Introduction

Filamentation temperature-sensitive H (FtsH) is an ATP-dependent zinc metalloprotease and chaperone protein family that belongs to the AAA (ATPases associated with diverse cellular activities) protein superfamily. It was originally identified in Escherichia coli and classified among the AAA proteases, which consist of ClpXP, Clpap, Hsuv, Lon, and FtsH (Santos et al. 1975; Tomoyasu et al. 1993; Confalonieri et al. 1995). Of these, FtsH is the only protease that plays an essential role in the life of Escherichia coli (Langer. 2000; Langklotz et al. 2012).

FtsH proteins are found ubiquitously in prokaryotes and eukaryotes. (Jayasekera et al. 2000; Bourdineaud et al. 2003; Kato et al. 2019). FtsH genes have been found in diverse plants, including Arabidopsis (Wagner et al. 2012), tobacco (Seo et al. 2000), alfalfa (Ivashuta et al. 2002), tomatoes (Sun et al. 2006), rice (Zhang et al. 2009), soybean (Yin et al. 2011), wheat (Li et al. 2015), and peanut (Zheng et al. 2016). Almost all the FtsH reported in higher plants have been localized in the mitochondria and chloroplast. The N-terminal transmembrane domain of the FtsH protein anchored membranes of mitochondria or thylakoid, whereas the C-terminal proteolytic domain contains a zinc-binding motif (Zaltsman et al. 2005).

In plants, FtsH protein not only functions as proteases, but also participates in protein assembly and folding as molecular chaperones, and plays roles in the responses to stress and plant development. For example, in Arabidopsis, inhibition of the expression of the DS9 gene accelerated the hypersensitive reaction (Seo et al. 2000). AtFtsH1 and AtFtsH2 were related to the degradation of the 23 kDa fragment of the D1 protein (Zaltsman et al. 2005; Yin et al. 2011). AtFtsH1, AtFtsH2, AtFtsH5, and AtFtsH8 were divided into two types (type A, FtsH1 and FtsH5; type B, FtsH2 and FtsH8) which form a hexameric ring to regulate the thylakoid structure and remove photodamaged D1 protein from photosystem II (PS II) under light stress (Yoshioka-Nishimura et al. 2014; Sedaghatmehr et al. 2016; Zhang et al. 2018). The expression of type A (AtFtsH1, AtFtsH5) and type B (AtFtsH2, AtFtsH8) were significantly altered in senescing and aging leaves (Sakamoto et al. 2003). AtFtsH6, AtFtsH11, and other FtsH proteins of Arabidopsis were involved in senescence, the responses to light, heat, and other functions (Sedaghatmehr et al. 2016; Wagner et al. 2016; Chen et al. 2006). These proteins also play important roles in other plants. For example, one FtsH gene was independently regulated either by either low temperature or light in alfalfa (Ivashuta et al. 2002). LeFstH6 was a typical heat shock gene and can interact with other heat shock factors (Nishimura et al.2016). GmFtsH9, an AtFtsH2-like protein, could be involved in regulating PSII function (Yin et al.2011). In peanut, FtsH genes may play an important role in resistance to salt stress (Zheng et al. 2016).

Previous studies have shown that FtsH is a relatively small and conserved family in many plants (Zhang et al. 2009) and the function of reported FtsH genes were predominantly related to stress. Several studies have reported the importance of FtsH proteases in development of plants, including the AtFtsH12 protease, which was an essential factor in the early development of the chloroplast. Arabidopsis FtsH4 mutant bloomed one week later than the wild type and flower development was also affected (Gibala et al. 2009; Zikhali et al. 2016). The interaction between WYMV CP (Wheat yellow mosaic virus coat protein) and FtsH2 in wheat may interfere with the greening of leaves of during plants’ development and their function (Liang et al. 2019). However, research specialized specific to the FtsH proteins of pear (Pyrus bretschneideri) has been limited to rare reports. To study the function of FtsH family in pear fruit development, we performed a comprehensive bioinformatic-based analysis of the FtsH genes in pear. The completion of pear genome sequence made it possible to identify 19 pear FtsH genes, and we made the comprehensive analysis including the exon–intron organization, motif compositions, gene duplications, chromosome distribution, and phylogenetic and synteny. The expression analysis was performed to identify the expression of specific FtsH genes during ‘Dangshansuli’ different fruit development. Our findings contributed to a basic understanding of the FtsH family and provided a foundation for further clarification the FtsH gene function during fruit development of in pear.

Materials and methods

Plant materials

The ‘Dangshansuli’ (P. bretschneideri Rehd.) pear cultivar was maintained at the Jiangpu orchard of Nanjing Agricultural University, Nanjing, China. Pear fruits were collected at fruitlet (30 days after full blooming, DAFB30), first enlargement (98 days after full blooming, DAFB98), interval enlargement (126 days after full blooming, DAFB126), second enlargement (140 days after full blooming, DAFB140), and maturation (168 days after full blooming, DAFB168), respectively. The collected pear fruits were frozen in liquid nitrogen and stored at -80 °C for analysis.

Gene identification

To identify the members of FtsH gene family in pear, multiple database searches were performed. First, the amino acid sequences of Arabidopsis FtsH genes were downloaded from Arabidopsis Information Resource (TAIR) (http://www.Arabidopsis.org) (Swarbreck et al., 2008) and used to perform BLAST searches in the pear genome database (http://www.peargenome.njau.edu.cn) (Wu et al. 2013) and together with those of other fruit trees, including apple, peach, orange, papaya, grape, and strawberry (https://phytozome.jgi.doe.gov/). The isolated genes were further analyzed by checking the core sequences of FtsH genes family using Pfam. The pear genes were then manually examined to ensure the conserved sequence using the domain analysis program Inter ProScan. The acquired web results with the output row format reported. The isoelectric points, molecular weights, and lengths of the FtsH proteins were obtained with the ExPasy website (https://web.expasy.org/protparam/). Prediction of transmembrane regions and orientation was obtained using TMHMM software (http://www.cbs.dtu.dk/services-/TMHMM).

Sequence and phylogenetic analyses of FtsH genes

The characterized FtsH protein sequences were aligned using program Clustal W with default parameters. After the null members were removed, the phylogenetic trees were constructed in MEGA version 7.0, based on the equally weighted neighbor-joining method, Bootstrap values of the phylogenetic tree were calculated with 1000 replicate analyses.

Gene structure and conserved motifs of FtsH genes.

To determine the gene intron/exon structure, pear FtsH genes information was implemented from the genome annotations using the online program Gene Structure Display Server (GSDS: http://gsds.cbi.pku.edu.cn) (Hu et al. 2015). MEME online program (http://meme.nbcr.net/meme/intro.html) was used to identify conserved motifs (Bailey et al., 2009). The gene structures and conserved motifs patterns were then drawn and visualized with the TBtools (Chen et al. 2006).

Chromosomal localization and gene duplication

All FtsH genes were mapped to the pear chromosomes based on the physical location information from the database of pear genome, and visualized using the TBtools. Gene family expansion models for the FtsH family were investigated based on the diverse chromosomal location of the genes, and included whole genome (WGD), and segmental, tandem, proximal, and dispersed duplications (Cannon et al. 2004). Multiple Collinearity Scan toolkit X (MCScanX) was used to analyze the gene duplication events, with the default parameters (Wang et al. 2012). To display the synteny analysis of the orthologous FtsH genes of pear, Arabidopsis, and other fruit trees, Syntenic analysis maps were constructed using the Dual Synteny Plotter software. Non-synonymous (Ka) and synonymous (Ks) substitution of each duplicated FtsH genes was calculated using TBtools.

Prediction and subcellular localization

Protein subcellular localization was predicted at the WOLF PSORT (http://wolfpsort.cbrc.jp/) (Horton et al. 2007) and BUSCA (http://busca.biocomp.unibo.it/) (Savojardo et al. 2018). We isolated three PbrFtsH genes which were cloned from the cDNA of flesh in different development stages. The open-reading frames (ORFs) of the three PbrFtsH genes were fused to the pCAMBIA1300-35S-GFP vector (Takara, Beijing, China) to produce the plasmids PbrFtsH7-GFP, PbrFtsH12-GFP, and PbrFtsH13-GFP. Tobacco leaves were infiltrated by Agrobacterium tumefaciens strain GV3101 harboring the recombinant constructs and then incubated for 3–4 days at 20–25℃. GFP fluorescence in the tobacco leaves was imaged using a Zeiss Axio LSM 700 inverted confocal laser scanning microscope (CLSM). All transient expression assays were repeated three times. Primers used for cloning genes were listed in additional file 8.

Expression analysis of FtsH genes in pear fruit

In a previous study, RNA-Seq was performed on fruit samples of pear (Pyrus bretschneideri) collected at five fruit developmental stages including the fruitlet (DAFB30), first enlargement (DAFB98), interval enlargement (DAFB126), second enlargement (DAFB140), and maturation (DAFB168) (Pei et al., 20,020). Based on the RNA-Seq data, the expression levels of FtsH genes were evaluated by calculating the Reads Per Kilobase per Million mapped reads (RPKM), and the expression patterns were visualized using heatmaps drafted in Cluster version 3.0 software (Stanford University, Palo Alto, CA).

Total RNA was extracted using the RNAprep Pure Plant Kit (Polysaccharides & Polyphenolics-rich) (Tiangen, Beijing, China). The first-strand cDNA was synthesized with TransScript One-Step gDNA Removal and cDNA synthesis Supermix (TransGen, Beijing, China) according to the manufacturer's instructions. qRT-PCR was carried out in the LightCycler 480® II/96 Thermal Cycler (Roche, USA), and the reaction mixture and cycling conditions were identical to a previous report (Hao et al. 2018) All analyses were performed with three biological replicates. The actin gene (Pbr038418.1) was used as the constitutive control in pear fruit. All of the primer sequences are listed in additional file 8.

Results

Phylogenetic analysis and classification of PbrFtsH genes

A total of 19 candidate FtsH genes were isolated from the pear genome (Pyrus bretschneideri) and designated PbrFtsH1 − PbrFtsH19 (Table 1). The CDS and protein sequences of these genes were available in Additional file 1. The proteins lengths ranged from 582 (PbrFtsH14) to 1265 (PbrFtsH5) amino acid (aa). The pI of the proteins ranged from 5.76 (PbrFtsH19) to 9.89 (PbrFtsH16, and their molecular weight ranged from 63,966.67 (PbrFtsH14) to 138,773.43 (PbrFtsH5).

Table 1 Basic information of the FtsH in pear
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To examine the evolutionary relationships among the FtsH genes of pear, Arabidopsis and other fruit species, the various FtsH genes were isolated from strawberry (10), peach (12), orange (12), papaya (8), apple (13), grape (6) and in Arabidopsis (12) (Fig. 1). The FtsH genes were used to construct a phylogenetic tree (neighbor-joining method). A total of 92 genes were clearly divided into eight group, A → H (Fig. 1). Group B was the smallest clade with 4 members and Group D is constituted with 5 members, whereas Groups E and F were both larger. Based on reliable bootstrap values (> 90%), Group C, Group E, Group F, Group G, and Group H were further divided into two subclusters, respectively (C1and C2; E1and E2; F1and F2; G1andG2; H1and H2, respectively). PbrFtsH15, 16, 17 were included in Group C, PbrFtsH1, 2, 3, 4, 5 in Group E, PbrFtsH6, 12, 13 were in Group F, PbrFtsH10, 11, 14 were in Group G, and PbrFtsH7, 8, 9 were in Group H. Only PbrFtsH18 was exhibited in group D and PbrFtsH19 was not exhibited to any group.

Fig. 1
figure 1

A phylogenetic tree of FtsH genes isolated from pear, peach, apple, strawberry, papaya, orange, and grape, Arabidopsis. The tree was generated using MEGA 7.0 with the Neighbor Joining method. The proteins were clustered into eight different groups and marked with different colors. Different species be distinguished: “” represented strawberry; “” represented papaya; “” represented orange; “” represented Arabidopsis; “” represented grape; “” represented peach; “” represented pear and “” represented apple

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To identify the PbrFtsH family members more clearly, another phylogenetic tree was constructed with protein sequences of pear and Arabidopsis. Five pairs of FtsH genes PbrFtsH1, 2and 4, PbrFtsH3and 5, PbrFtsH7and 8, PbrFtsH12and 13, PbrFtsH15and 16 showed high degrees of similarity (Fig. 2). Compared with Arabidopsis FtsH genes, AtFtsH2/8 with PbrFtsH7/8, AtFtsH7/9 with PbrFtsH12/13, and AtFtsH3/10 with PbrFtsH3/5 have more than 99% bootstrap values inferring they have more larger homology, respectively.

Fig. 2
figure 2

The phylogenetic analysis and schematic diagram for motif of FtsH genes in pear and Arabidopsis. The phylogenetic tree was constructed from a complete alignment of 31 FtsH proteins by maximum-likelihood method with bootstrapping analysis (1000 iterations). The numbers beside the branches indicate the bootstrap values that support the adjacent node. The gene pairs are bolded and different color boxes marked three similar gene pairs of pear and Arabidopsis are by different color boxes exons, introns, and UTR, respectively. Motif models are drawn to scale as indicated on the bottom

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Sequence alignment and transmembrane region of PbrFtsH genes

Multiple sequence alignment and visualization by Jalview revealed the five conserved domains: Walker A can be represented as GX1X2X3PX4GX5 LLX6GX7PPGTGKT and Walker B was represented as PX1X2X3FIDEIDA (where X is an uncharged residue,). The second region of homology (SRH), a characteristic domain of FtsH, can be represented as TNX1X2X3X4LDX5X6X7X8RX9GRX10DR. The SRH can be used to distinguish the FtsH proteins from other AAA proteases. The Zinc-binding motif HEX1X2H was the active site of the protease, and the only variant detected was at residue X1 of the PbrFtsH1-14 proteins, in which residue X2 is conserved as ‘G’. No zinc-binding site presented in the sequence of PbrFtsH15-19 (Fig. 3). In addition, the non-named region that occurred between the SRH and zinc-binding motifs was described as TX1GFX2GAX3X4X5NX6X7NX8AA, and the only the variant detected was a residue in the PbrFtsH17and 19 proteins. These conserved sequences provided the criterion for the isolation and identification of the FtsH genes from other plants.

Fig. 3
figure 3

Multiple sequence alignment of the FtsH from pear. SRH the second region of homology; NM non-named region of homology. ZB Zn binding. JNetPRED is a prediction, the α-helices are represented by red tubes, and the β-folds are represented by green

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FtsH family is an independent member of AAA proteases super family and its members have their own sequence specificity and characteristics. The transmembrane regions of PbrFtsH proteins were predicted sing TMHMM. Most of them contained two transmembrane domains; a few of them (PbrFtsH1, PbrFtsH10, PbrFtsH14, and PbrFtsH19) have one transmembrane; and only PbrFtsH9 has no transmembrane (Additional file 4), suggesting that the functions of their transmembrane domains differ.

Conserved motifs and gene structure analysis of the PbrFtsH genes

To further identify the conserved motifs of the PbrFtsH, the MEME program and TBtools were used in this study. As shown in Fig. 4b, 10 conserved motifs with were found. The number of motifs in the individual FtsH sequences were vary (Additional file 5). Motifs 3,1,4,6,2,5,7 and 8 were commonly distributed in the PbrFtsH genes, except in PbrFtsH15-19. Motif 10 only existed in PbrFtsH1-5, which encoded FtsH-ext domain (Fig. 4d)., PbrFtsH5 was clearly longer than PbrFtsH3, and motif 10,3,6,2,5,7,8 and 9 were repeated in its N-terminal, t meaning two FtsH domains, which may indicate functional redundancy. Motif 9 was only existed in FtsH1-6, FtsH9, and FtsH10, which indicated different FtsH functional motifs arose during the evolution of the FtsH genes.

Fig. 4
figure 4

Phylogenetic relationships, motif pattern, gene structure, and architecture of domains in FtsH genes from pear. a The phylogenetic tree was constructed based on the full-length sequences of pear FtsH proteins using MEGA7 software. Details of clusters are shown in different colors. b The motif composition of pineapple FtsH proteins. The motifs, numbers 1–10, are displayed in different colored boxes. The sequence information for each motif is provided in Additional file 2. The length of protein can be estimated using the scale at the bottom. c Exon–intron structure of pear FtsH genes. Pink boxes indicate untranslated 5′- and 3′-regions; yellow boxes indicated exons; black lines indicated introns. d The FtsH domains are highlighted by green boxes. The number indicates the phases of corresponding introns

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The schematic gene structures of FtsH genes were analyzed using the GSDS (Fig. 4b). The number of exons ranged from 3 to 18 (Table 2 and Fig. 4c, Fig.S1). PbrFtsH11 had 18 exons with the most numbers. In Group C, PbrFtsH15 and PbrFtsH16 had four exons, and PbrFtsH17 had eleven exons. PbrFtsH12 and PbrFtsH13 in Group F, respectively, have 5, 14, and 13 exons. PbrFtsH7, PbrFtsH8, and PbrFtsH9 had 3, 3, and 5 exons in the subgroup H1, respectively. All Group E members contained 8 exons. PbrFtsH18 was distinguished from the other clades in having 15 exons, suggesting an independent evolutionary line. PbrFtsH5 was an exception with 14 exons, because it encoded one domain more than PbrFtsH3.

Table 2 Groups classification and predicted subcellular localization of FtsH proteins of pear and Arabidopsis
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Chromosomal distribution and synteny analysis of FtsH genes

The PbrFtsH genes were unevenly distributed on the chromosomes (Fig. 5). Among the 17 chromosomes of pear genome, most of the FtsH members were located on chromosome 3 (three members), chromosome 9 (four members), and chromosome 15 (three members). Some chromosomes possessed only one gene. Chromosome 9 contained most members, indicating a hot spot in FtsH gene distribution. No FtsH gene was distributed on chromosome 1,5,7,8,10,12,13,14, or 16. These results indicated that the chromosomal locations of the PbrFtsH genes were preferential (Fig. 5). Furthermore, PbrFtsH1 and PbrFtsH4 were tandemly arrayed on the scaffold 490.0, and PbrFtsH2 was located on unassembled scaffold823.0 (Fig.S2). No positive correlation could be detected between the chromosomal length and the number of FtsH genes.

Fig. 5
figure 5

Schematic representations for the chromosomal distribution and inter chromosomal relationships of FtsH genes. The circular forms of chromosomes are shown in green color. Gray lines indicated all synteny blocks in the pear genome, and the green and purple lines indicate syntenic relationships gene pairs. Bar in the blue and Heatmap displayed gene density. The positions of the PbrFtsH genes are marked with short lines on the circles

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We analyzed the mode of duplication for the FtsH genes using MCScanX. As a result, we detected two syntenic gene pairs, PbrFtsH3 and PbrFtsH5, PbrFtsH15 and PbrFtsH16, which probably derived from recent WGD or Segmental events (Fig. 5). It was noteworthy that PbrFtsH genes arose from singleton events (except PbrFtsH1, PbrFtsH2, and PbrFtsH4), six PbrFtsH genes were attributed to dispersed events, only two genes, PbrFtsH10 and PbrFtsH14, were attributed to proximal events (Table 3), and no tandem duplication had not been occurred in pear FtsH family.

Table 3 The duplication events in pear
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To further explore the phylogenetic evolutionary mechanisms of PbrFtsH genes, comparative syntenic maps were constructed with five representative species Arabidopsis, strawberry, grape, peach, and apple (Fig. 6). A total of 19 PbrFtsH genes showed syntenic relationships with strawberry (2), grape (1), peach (2), apple (14), and Arabidopsis genes (1) (Additional file 6). The numbers of orthologous pairs between the pear genes and those of the other five species r were: strawberry 16, grape 12, peach 16, apple 28, and Arabidopsis 12. PbrFtsH3 and PbrFtsH5 genes were found to be associated with at least three syntenic gene pairs (particularly between pear and apple FtsH genes). Significantly, the results showed that some collinear FtsH gene pairs shared by pear and apple were anchored to the highly conserved syntenic blocks (Fig. 6), which spanned more than 20 genes. In contrast, pear and grape had only 12 orthologous gene pairs, as in Arabidopsis (Additional file 6). Similar patterns were also observed between pear-peach with pear-strawberry. Interestingly, PbrFtsH18 and corresponding collinear pairs were showed between pear and strawberry, peach, and apple, respectively, but between pear and grape (Additional file 6). The syntenic PbrFtsH11 and MD06G1038500 identified only between pear and apple. However, orthologues of PbrFtsH7 and PbrFtsH8 were identified in pear and strawberry, peach, apple, and grape, but were not in Arabidopsis.

Fig. 6
figure 6

Synteny analysis of FtsH genes between pear and five representative plant species. Gray lines in the background indicate the collinear blocks within pear and other plant genomes, while the red lines highlight the syntenic FtsH gene pairs. The specie names with the prefixes ‘P.bretchneideri’, ‘A.thaliana’, ‘F.vesca’, ‘V.vinifera’, ‘P.persica’, and ‘M.domestica’ indicated Pyrus bretschneideri, Arabidopsis thaliana, Fragaria vecsa, Vitis vinifera, Prunus persica, and Malus domestica, respectively

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To better understand the evolutionary constraints that have acted on FtsH family, the classical measure nonsynonymous (Ka)/synonymous substitution (Ks) ratios of the FtsH gene pairs were calculated (Additional file 7). The ratio is associated with the magnitude and direction of selective constraints (Ka/Ks > 1, = 1, and < 1) and indicated neutral evolution, purifying selection, and positive diversifying selection, respectively. The results showed all PbrFtsH genes, and those of the other five species, had Ka/Ks < 1, suggesting that the pear FtsH family might have experienced strong purifying selective pressure during evolution.

Subcellular localization of FtsH proteins for pear

The predicted subcellular location of PbrFtsH genes showed that seven PbrFtsH genes were located in the mitochondrion or plasma membrane (PbrFtsH1-5, PbrFtsH15, and PbrFtsH16), whereas 10 genes were located in the chloroplast (PbrFtsH6-10, PbrFtsH12-14, PbrFtsH18, and PbrFtsH19) in Table 2.

To further verify the location of FtsH, we performed three PbrFtsH genes which were cloned from the fruit flesh in different developmental stages. The ORFs of three PbrFtsH were fused to the pCAMBIA1300 vector to express fusion proteins with green fluorescent protein (GFP) under the control of the CaMV 35S promoter. The fusion proteins (PbrFtsH-GFP) and control (35S-GFP alone) were separately transformed into Tobacco leaves. As shown in Fig. 7, PbrFtsH7, PbrFtsH12, and PbrFtsH13 were successfully expressed as PbrFtsH–GFP fusion proteins in chloroplasts. These observation were consistent with the predictions, indicating roles for these proteins in chloroplast or mitochondrion.

Fig. 7
figure 7

Subcellular localization of PbrFtsH proteins. Selected PbrFtsH genes were cloned from pear fruit (Pyrus bretchneideri Rehd.). The PbrFtsH-GFP fusion proteins (PbrFtsH7-GFP, PbrFtsH12-GFP and PbrFtsH13-GFP) and the GFP control were transiently expressed in transformed tobacco leaf epidermal cells and were then observed using confocal laser scanning microscopy. Merged pictures include the green fluorescence channel (first panels), Chloroplast (second panels), and the bright channel (third panels); Bar = 20 μm

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Expression patterns of FtsH genes in different development stages of pear fruit

To understand the functions of FtsH genes during different developmental stages of pear fruit, available transcriptome data and the RT-qPCR analysis were used to analyze their expression patterns (Fig. 8). As shown in Fig. 8, the expression patterns of the FtsH genes were investigated in five stages and varied greatly across them, which indicated that they may have different functions in the diverse growth stages of the pear fruit. On the basis of transcriptome data, the expression of PbrFtsH11, PbrFtsH16, PbrFtsH18, and PbrFtsH19 were high in fruitlet, but decreased gradually in the subsequent developmental stages. The expression of PbrFtsH2 and PbrFtsH4 increased gradually and these genes exhibited preferential expression in the later stages, implying their roles in later fruit development and ripening. The expression levels of other genes (except PbrFtsH11/16/18/19) tended to increase early and decline (Fig. 8a). For instance, PbrFtsH8, PbrFtsH9, PbrFtsH13, and PbrFtsH14 displayed relatively strong expression at DAFB98, indicating that they participated in the initial stages of fruit enlargement (Fig. 8a, b). PbrFtsH1 and PbrFtsH15 were observed relatively highest expression level during the middle stages of fruit enlargement, suggesting that they might play a role during this stage (Fig. 8a). PbrFtsH7, PbrFtsH11, PbrFtsH12, PbrFtsH13, PbrFtsH14, PbrFtsH18, and PbrFtsH19 were relatively highly or moderately expressed during fruit development (Fig. 8b). These analyses showed that PbrFtsH1, PbrFtsH3, PbrFtsH5-14, and PbrFtsH17 were more strongly expressed during fruit enlargement (including first enlargement, interval enlargement, and second enlargement) than the other stages, indicating that these genes were involved in pear fruit development, especially enlargement.

Fig. 8
figure 8

Expression pattern analysis of PbrFtsH genes during ‘Dangshansuli’ pear fruit development stages. a Heat maps showed the hierarchical clustering of 19 PbrFtsHs based on their expression patterns by Transcriptome analysis. b 7 selected PbrFtsH genes expression patterns determined by qPCR assay

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Discussion

Discovery of FtsH family

In recent years, genome-wide identification and functions of FtsH family have been widely carried out in many plants (Sun et al. 2006; Yin et al. 2011; Li et al. 2015,). However, research on the FtsH family in pear were not covered. The release of the whole-genome sequence of pear allowed us to perform a genome-wide investigation and evolution of pear FtsH genes. In this study, we identified 19 members of pear FtsH family, which designated PbrFtsH1-19. Phylogenetic analysis of FtsH genes was performed between the pear and Arabidopsis, strawberry, peach, orange, papaya, apple, and grape. These FtsH genes were classified into eight subfamilies which is consisted with the classification of Arabidopsis FtsH genes (Zhang et al. 2009). However, Group A members were all strawberry genes, implying that these genes had been amplified in a different direction. PbrFtsH19 was not assigned to any group, implying that different characteristics have arisen within the FtsH family during evolution. Additionally nor could evm.TU.supercontig 6.170, PbrFtsH19, or orange1.1g035561m.g be classified into any of the groups. A comparative analysis of FtsH genes of pear and Arabidopsis identified highly similar gene structure and functional domains within the same group. Three sets of genes, AtFtsH7/9 and PbrFtsH12/13, AtFtsH3/10 and PbrFtsH3/5, AtFtsH2/8 and PbrFtsH7/8 were assigned to the same cluster, and shared high similarity in motif and functional domain, respectively (Fig. 2 and Fig. 4).

It has been reported that most of AtFtsH proteins had two transmembrane domains at the N-terminus anchoring the FtsH proteins to the thylakoids or mitochondria membranes. The exception is AtFtsH11, which contained only one transmembrane domain (Chen et al. 2006). The transmembrane region and subcellular location of the PbrFtsH proteins were assessed in this study. One or two transmembrane regions were identified at the N-terminus in the majority of pear FtsH genes, anchoring the proteins to the membranes of the chloroplast or mitochondrion. PbrFtsH9 had no transmembrane region and the loss of a transmembrane region may lead to the loss of enzyme activity. Multiple sequence alignments revealed that five PbrFtsH proteins (PbrFtsH15, PbrFtsH16, PbrFtsH17, PbrFtsH18, and PbrFtsH19) had no zinc-binding domain, presumably indicating that they were inactive in proteolysis. The non-named region of homology was very similar to those in the rice and Arabidopsis, but the function remained unclear. Furthermore, the subcellular localization of PbrFtsH7, PbrFtsH12, PbrFtsH13 proteins in chloroplasts was consistent with published studies of Arabidopsis FtsHs. Therefore, the PbrFtsH genes have a similar subcellular localization pattern as the Arabidopsis AtFtsH genes, so the proteins may play important role in regulating chloroplast and mitochondria development, or photosystem II (PSII) reaction. (Zaltsman et al. 2005; Kato et al. 2009; Zhang et al. 2010).

Expression patterns of FtsHs in different development of pear fruit

We aimed to study its role in fruit developmental different stages. Based on the transcriptome data and RT-qPCR analysis, the expression of PbrFtsH genes during fruit development and enlargement were obtained. In this study, PbrFtsH11, PbrFtsH16, PbrFtsH18, and PbrFtsH19 were strongly expressed in the fruitlet (28 DAFB), implying that the encoded proteins function in the fruitlet (Fig. 8a). The high expression of PbrFtsH7, PbrFtsH12, PbrFtsH13, and PbrFtsH14 implied their key roles in enlargement stages. PbrFtsH8 also showed the high expression in early (98 DAFB) and middle enlargement (126 DAFB) (Fig. 8a), which was consistent with the previous studies, in which PbrFtsH8 was located within the Model I of the quantitative trait locus (QTL) (Pei et al. 2020). Pei et al. reported that pear fruit showed a single sigmoid pattern during development and single sigmoid patterns are coordinately mediated by Model I and II genes. It was indicated that PbrFtsH8, an AtFtsH2-like gene, was not only involved in chloroplast development, but also in fruit enlargement. Overall, these findings provided insight into the potential functions roles of PbrFtsH genes. However, the detailed biological functions of pear FtsH genes are still unclear. More in-depth studies about the PbrFtsH genes are required to better understand the roles in pear development.

Conclusions

A comprehensive analysis of FtsH family in pear was carried out in the present study. Nineteen full-length FtsH genes were characterized and further classified into eight main groups, with highly similar exon–intron structures and motif compositions within the same groups and subgroups. Distribution of PbrFtsH genes on chromosome displayed a clear preference. Synteny analysis and phylogenetic comparison of the FtsH genes from several different plant species, including Arabidopsis, provided valuable clues to the evolutionary history of pear FtsH genes. Subcellular localization showed that PbrFtsH were located in the mitochondrion or chloroplast. PbrFtsH genes played important roles in pear fruit growth and development as indicated by their expression patterns in different development stages of pear fruit. These results provide a valuable resource for better understanding the biological roles of individual FtsH genes in pear.