (v1.120) software according to the GFF file of the passion fruit genome. The gene duplication events of the PeCaM/PeCML genes were analyzed by MCScanX [49] and graphics were displayed using Advanced Circos and Dual Synteny Plot for MCScanX in TBtools (v1.120) software [39]. Based on screening criteria of > 75% identity and query coverage over 75% gene length, the Ka (non-synonymous substitution) and Ks (synonymous substitution) of PeCaM/PeCML genes were calculated using Simple Ka/Ks Calculator in TBtools (v1.120) software [39].

Protein-protein interaction network prediction and homology modeling analysis.

All PeCaM/PeCML protein sequences were submitted to the STRING online website (http://string-db.org). Based on Arabidopsis homologous proteins, the protein–protein interaction network of PeCaM/PeCML proteins was predicted and constructed using the medium confidence parameter (0.4). Proteins that do not interact with other proteins were removed. The secondary structure of PeCaM/PeCML proteins was predicted using SOPMA secondary structure prediction (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa%20_sopma.html). The PDB database (http://www.rcsb.org/) was used to find the most similar homology to the corresponding PeCaM/PeCML protein. Then, the 3D structure of the PeCaM/PeCML proteins was predicted by the Swiss-Model interactive tool (https://swissmodel.expasy.org/interactive/) with default parameters.

Prediction of transcription factor networks.

The Transcription factors prediction and regulatory network analysis as described by Rizwan et al. [50, 51]. The upstream 1000 bp sequences of the PeCaM/PeCML were extracted by TBtools (v1.120) software [39]. Then the extracted sequences were submitted to the plant transcriptional regulation map (PTRM) [52] and the transcription factor prediction was performed with p-value ≤ 1e− 6. Cytoscape software [53] was used to visualize the transcription factor regulatory network.

Expression patterns analysis based on RNA-seq data.

The purple passion fruit (Passiflora edulis sim) used for transcriptomic analysis was planted in the orchard of the Institute of Horticulture, Guangxi Academy of Agricultural Sciences. The RNA-sequencing data of the different floral tissues, including two stages of bract tissues (br1 and br2), two stages of corona filament tissues (ca1 and ca8), two stages of the petal (pe1 and pe8), two stages of sepal tissues (se1 and se8), two stages of stigma tissues (sg1 and sg1), three stages of stamen tissues (st1, st8, and st9), and seven stages of ovule tissues (ov2-ov8), were downloaded from China National Gen-Bank (CNGB) (accession number CNP0005768) [54, 55]. Additionally, the RNA-seq data of four different tissues (roots, flower, leaf, and seed) were obtained from China National Center for Bioinformation (CNCB) (accession number CRA003773) [38]. We used ComBat-seq to remove the batch effect [56]. The heatmap was illustrated by pheatmap packages using R software based on log2 (FPKM + 0.01).

Additionally, fruit samples were gathered during the fruit juice formation period (53 days after pollination/DAP), fruit juice color transformation period (60 DAP), peel color transformation period (100 DAP), and fruit ripening period (128 DAP). Detailed sample information is also listed in Supplementary Table S9. RNA extraction and Illumina sequencing were performed as previously described [57]. The TPM (Transcripts Per Kilobase Million) value of the PeCaM/PeCML genes at different developmental stages of passion fruit was calculated. The expression profile of PeCaM/PeCML genes at different senescence stages was measured by their TPM value, and the heat map was performed by TBtools (v1.120) software [39].

Abiotic stress treatments.

The plants used for stress treatments were grown in a growth chamber under long-day conditions (16 h light/8 h dark) at 27/22°C (day/night) at the Center for Genomics and Biotechnology, Fujian Agriculture and Forestry University. The 3-week-old passion fruit seedlings were treated with cold (8 ± 2 ℃), heat (45 ± 2 ℃), osmotic (200 mM mannitol), and ABA (100 mM ABA) stress, respectively [54, 55]. For cold and heat stress treatment, healthy seedlings in soil were placed in the growth chamber at 8 ℃ or 45 ℃; For osmotic and salt stress, the passion fruit seedlings were first cultured in 1/2 MS liquid medium for 7 days, and then transferred to fresh 1/2 MS liquid medium containing 100 mM ABA or 200 mM mannitol for stress treatment. Plant leaves were collected from at least three independent plants at 0 h, 24 h, and 48 h time points after stress treatment, and untreated plant samples were collected as the control. Then, all collected samples were rapidly frozen with liquid nitrogen and stored at − 80 ℃ for RNA extraction.

RNA extraction and quantitative real-time PCR.

The total RNA was extracted by the Trizol method (Invitrogen, Carlsbad, CA, USA), and the HiScript III Reverse Transcriptase (Vazyme Biotech, Nanjing, China) was used for the reverse-transcription experiment. The qRT-PCR was performed in the Bio-Rad Real-time PCR system (Foster City, CA, USA), using the SYBR Premix Ex Taq II system (TaKaRa Perfect Real Time) with a 20 µL sample volume, and the primers used has been listed in Supplementary Materials Table S10. Each sample was performed three times technically using a replicate, with a total of three biological replicates for each sample. EF1a was used as an internal control to normalize the mRNA levels [58, 59]. The fold change of genes was calculated using the 2−ΔΔCT method [59].

Results

Identification of CaM/CML TFs in passion fruit.

According to the hidden Markov model (HMM) profile of the EF-hand domain, candidate genes with EF-hand domain were preliminarily screened from the genome database of passion fruit. The redundant sequences were removed manually. Subsequently, EF-hand domain was validated by SMART and NCBI-CDD. As a result, a total of 32 PeCaM/PeCML genes were identified, which included 1 PeCaM genes and 31 PeCML genes. All of the PeCaM and PeCML were named according to the position of genes on chromosomes. It is noteworthy that PeCML26 is not only highly homologous to PeCaM1 but also closely related to AtCaMs phylogenetically, but it lacks a typical EF-hand domain. Therefore, it was excluded from the PeCaM group and defined as the PeCML gene.

Then, the biochemical properties of PeCaM and PeCML proteins were predicted using the ExPASy and Cell-PLoc 2.0 (Table 1). As shown in Table 1, PeCaM1 contains four typical EF hand motifs, and PeCMLs contained two to four EF-hands motifs. The lengths of the protein sequences of PeCaM and PeCML ranged from 84 (PeCML16) to 365 (PeCML28) amino acids, and molecular weight (MWs) varied from 9.23 kDa (PeCML16) to 39.72 kDa (PeCML28). Moreover, the theoretical pI of PeCaM/PeCMLs was from 3.95 (PeCML17) to 9.2 (PeCML20). Most of the predicted grand average of hydropathicity (GRAVY) of PeCaM/PeCMLs was negative, and only a few (3/30) were positive, indicating that most of the PeCaM/PeCMLs were hydrophilic proteins and a few were hydrophobic proteins. Most of PeCaM/PeCML proteins were predicted to be located on the cell membrane, but some were also located on cytoplasm, vacuole, nucleus and chloroplast. The diversity of PeCaM/PeCML proteins localization indicates the different functions of these proteins in calcium signal transduction.

Table 1 Physiochemical properties of PeCaM/PeCML genes
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Chr*-chromosome NO.; AA*-amino acid/protein length; AI*--aliphatic index; GRAVY*-grand average of hydropathicity; pI*-isoelectric point; MW*-molecular weight (Da); SCL*-Sub-cellular localization.

Phylogenetic analysis of CaMs/CMLs.

To analyze the phylogenetic relationship and gene function of PeCaMs and PeCMLs, a neighbor-joining (NJ) tree containing 32 PeCaM/PeCMLs and 57 AtCaMs/AtCMLs was constructed by MEGA software (Fig. 1). According to the topological structure of the phylogenetic tree and the classification of AtCaMs/AtCMLs [30], PeCaM/PeCML proteins and AtCaM/AtCML proteins were clustered into nine groups (Groups I–IX), and each group contained different amounts of PeCaM/PeCML proteins. Group II had the largest number of PeCML members (8), followed by Group VII (7) and Group III (4). Group V is the smallest, which contains only one member (PeCML2). Among them, PeCML26 is most closely related to CaMs, suggesting its potential function as CaM. At the same time, we also performed phylogenetic analysis of PeCaM/PeCML with CaM/CML proteins in Arabidopsis, rice, and Populus trichocarpa (Supplementary figure S1). Phylogenetic analysis showed that CaM/CML proteins from Arabidopsis, rice, Populus trichocarpa and passion fruit were highly similar, indicating that homologous members have similar functions.

Fig. 1
figure 1

Phylogenetic tree of CaM and CML proteins from passion fruit (Pe) and Arabidopsis thaliana (At). The white and red triangles represented genes from Arabidopsis and passion fruit, respectively. All 9 groups of CaM/CML were well separated in different clades and represented by different colors

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Gene structure, conserved motifs, and cis-elements analysis of PeCaM/PeCML.

To understand the structural and genetic diversity of PeCaM/PeCML genes, the gene structure and the composition of conserved motifs of 32 PeCaM/PeCMLs and 57 AtCaMs/AtCMLs proteins were analyzed and displayed according to their phylogenetic relationships (Fig. 2A-D). The exon-intron structure 89 CaMs/CMLs was analyzed and visualized using the TBtools software (Fig. 2B). The result showed that the number of exons varied from 1 to 7 in CaM/CML genes. Among the 89 CaMs/CMLs, 49 CaMs/CMLs contained a single exon without intron, including 16 PeCMLs. These intronless CaM/CMLs were generally distributed in groups IV to IX. In addition, the genes with similar exon-intron structures, especially in terms of the number of introns, tended to cluster in the same subgroup in the phylogenetic tree. At the same time, we analyzed the domains of PeCaM/PeCMLs and AtCaMs/AtCMLs, and the results showed that most of CMLs belonged to the PTZ00184 superfamily and a few belonged to other superfamilies (Fig. 2C). MEME analysis also proved this similarity (Fig. 2D). The results indicated that motif 1, motif 2, motif 3, and motif 4 represent four different types of EF hands motifs. PeCaM1 contains four types of motifs, which constitute a typical EF-hand domain. At the same time, PeCMLs contain two to four different EF hands motifs. Overall, genes in the same group showed a similar motif composition, however, there were noticeable differences between subgroups suggesting that the motif pattern may be related to the function of the CaM/CML proteins. For instance, motif 6 and motif 8 only appeared in group IX, it may be possible that some genes in the group IX have changed during evolution. The motif analysis of CaMs/CMLs revealed that the members under the same group were highly conserved, further proving the proximity of their evolutionary relationship in the phylogenetic tree.

Fig. 2
figure 2

Distributions of gene structure and conserved motifs in CaMs/CMLs. (A) Phylogenetic relationship of PeCaM/PeCMLs and AtCaMs/AtCMLs. (B) Gene structure of PeCaM/PeCMLs and AtCaMs/AtCMLs. (C) The domain distribution of PeCaM/PeCMLs and AtCaMs/AtCMLs. (D) The conserved motifs of PeCaM/PeCMLs and AtCaMs/AtCMLs.

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Gene promoters contain many essential cis-acting elements that regulate the expression of corresponding genes at the transcriptional level [60]. To explore the transcriptional regulation of PeCaM/PeCMLs, the PlantCARE tool was used to predict the cis-elements in 2000 bp upstream sequences of the PeCaM/PeCMLs (Fig. 3). As a result, a total of 15 cis-regulatory elements were identified in the promoter, which were divided into three categories: hormone responsiveness, growth and development, stress responsiveness (Supplementary Materials Table S2). Among them, light responsive element was most commonly present in the promoter regions of PeCaM/PeCML genes, followed by the MeJA-responsive element and ABA-responsive element (ABRE). At the same time, all PeCaM/PeCML gene promoters contained at least two hormone responsive elements and stress responsive elements, suggesting that PeCaM/PeCMLs may respond to plant hormones and abiotic stresses. Some elements involved in plant growth and development were observed in a few genes, such as circadian control, endosperm expression, and seed-specific regulation element. These results suggest that the expression of PeCaM/PeCML genes might be regulated by various cis-acting elements related to hormone responsiveness, growth and development, stress responsiveness.

Fig. 3
figure 3

Regulatory elements in the promoter region of CaM/CML genes in passion fruit. (A) Distribution of cis-acting elements identified in the 2000 bp upstream region of PeCaM/PeCMLs genes. Different colors represent the different types of cis-elements. (B) The number of cis-acting elements on putative promoters of PeCaM/PeCML genes

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Chromosome localization and collinearity analysis of PeCaM/PeCML genes.

To study the genetic divergence of the CaM/CML family of passion fruit, the chromosome localization of the PeCaM/PeCML genes was analyzed (Fig. 4). As a result, the 30 PeCaM/PeCML genes were unevenly mapped on nine chromosomes, while two were distributed on unassembled scaffolds. Chromosome 6 has the maximum number of PeCaM/PeCML genes (13 genes, 40.6%), followed by chromosome 1, which contains 4 (12.5%) genes. The remaining chromosomes had between 1 and 3 genes, indicating no correlation between chromosomes and gene number.

The gene duplication event analysis revealed 7 pairs of segmentally duplicated genes and 1 pairs of tandemly duplicated genes were identified in PeCaM/PeCMLs (Fig. 4, Supplementary Materials Table S3-1). The only tandem duplication event occurred on chromosome 6, whereas segmental duplication events occurred on chromosomes 1, 3, 4, and 6. Of the 32 genes, 8 (25.0%) produced 7 segmental duplication gene pairs suggesting that gene duplication events (segmental duplications) contributed significantly to the diversity and evolution of PeCaM/PeCMLs. Among the 7 pairs of segmental duplication genes, 6 pairs of duplication genes were distributed in group II, indicating that the PeCML genes in group II may have functional redundancy. The ratio of Ka (non-synonymous substitution) and Ks (synonymous substitution) can reflect the selection pressure during organism evolution. Therefore, to explore the role of selection pressure in the evolution of the CaM/CML gene family, the Ka/Ks ratios of homologous genes were obtained (Supplementary Materials Table S3-2). The Ka/Ks ratios of all PeCaM/PeCML gene pairs were less than 1, indicating that purification selection played a role in the evolution of PeCaM/PeCML genes.

Fig. 4
figure 4

Chromosome location and gene duplication of PeCaM/PeCML genes on 9 chromosomes. PeCMLs marked in red have collinearity, while PeCMLs marked in black lack collinearity. The inner rings represent the gene density of each chromosome. The red line represents the segmented and tandem duplication gene pairs among PeCMLs.

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To further explore the evolutionary mechanisms of PeCaM/PeCML genes, the synteny and collinearity of passion fruit and five representative plants (Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera, Oryza sativa, and Zea mays) were analyzed (Fig. 5, Supplementary Materials Table S4). As expected, the genomes of dicotyledon plants P.trichocarpa (57 orthologous gene pairs scattered on all chromosomes except chromosome 2), V.vinifera (32 orthologous gene pairs scattered on all chromosomes except chromosome 2), and A.thaliana (20 orthologous gene pairs scattered on all chromosomes except chromosome 2/7/9) have more collinear gene pairs with the genome of passion fruit, while monocotyledon plants O.sativa (7 orthologous gene pairs scattered on chromosomes 1/3/6) and Z.mays (4 orthologous gene pairs scattered on chromosomes 1/3/6/8) have less homologous gene pairs. These results show that the divergence of passion fruit occurs after the common ancestor divergence of monocotyledons and dicotyledons. The number of collinear gene pairs between passion fruit and Populus trichocarpa was far greater than that among other species, indicating that the genetic relationship between passion fruit and Populus trichocarpa is closer than that of the other four species. Among all the PeCaM/PeCMLs, two genes (PeCML7 and PeCML15) had collinearity with all five species, suggesting that PeCML7 and PeCML15 played an important role in the evolution of CaM/CML gene family.

Fig. 5
figure 5

Interspecific collinearity analysis of PeCaM/PeCMLs and five representative plants (Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera, Oryza sativa, and Zea mays). Grey lines in the background indicate collinear blocks in passion fruit and other plant genomes, while red lines highlight syntenic PeCaM/PeCML gene pairs

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Protein interactions and homology modeling analysis of PeCaM/PeCML protein.

In order to better study the biological function and regulatory network of PeCaM/PeCMLs, the protein-protein interaction network was predicted based on known proteins in Arabidopsis (Fig. 6). Among them, 26 PeCaM/PeCML proteins are related to known Arabidopsis proteins (Supplementary Table S5). The results showed that there were 14 nodes in the PeCaM/PeCMLs protein interaction network, and most of the nodes interacted with multiple other nodes. In addition to PeCML1, PeCML5, and PeCML9 proteins interacting with only one protein, other proteins showed more complex protein interactions, of which PeCML29 interacted with the largest number of proteins. The co-expression of PeCML13 with PeCML2 and PeCML22, and PeCML2 with PeCML7 indicates that they can form a complex biological network to further regulate cell function. The prediction of the protein interaction network showed the interaction between multiple proteins, which indicated the functional diversity of PeCaM/PeCML proteins.

The secondary structure of the protein mainly includes the α-helix, extended strand, β-turn and random coil. We analyzed the secondary structure of all PeCaM/PeCML proteins (Supplementary Table S6-1), and found that the α-helix had the largest proportion in the secondary structure, such as PeCML26 (65.96%); followed by random coil, such as PeCML30 (49.61%); followed by extended strand, such as PeCML10 (12.79%); the proportion of β-turn is the smallest, such as PeCML12 (12.06%). Since each EF-hand motif is composed of α-helix connected by 12 amino acid residues, while PeCaM/PeCML protein contains two to four EF-hand motifs, which may be the reason for the high proportion of α-helix in the secondary structure. In addition, we also predicted the 3D structure of PeCaM/PeCML proteins by SWISS-MODEL database (Supplementary Figure S2). The highest GMQE was selected as the best structure of PeCaM/PeCML proteins (Supplementary Table S6-2). The results showed that most PeCaM/PeCML proteins contain classical EF-loops, which is the core region of the EF-hand domain binding Ca2+. PeCaM/PeCML proteins can bind to Ca2+ through the EF-loop and enter the activated state, resulting in changes in its conformation, thereby specifically activating the target protein to exert cell function. In addition, some PeCML proteins exhibit different 3D structures, indicating the functional diversity of PeCML proteins.

Fig. 6
figure 6

Protein–protein interaction and predicted 3D models of PeCaM/PeCML proteins

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Prediction of transcription factor regulatory network of PeCaM/PeCML genes.

In order to explore the potential regulatory network of PeCaM/PeCML gene family, the potential TFs were predicted for 1000 bp upstream of all PeCaM/PeCML genes, and the TF regulatory network was constructed by Cytoscape. The results showed that the PeCaM/PeCML genes were regulated by a variety of transcription factor families, including ERF, MYB, bHLH, BBR-BPC, AP2 and bZIP (Fig. 7 and Supplementary Table S7). Among them, ERF family had the most members (260), followed by BBR-BPC (154), Dof (71), MIKC_MADS (42) and bZIP (38). Among all 32 PeCaM/PeCML genes, PeCML25 was targeted by the most TFs (186), followed by PeCML22 (62), PeCML24 (60), and PeCML9 (50), while PeCML2, PeCML6, PeCML14, and PeCML31 were targeted by only one TF. Different PeCaM/PeCML genes were targeted by diverse types and numbers of TF families. For example, the families targeting PeCML25 include ERF (126), Dof (26) and MIKC_MADS (15); the families targeting PeCML23 gene were BBR-BPC (35) and AP2 (3). Among the TFs targeting PeCaM/PeCML genes, there were many TFs related to plant growth and development and response to biological and abiotic stress, such as ERF, bZIP, BBR-BPC, WRKY, bHLH, and AP2.

Fig. 7
figure 7

The putative transcription factor regulatory network of PeCaM/PeCML genes. Green oval nodes represent transcription factors; the orange round nodes represent the PeCaM/PeCML genes

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Expression analysis of PeCaM/PeCML genes based on transcriptomic data.

To explore the possible functions of the PeCaM/PeCMLs, the expression profiles of the 32 PeCaM/PeCMLs in the various tissues at different developmental stages were characterized using RNA-seq data (Fig. 8). Genes with low expression levels in all samples were filtered out. The results showed the expression variation of PeCaM/PeCML genes at different stages of flower development. For example, PeCML6, PeCML9, and PeCML26 were highly expressed throughout the ovule development stage. PeCML12, PeCML13, PeCML23, and PeCML24 were highly expressed in the early stages of sepal, corona filament, stigma and stamen development. PeCaM1 was highly expressed in all vegetative tissues and various developmental stages of floral tissues, with higher expression patterns in sepal and ovule development stages, indicating that PeCaM1 is ubiquitous in the growth and development of passion fruit and involved in early flower development. In addition, seven genes (PeCML7, PeCML10, PeCML11, PeCML16, PeCML22, PeCML27, and PeCML30) showed high expression levels in the late stage of stamen development, suggesting that these genes may play essential roles in stamen development. Furthermore, we also found that some PeCML genes were specifically expressed in specific tissues. For example, PeCML19 and PeCML21 were highly expressed in roots. Interestingly, some PeCML genes were preferentially expressed in multiple vegetative tissues, such as PeCML17 and PeCML18, which were highly expressed in root, leaf, and flower tissues. These results indicated that PeCaM/PeCML genes exhibited a certain degree of tissue expression specificity.

Fig. 8
figure 8

The expressional pattern of PeCaM/PeCML genes in passion fruit. The expression of PeCaM/PeCMLs in diverse tissues at different developmental stages (bract (br); sepal (se); petal (pe); corona filament (ca.); stamen (st); stigma (sg); ovule (ov); numbers represent developmental stages, 1 and 2 was early stage, 8 was the later stage. Blue or orange colors represent the difference in expression levels, respectively. The heatmap was created according to the log2(FPKM + 0.01) value of PeCaM/PeCMLs and normalized by row. The FPKM value higher than 50 was shown as abundant genes, and marked with “*”

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In order to verify the expression of PeCaM/PeCML genes in floral tissues development and various organs, qRT-PCR was used to study the expression profiles of seven representative PeCML genes (PeCML6, PeCML9, PeCML11, PeCML15, PeCML17, PeCML22 and PeCML26) in the same flower and organ sample (Fig. 9). Overall, the trends of the qRT-PCRs for the six PeCML genes were consistent with the results of RNA-seq analysis. PeCML11 was highly expressed at the late developmental stages in multiple floral tissues such as petals, ovules, stamens and corona filament, and PeCML22 was significantly highly expressed during late stamen development. PeCML15 was highly expressed in late developmental stages of petals and ovules and specifically expressed in root organ. In addition, PeCML6 and PeCML9 were highly expressed in the late developmental stages of petals and ovules.

Fig. 9
figure 9

qRT-PCR analysis of 7 genes (PeCML6, PeCML9, PeCML11, PeCML15, PeCML17, PeCML22 and PeCML26) in 4 floral tissues (petal, ovule, corona filament, and stamen) and three organs. All experiments were repeated three times. The error bar represents the standard deviation (SD) of three replicates. Asterisks indicate significant differences in transcript levels compared with those of early stage of petal development (pe1). (*P < 0.05, **P < 0.01, ***P < 0.001)

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We also analyzed the expression patterns of PeCaM/PeCML genes at different developmental stages after the pollination based on RNA-seq (Fig. 10). Among these PeCaM/PeCML genes, the expression levels of PeCML3, PeCML27, and PeCML30 were continuously increased with the prolongation of the time after pollination, while the expression levels of PeCML15 were decreased. In addition, other PeCML genes such as PeCML6 and PeCML26 exhibited fluctuating expression patterns. Interestingly, compared to other PeCML genes, PeCML26 had extremely high expression levels during fruit development, suggesting that PeCML26 may be involved in the fruit development of passion fruit. Besides, we verified the expression level of PeCML26 at different stages of passion fruit by qRT-PCR, and the result was consistent with RNA-seq results (Supplementary figure S3). These results indicate that different PeCMLs play vital roles in various stages of passion fruit development.

Fig. 10
figure 10

The expression profiles of PeCaM/PeCML genes at different stages of passion fruit. DAP, the day after pollination. Blue or orange colors represent the difference in expression levels, respectively

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Expression pattern of PeCaM/PeCML genes in response to abiotic stress.

In order to explore whether the PeCaM/PeCML genes are involved in the response of passion fruit to abiotic stress, seven PeCML genes (PeCML5, PeCML6, PeCML8, PeCML12, PeCML17, PeCML23, and PeCML24) were randomly selected to analyze their expression patterns under temperature stress, ABA stress and osmotic stress using qRT-PCR methods (Fig. 11). Under low-temperature treatment, five genes (PeCML5, PeCML8, PeCML12, PeCML23, and PeCML24)) had similar expression patterns and showed a continuous increase with the treatment time. The relative expression level of PeCML6 and PeCML17 first decreased and then increased with the treatment time. Whereas under heat treatment, only the expression levels of PeCML12 significantly up-regulated, and other PeCML genes (PeCML5, PeCML6, PeCML8, PeCML17, PeCML23, and PeCML24) showed fluctuated expression patterns. In terms of ABA stress, all the seven genes showed a fluctuating expression pattern, but the expression levels of the other six genes except PeCML17 were increased compared with the control at different times, while the expression level of PeCML17 was decreased compared with the control. During the osmotic treatment, PeCML6, PeCML8, PeCML12, PeCML23, and PeCML24 exhibited fluctuated expression patterns, the expression level of PeCML5 was significantly up-regulated and the expression level of PeCML17 was down-regulated. Interestingly, PeCML5 exhibited significantly up-regulated expression levels in response to all the stresses. The expression level of PeCML12 showed an increasing trend only under cold and heat treatments, while PeCML23 and PeCML24 increased under only cold stress conditions.

Fig. 11
figure 11

Expression patterns of seven PeCML genes under cold, heat, osmotic, and ABA stress. The ordinate is the relative expression level (multiple) of PeCMLs compared with the internal reference gene (EF1a). All experiments were repeated three times. The error bar represents the standard deviation (SD). Asterisks indicate significant differences in transcript levels compared to control values (*P < 0.05, **P < 0.01, ***P < 0.001)

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Fig. 12
figure 12

The potential function of CaM/CML genes in passion fruit. The schematic diagram model of PeCaM/PeCML gene expression patterns during flower and fruit development and its application. The PeCML genes marked in red were highly expressed in the corresponding tissue

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Discussion

Increasing investigations suggests that Ca2+ is a second messenger involved in plant growth and development and in response to various biotic and abiotic signals [1, 2, 61]. Plants have evolved a variety of Ca2+ sensors that bind to calcium ions. Calmodulin (CaM) and Calmodulin-like (CML) proteins are the main calcium sensors in eukaryotes. They have been extensively studied in a variety of plants, such as Arabidopsis [30], rice [31], apple [34], grape [32]. Passion fruit is a famous and economically valuable fruit crop whose growth and development processes are susceptible to various environmental factor changes [62]. Although many PeCaM/PeCML genes have been reported in numerous plants, limit information is available about the passion fruit PeCaM/PeCML genes. For the first time, we systematically studied the CaM/CML gene family of passion fruit with bioinformatics tools and expression profiles.

In this study, a total of 32 PeCaM/PeCML genes were identified in the passion fruit genome, including 1 PeCaM gene and 31 PeCML genes. Compared with other plants, the number of PeCMLs was more than Ginkgo biloba (21) [63] and Chinese jujube (23) [64] but was less than that in Arabidopsis (50) [30], rice (32) [31], grape (62) [32] and apple (58) [34]. Notably, the number of PeCMLs genes was not significantly correlated with genome size. For example, Arabidopsis has 50 CML gene family members with a 125 Mb genome size, while the genome size of passion fruit is more than 9 times that of Arabidopsis with fewer number of PeCMLs. The number of PeCMLs in grape was about twice to that of in passion fruit. In the process of evolution, grape experienced three genome-wide replication events while passion fruit experienced only twice [38, 65]. This indicates that the number of PeCML genes in plants is not related to genome size, but closely related to the evolution of species. According to the phylogenetic analysis results, all the CaM/CML genes from passion fruit (32 PeCaM/PeCMLs) and Arabidopsis (57 AtCaM/AtCMLs) were divided into 9 groups. All groups contained the CaM/CML members from both passion fruit and Arabidopsis, suggesting that they might be derived from a common ancestor. In addition, the neighbor-joining phylogenetic tree constructed with Arabidopsis, rice, and P. trichocarpa CaM/CMLs showed their evolutionary relationships and potential functional similarities (Supplementary Fig. 1).

The members clustered in the same subgroup share similar exon-intron structure, motif composition, and distribution order. Previous studies have shown that most CML genes have no intron, while some genes have less than ten introns [30, 31, 66]. As previously other plants, most of the PeCaM/PeCML genes were intron-less, and other genes had no more than 6 introns, which could indicate their conserved functions in different species. Motif 1, motif 2, motif 3, and motif 4 were highly conserved in most PeCaM/PeCML proteins and represented different types of EF hands motifs, which were critical for the functional specificities of these TFs [66]. The composition of the EF-hand motif also reveals that PeCaM/PeCMLs has a certain degree of diversity. Most of them (20 out of 32) have four different or identical EF-hand motifs, and the remaining members have at least one EF-hand motif. The number of EF-hand motifs of PeCaM/PeCML genes was consistent in the same group. In contrast, the structure of different groups varied considerably. For instance, group III and group IV have considerably different motif compositions from other subgroups, and motif 10 is unique to group III, suggesting that the of PeCML genes in group III may have undergone distinct evolutionary development and have specific functions. The expression profiles of PeCML genes in group III were different. For example, PeCML25 was highly expressed in root and seed tissues, while PeCML28 was highly expressed in sepals. Thus, the structural diversity among different subgroups might contribute to the functional diversity of the CaM/CML gene family.

Gene replication events play an important role in the expansion of gene family members, the segmental duplication and tandem duplication are the main reasons for the expansion of gene families in the genome [67]. Here, in the passion fruit PeCaM/PeCML gene family, 1 pairs of tandem duplication and 7 pairs of segmental duplication genes were identified, indicating that the segmental duplication events were the main source for the expansion of the CaM/CML gene family of passion fruit. The Ka/Ks value of all duplicated gene pairs are less than 1, indicating that PeCaM/PeCML genes have gone through purifying selection during evolution. Some duplicated genes showed similar expression patterns and clustered in the same subfamily, such as PeCML6 and PeCML9 from the group II were both highly expressed throughout the ovule development stage, indicating that they might have functional redundancy. However, most duplicated gene pairs exhibited diverse expression patterns, which indicates that the gene members of the CaM/CML family of passion fruit may have functional differentiation. For example, although PeCML19 and PeCML23 were in group II, they showed completely different expression patterns. PeCML19 showed low expression levels at different developmental stages of various floral tissues, while PeCML23 was highly expressed in the early developmental stages of the sepal, corona filament, stigma, and ovule. The difference in the expression patterns of these duplicate genes infers the functional diversity of PeCaM/PeCML genes. According to the collinearity analysis, the collinearity relationship between passion fruit and dicotyledon plants is much closer than that between passion fruit and monocotyledon plants. Among them, the number of homologous genes between passion fruit and Populus trichocarpa was the maximum, indicating that these homologous genes may have the same ancestors and retain the corresponding functions.

Interaction network analysis can better understand the biological functions and molecular pathways of proteins. In this study, protein-protein interaction analysis showed that most PeCaM/PeCML proteins were homologous and interacted with known Arabidopsis proteins, indicating that PeCaM/PeCML may have similar functions to the corresponding proteins. Homologous modeling results also showed that gene members from the same group shared similar protein secondary structure and 3D structure features, while PeCaM/PeCML proteins from different groups present different 3D structure features, which supports the classification results. For example, PeCML23 and PeCML24 in II group not only have the same 3D structure but also are highly expressed in the early stage of flower tissue. In summary, the diversity of PeCaM/PeCML protein structure also indicates the difference between protein functions. In addition, we also predicted the TFs in the promoter region of the PeCaM/PeCML genes and constructed a transcription factor regulatory network. The results showed that ERF, BR-BPC, Dof, MIKC_MADS, bZIP, and AP2 families accounted for the largest proportion. BBR-BPC1 is involved in the regulation of Arabidopsis ovule development by regulating SEEDSTICK (STK) [68]. The bZIP protein plays an important role in various biological functions such as plant growth and development, seed maturation, response to light signals and environmental stress [69, 70]. It is worth noting that PeCaM/PeCML proteins that interact with these transcription factors may have similar functions. PeCaM1 showed high expression levels in various developmental stages of floral tissues and various vegetative tissues. At the same time, it mainly interacted with various transcription factors of bZIP and BBR-BPC families, indicating that PeCaM1 was mainly involved in plant development through these transcription factors. PeCML26 also has a similar expression pattern with PeCaM1 and interacts with multiple BBR-BPC TFs, further illustrating the importance of PeCML26 in the growth and development of passion fruit. Moreover, PeCML26 was highly expressed during ovule development and fruit development, further indicating that PeCML26 plays a crucial role in the development of flowers and fruits in passion fruit. Through qRT-PCR expression analysis, it was found that the expression levels of PeCML genes were positively or negatively regulated under various stress conditions, and it interacted with a large number of TFs (Fig. 11). Consistent with the predicted function, these results indicate that PeCaMs/PeCMLs play important roles in various growth and development processes of plants and in response to various biotic and abiotic stresses.

As a popular fruit and ornamental crop, it is of great significance to exploring the regulatory mechanism of flower and fruit development of passion fruit. The biological functions of most PeCaM/PeCMLs remain unclear, and the identification of putative orthologs in different species will provide valuable reference for the functional study of these genes. Previous studies suggested that AtCML24 and AtCML25 were involved in the regulation of pollen germination and pollen tube growth in Arabidopsis [14, 71]. PeCML11 clustered with AtCML24 and AtCML25 showed a high expression pattern at the late developmental stage of stamen and stigma, suggesting that PeCML11 may have similar functions in the regulation of pollen germination and pollen tube growth. PtCML20 is involved in the development of leaves in Populus trichocarpa [44]. PeCML8 clustered in the same subgroup was highly expressed in leaf, suggesting that PeCML8 may be involved in the leaf development of passion fruit. PtCML23 shows a gradually upregulated expression trend with flower development [44], and the expression level of PeCML7 clustered with PtCML23 increases with flower development, indicating that PeCML7 may be involved in flower development. AtCML42 is involved in trichome formation and is broadly expressed in plant tissues (such as flowers, leaves and roots) [72], the homologous PeCML2 expressed highly in flower, leave and root tissues, suggesting that PeCML2 may play similar roles in the development of passion fruit tissues. In addition, the tissue expression pattern indicated that PeCMLs played an important role in the reproductive development of passion fruit, as shown in Fig. 12. This also provides an important basis for understanding the role of CML genes in regulating the development of passion fruit flowers and fruits.

The growth and development of passion fruit are susceptible to the alterations of environmental factors including light, temperature and water [73]. CaM/CML genes have also been proven to be related to the abiotic stress response [12]. For example, ectopic expression of soybean GsCML27 in Arabidopsis enhances the tolerance of plants to bicarbonate stress [74]. The expression of seven PeCML genes was analyzed by qRT-PCR under four abiotic stress conditions, including cold, heat, ABA, and osmotic stress. OsCML8 showed high expression levels under osmotic and salt stress in rice [75]. PeCML6 and OsCML8 were clustered in the same group, and the expression level of PeCML6 was upregulated under osmotic and salt stress, indicating that PeCML6 may be involved in Ca2+-mediated responses to stimuli. Studies have shown that AtCML9 is involved in response to cold, ABA and drought treatments [76]. PeCML5 in the same group as AtCML9 responded to various stresses. At the same time, PeCML5 also has the most five MYB binding sites involved in drought inducibility, and its expression was significantly increased under drought stress, indicating that PeCML5 plays a crucial role in the response of passion fruit to drought stress. PeCML23 and PeCML24 contain the most ABA responsive elements and some low-temperature responsive element, and they are highly sensitive to cold and drought stress, indicating their potential functions in the process of cold and drought. These results indicate that the results of PeCML cis-elements are consistent with the results of qRT-PCR expression analysis, and PeCML genes containing more stress-related cis-elements not only respond to multiple stresses but also their expression levels are highly up-regulated. The complex interactions with multiple hormone regulations might enable these PeCMLs to be involved in diverse biological processes. The response of PeCMLs to abiotic stress can alleviate the further damage of abiotic stress to the normal growth of passion fruit, which plays an important role in the development of passion fruit.

Conclusions

Here, a total of 32 PeCaM/PeCML genes were identified in the passion fruit (Passiflora edulis) genome, including 1 PeCaM gene and 31 PeCML genes. Based on the phylogenetic relationships, 32 PeCaM/PeCML genes were divided into 9 groups. The PeCaM/PeCML genes were randomly distributed on 9 passion fruit chromosomes. Gene structure and motif composition analysis showed that PeCaM/PeCMLs in the same subgroup were highly conserved. Collinearity analysis showed that segmental duplication events contributed to the expansion of the CaM/CML family of passion fruit. Cis-regulatory elements of promoters were involved in growth and development, Hormone responsiveness, and stress responsiveness. The interactions among PeCaM/PeCML proteins or with other transcription factors may make the regulatory network of PeCaM/PeCMLs more diverse and complex. The PeCaM/PeCML genes showed differential expression patterns in floral tissues at different development stages. It is worth noting that PeCML26, which is highly homologous to AtCaM2, not only interacts with multiple BBR-BPC TFs, but also has high expression levels during ovule and fruit development, suggesting that PeCML26 has potential functions involved in the development of passion fruit flowers and fruits. Additionally, seven PeCML genes also showed a differential response under various abiotic stresses (cold, heat, ABA, and drought). This research lays the foundation for future investigations and validation of the potential function of PeCaM/PeCML genes in the growth and development of passion fruit.

Data availability

The data presented in this study are available in the article, Supplementary Materials, and online repositories. RNA-seq data used in this work were deposited in China National GenBank (CNGB) under (accession number CNP0005768 and CNP0005717) and China National Center for Bioinformation (CNCB) (accession number CRA003773).

Abbreviations

ABA:

Abscisic acid

CaM:

Calmodulin

CML:

Calmodulin-like

FPKM:

Fragments Per Kilobase of exon model per Million mapped fragments

Gravy:

Grand average of hydropathicity

HMM:

Hidden markov mode

MEME:

Multiple EM for motif elicitation

qRT-PCR:

Quantitative real-time PCR

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Acknowledgements

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Funding

This work was supported by the Natural Science Foundation of Guangxi (2022GXNSFAA035535); the China Postdoctoral Science Foundation (2022MD713731) to LW; and the Science and technology innovation project of Pingtan Science and Technology Research Institute (PT2021007, PT2021003).

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Author notes

  1. Dan Zhang, Lumiao Du and Jinting Lin contributed equally to this work.

Authors and Affiliations

  1. Horticulture Research Institute, Guangxi Academy of Agricultural Sciences, Nanning Investigation Station of South Subtropical Fruit Trees, Ministry of Agriculture, Nanning, 530007, China

    Dan Zhang, Lumiao Du, Biao Deng, Weiqiang Su, Yuming Lu & Xiaomei Wang

  2. College of Life Science, Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Pingtan Institute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou, 350002, China

    Dan Zhang, Lumiao Du, Jinting Lin, Lulu Wang, Ping Zheng, Yuan Qin & Xiaomei Wang

  3. Fine Variety Breeding Farm in Xinluo District, Longyan, 364000, China

    Wenbin Zhang

  4. College of Life Sciences, Longyan University, Longyan, 364000, China

    Yanhui Liu

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  1. Dan Zhang
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Contributions

DZ, YQ, and XM conceived and designed the experiments; DZ, JL, and LD authored and reviewed drafts of the paper, LW, PZ and BD prepared figures and tables, WZ and WS performed qRT-PCR analysis and prepared the figures, YL and YL helped analyzed the data and revised the manuscript, and all authors approved the final manuscript.

Corresponding authors

Correspondence to Yuan Qin or Xiaomei Wang.

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Zhang, D., Du, L., Lin, J. et al. Genome-wide identification and expression analysis of calmodulin and calmodulin-like genes in passion fruit (Passiflora edulis) and their involvement in flower and fruit development. BMC Plant Biol 24, 626 (2024). https://doi.org/10.1186/s12870-024-05295-y

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Keywords

BMC Plant Biology

ISSN: 1471-2229

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