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Polygalacturonase gene family analysis identifies FcPG12 as a key player in fig (Ficus carica L.) fruit softening

Abstract

Background

The fig (Ficus carica L.) tree has high economic value. However, its fruit have a short shelf life due to rapid softening. Polygalacturonases (PGs) are essential hydrolases, responsible for the pectin degradation that plays a key role in fruit softening. However, fig PG genes and their regulators have not yet been characterized.

Results

In this study, 43 FcPGs were identified in the fig genome. They were non-uniformly distributed on 13 chromosomes, and tandem repeat PG gene clusters were found on chromosomes 4 and 5. Ka/Ks calculation and collinear analysis indicated negative selection as the main driver of FcPG family expansion. Fourteen FcPGs were found expressed in fig fruit with FPKM values > 10, of which seven were positively correlated, and three, negatively correlated with fruit softening. Eleven FcPGs were upregulated and two downregulated in response to ethephon treatment. FcPG12, a member of the tandem repeat cluster on chromosome 4, was selected for further analyses due to its sharp increment in transcript abundance during fruit softening and its response to ethephon treatment. Transient overexpression of FcPG12 led to decreased fig fruit firmness and increased PG enzyme activity in the tissue. Two ethylene response factor (ERF)-binding GCC-box sites were found on the FcPG12 promoter. Yeast one-hybrid and dual luciferase assays showed that FcERF5 binds directly to the FcPG12 promoter and upregulates its expression. Transient overexpression of FcERF5 upregulated FcPG12 expression, thereby increasing PG activity and fruit softening.

Conclusions

Our study identified FcPG12 as a key PG gene in fig fruit softening, and its direct positive regulation by FcERF5. The results provide new information on the molecular regulation of fig fruit softening.

Peer Review reports

Introduction

Textural change is a characteristic and irreversible phenomenon of fruit ripening, accompanied by a series of other physiological and biochemical changes, such as sugar accumulation, color transformation, and aroma development. These changes result from the spatiotemporal expression of a series of fruit ripening-related genes [1]. The texture of the ripening fruit changes mainly due to pectin degradation, cell wall relaxation and expansion, and cellulose and hemicellulose disaggregation [2]. The decomposition of cell wall polysaccharides involves the synergistic effects of multiple enzymes and proteins, including polygalacturonase (PG), pectate lyase, endotransglycosylase/hydrolase, expansin, and others [3,4,5].

PG is one of the largest hydrolase families, belonging to glycoside hydrolase family 28 (GH28) [6, 7]. The PG gene family has been identified in many fruit species, including 45 PGs in sweet cherry (Prunus avium) [8], 51 PGs in kiwifruit (Actinidia chinensis) [9], 35 PGs in grape (Vitis vinifera) [10], 61 PGs in pear (Pyrus pyrifolia) [11], and 45 PGs in peach (Prunus persica) [12]; 66 PGs have been identified in Arabidopsis [6]. The PG family is widely involved in the cell wall modifications that are required for various tissues’ growth and expansion, such as shoots, leaves and roots, and for organ abscission. The transcription levels of different PGs are directly or indirectly regulated by internal and external factors, such as phytohormones, temperature and ultraviolet light [13].

PG family members are of interest in fruit development and ripening. Five PGs were identified as related to fruit development out of a family of 54 members in Solanum lycopersicum [14]. Two PGs were strongly related to fruit ripening in Actinidia arguta, and AaPG18 was found to be involved in kiwifruit ripening processes [15]. In papaya (Carica papaya), CpPG1 was revealed to play a key role in fruit softening [16]. The expression of FaPG1 increased during strawberry fruit softening, and antisense FaPG1 delayed softening [17]. In sweet cherry, transient overexpression of PavPG38 reduced fruit firmness and the contents of ion-linked pectin (ISP) and covalent pectin (CSP) content, and increased the content of water-soluble pectin (WSP) [8].

The expression of PGs that are related to fruit ripening and softening is regulated by a number of transcription factors, including APETALA2/ethylene response factor (AP2/ERF) family transcription factors, the NAC family (NAM, ATAF1, ATAF2 and CUC2) and MADS-box family (MCM 1, AGAMOUS, DEFICIENS, SRF-box) [18, 19]. GCC-box, with core sequence AGCGCCC, is usually a cis-acting element that is directly bound by AP2/ERF protein on the target gene promoter [20]. In papaya, electrophoretic mobility shift assay demonstrated specific binding of CpERF9 to the promoter of CpPG5 via the GCC-box motif [21]. Knockout of SlERF52 in tomato resulted in downregulation of TAPG1, TAPG2 and TAPG4 in the abscission layer [22]. In peach, PpERF2 directly binds to the PpPG1 promoter and inhibits PpPG1 expression during fruit softening [23].

Fig (Ficus carica L.) is one of the earliest domesticated fruit trees. It is highly adaptable to planting environments, and produces considerable economic value [24]. The fig fruit is climacteric in nature, and fruit firmness rapidly decreases after ripening, making it extremely intolerant to storage and transportation, which hinders its use as a fresh fruit [25, 26]. The content and state of pectin and hemicellulose in the fig receptacle and flesh change when the fruit are physiologically ripe and begin to soften. Pectin solubilization is associated with higher activity of PG and pectin methylesterase (PME) enzymes [27]. The expression patterns of FcPG1 and FcPG2 have been isolated and identified, but in that study, the information on gene structure and sequence was incomplete, and the genes’ functions were not verified [28]. Thus, although the change in texture is an important biological characteristic that affects the shelf life of fresh figs, there is a lack of in-depth research on PG in figs, and the key PG in fruit ripening and softening has not yet been identified.

Table 1 Physical and chemical properties of FcPGs.
Fig. 1
figure 1

Chromosomal location and phylogenetic tree of FcPGs. (A) FcPGs are marked on chromosomes; scale bar on the left indicates length of fig chromosome (Mb). The color of the gene background corresponds to the clade color in the phylogenetic tree of panel B. (B) Phylogenetic tree showing the relationships of the 43 FcPGs in F. carica, and PGs in A. thaliana and other species. Four color triangles at the bottom right indicate identified biological functions of the PG genes

In this study, the PG gene family and PG expression in fig fruit were analyzed. FcPG12 (FCD_00027285), a member of a small PG cluster on chromosome 4, was identified as a key PG in fruit softening. FcERF5 was found to regulate FcPG12, activating its transcriptional expression and promoting fig fruit softening.

Results

Identification of PG gene family in F. carica

A total of 67 PG candidate sequences were obtained by pooling 65 sequences recruited using the Arabidopsis PG protein sequence as the query object; 56 sequences were screened by searching the GH28 HHM map of the fig genome database, and repetitions were deleted. After eliminating the protein sequences lacking PG domain characteristics by CDD database search and motif test, 43 PGs were identified. The PG genes were named FcPG1 to FcPG43 according to their position on the chromosomes (Fig. 1A). The FcPGs were 252–974 amino acids long, with a calculated relative molecular mass of 2.75–10.38 kDa and a predicted pI of 4.86–9.82. About half of the FcPG proteins contained the signal peptide sequence predicted by SignalP (Table 1).

Table 2 Ka/Ks analysis of FcPG tandem repeat genes

To clarify the evolutionary relationship and functional differences between FcPGs, a phylogenetic tree of fig, Arabidopsis and 19 PG genes identified in other plants was constructed by the ML method. The FcPGs could be divided into seven clades, of which clade D, with 14 genes, was the largest (Fig. 1B). FcPGs were non-uniformly distributed on the chromosomes. Two tandem repeat regions, including FcPG9, 10, 11, 12, 13 and FcPG17, 18, 19, 20, 21, 22, 23, respectively, on chromosomes 4 and 5 (Fig. 1A) were separately located on clades C and D in the phylogenetic tree (Fig. 1B). The sequences of FcPG1, 9, 10, 11, 12 and 13 were similar and among them, FcPG9, 10, 11, 12, 13 clustered together on the chromosome. Moreover, they were closely clustered with PG genes functioning in fruit softening in apricot, plum, peach and pear. On clade B, FcPG4, 37, 31 and 39 were also closely clustered with fruit softening-related PGs in grape, kiwifruit, peach, pear, apple and orange. Clade A, which clustered FcPG34, 16, 36, 7, 8 and 33, contained known PG genes of peach and banana (Fig. 1B).

The Ka/Ks ratios of FcPG1, 9, 10, 11, 12 and 13 were 0.17–0.54; the values < 1 indicate that these genes were under purified selection, and the function of the encoded proteins was retained. The Ks of FcPG1 was higher than that of the others (Table 2), suggesting that FcPG1 was the first gene to be segregated and to differentiate. We estimate that the tandem duplication of FcPG9, 10, 11, 12 and 13 occurred 2.8–19.3 million years ago, whereas that of FcPG1 occurred at least 10 million years earlier (Table 2).

Gene structure, conserved motif and domain analysis of FcPGs

The amino acid sequence of PG proteins usually contains four conserved domains—SPNTDG, GDDC, CGPGHG and RIK, and three cysteine (Cys) sites. Multiple sequence alignment showed that most FcPGs have the four conserved domains (motifs I to IV) that are critical for PG’s hydrolytic activity. FcPG38, 40, 14, 27, 24, 29 and 5 lacked one or two of the typical PG domains (Fig. 2).

Fig. 2
figure 2

Multiple sequence alignments of the conserved domains (I–IV) of the FcPGs. Similar domains are indicated in red font and framed by blue boxes. Identical amino acid positions are highlighted by a red background. The gene cluster of FcPG12 is circled in red

Similar gene structure and composition were found for FcPG members of the same clade. FcPGs had 3–15 exons and 2–14 introns; fewer exons/introns were found in the members of clades A and G, whereas 4 exons/introns were identified in most other FcPGs (Fig. 3B). In addition, most of the FcPGs did not have a non-coding region structure; FcPGs with a non-coding region structure were mainly concentrated on clades E and F (Fig. 3B).

Fig. 3
figure 3

Structure of the FcPGs. (A) Motifs. (B) Gene structure. (C) Domains. The x-axis represents gene length. Gene background color corresponds to its clade color in Fig. 1B

Eight conserved motifs were obtained when the 43 FcPG protein sequences were aligned (Fig. 3A). Within each clade of the phylogenetic tree, the composition and positional order of the conserved motifs were usually similar. Motifs 1 and 2 were present in all 43 PG protein sequences; motif 5 was absent in FcPG5 and FcPG29, and motif 3 was absent in members of clade F (Fig. 3A). According to the CDD database in NCBI, most of the PG protein domains belong to PL-6 (polysaccharide lyase family 6), the members on clade D belong to the PLN02218 superfamily, and the members on clade F are in the Pgu1 family (Fig. 3C).

Expression patterns of FcPGs in fig fruit

Among the 43 FcPG genes, 14 were detected during fig fruit development and ripening (FPKM > 10 in at least one sample) (Fig. 4), and had different expression patterns. FcPG1, 9, 10, 11, 12, 13 and 14 showed upregulation during fruit softening, whereas FcPG27, 29 and 38 were downregulated (Fig. 4). The expression of other FcPGs fluctuated during fruit development and ripening. The expression of FcPG39 was rather flesh-specific: it increased in the early stages of fruit development, then decreased, and increased again at fruit ripening, whereas almost no expression was found in the peel (Fig. 4).

Fig. 4
figure 4

Expression patterns of FcPGs during fig fruit development in (A) flesh and (B) peel. Color scale represents FPKM value normalized by log10 counts. The histogram indicates the number of genes in the FPKM expression range. Photographs show fig fruit syconia and internal structure on the six sampling dates

The expression pattern of transcript c43883_g4 (sequence ID in the transcriptome database) was very similar in the flesh and peel of the fig fruit. Almost no expression was detected in the early stage of fruit development, but the expression level increased sharply when the fruit started ripening. The expression of c43883_g4 in the flesh of stage 5 fruit was 268 times higher than that of stage 4 (Fig. 5A). The expression of c43883_g4 was also upregulated (162-fold) in the peel, and upregulation in the flesh and peel continued in stage 6, being 2-fold that in stage 5 (Fig. 5B). The results suggested that the transcript could play an important role in fruit softening. However, c43883_g4 was annotated as a transcript of 6 PG genes due to similar sequences in the genome; the cis-acting elements of the promoters of transcript c43883_g4 were annotated as 6 predicted FcPG genes. Phytohormone-related response elements such as abscisic acid (ABA), light, low temperature and wound signal stress response elements were found, suggesting FcPG regulation by phytohormones and a variety of environmental factors. Among them, the light response elements were the most abundant (Fig. 5C).

Fig. 5
figure 5

Expression pattern of c43883_g4 and promoter region analysis of FcPGs significantly correlated with fruit ripening. Absolute FPKM value of c43883_g4 in (A) flesh and (B) peel. (C) Promoter region analysis of FcPGs significantly correlated with fruit ripening

Expression patterns of FcPGs in fig fruit treated with ethephon

The expression pattern of FcPGs in response to ethephon treatment was data-mined (Fig. 6). Ethephon did not induce FcPG member expression, and it only affected the 14 FcPGs that had been previously detected in the fruit. The expression patterns of FcPGs in the flesh and receptacle were consistent in a comparison of treatment time (2, 4 and 6 d) with the control.

Fig. 6
figure 6

Expression patterns of FcPGs in fig fruit upon ethephon treatment in (A) flesh, and (B) peel. Color scale represents FPKM value normalized by log10 counts. Histogram indicates the number of genes in the FPKM expression range. Sketch shows the changes in fig fruit after ethephon treatment

After ethephon treatment, 11 FcPGs (FcPG1, 4, 9, 10, 11, 12, 13, 14, 24, 27 and 39) were upregulated, while FcPG36 and 40 were downregulated (Fig. 6). The expression of c43883_g4 was upregulated after ethephon treatment, 1700- and 590-fold in the flesh and receptacle, respectively, compared to their corresponding controls 4 d after treatment. The expression difference of c43883_g4 between the treated and control group became smaller 6 d after treatment, as the control fruit entered the ripening stage naturally. The expression of c43883_g4 in the ethephon-treated flesh 6 d after treatment was more than 2-fold that 4 d after treatment (Fig. 6).

FcPG12 is a major PG in fig fruit softening

We cloned FcPG1, 9, 10, 11, 12 and 13 and compared their sequences with c43883_g4. A difference of only two single-nucleotide polymorphisms was found between FcPG12 and c43883_g4 (Fig. 7A). The expression pattern of FcPG12 during ‘Purple Peel’ fig fruit softening as determined by qRT-PCR was consistent with that of c43883_g4 determined by RNA-Seq (Supplemental Fig. S1). FcPG12 protein was predicted to be located on the cell membrane, and this was verified by fusing FcPG12 with EGFP under the control of the CaMV 35 S promoter, infiltrating it into leaves of N. benthamiana and locating the signal (Fig. 7B).

Fig. 7
figure 7

Subcellular localization and transient overexpression of FcPG12. (A) Sequence alignment of FcPG1, 9, 10, 11, 12, 13 with c43883_g4. (B) Subcellular localization of FcPG12–EGFP fusion protein in N. benthamiana leaves. Scale bar, 50 μm. (C) Fruit tissue firmness with transient overexpression of FcPG12. Error bars represent ± SD of three biological replicates. Significance of differences in values (*P < 0.05, **P < 0.01) was determined by Student’s t-test. (D) Semi-quantitative RT-PCR and (E) qRT-PCR analyses of FcPG12 expression in fig fruit transiently overexpressing FcPG12. The image is an adjacent lane of the same gel (1.8%), and only the image size is cropped. FcActin was used as the housekeeping gene. (F) PG enzyme activity in fig fruit tissue with transient FcPG12 overexpression

FcPG12 and empty plasmid were injected into each of two equal halves of a fig fruit before ripening. Three days later, the firmness of the side with FcPG12 transient overexpression was lower than that of the control side (Fig. 7C). The relative expression of FcPG12 at the transcriptional level was higher in the transgenic fruit side than in the control side, as determined by semi-quantitative PCR and qRT-PCR (Fig. 7D, E). PG enzyme activity was also higher in the treated vs. control side (Fig. 7F).

FcERF5 upregulates FcPG12 by binding to its promoter

Coexpression pattern analysis revealed 593 and 141 genes with expression patterns similar and opposite to that of FcPG12, respectively, by including multiple transcription factors, such as FcERF, FcMYB, FcbHLH. Among them, the expression pattern of FcERF5 (FCD_00009851) was similar to that of FcPG12, increasing sharply when fruits were ripening, as verified by qRT-PCR (Supplemental Fig. S2).

The open reading frame (ORF) of FcERF5 is 906 bp, encoding 301 amino acids with a molecular mass of 32.78 kDa. FcERF5 contains a conserved AP2/ERF domain and belongs to the fig ERF-Va (IXa) subfamily [29]. Subcellular localization showed FcERF5’s position in the nucleus (Fig. 8A). Two GCC-boxes were predicted on the FcPG12 promoter (relative profile score threshold > 90%), the first motif ‘aagccgacatg’ located at -1272 bp to -1278 bp upstream of the start codon (ATG), and the second motif ‘cgccgccc’ located -222 to -230 bp upstream of ATG (Fig. 8B). In addition, there was a GCM motif ‘ataacgcaat’ (relative profile score threshold > 95%) that may bind to NAC transcription factors, located at -627 bp to -636 bp upstream of the ATG.

Fig. 8
figure 8

FcERF5 positively regulates FcPG12 through binding to promoter of FcPG12 (ProFcPG12) in the nucleus. (A) Subcellular localization of FcERF5–EGFP fusion protein in mCherry-stained N. benthamiana leaves. Scale bar, 50 μm. (B) Y1H assay showing FcERF5 binding to the first GCC motif in the FcPG12 promoter (P1). GCC-box: dehydration-responsive element/C-repeat element. Bold gray line, promoter of FcPG12 (-1500 to -1 bp) used for cis-element analysis; bold orange line, FcPG12 promoter used for Y1H. (C, D) Dual-luciferase activity assay showing that FcERF5 transactivates the FcPG12 promoter in N. benthamiana leaves. Values are mean ± SD. Imaging of N. benthamiana leaves infiltrated with A. tumefaciens harboring FcERF5 and ProFcPG12: LUC.

Two GCC-box fragments of the FcPG12 promoter (P1 and P2) were cloned as respective baits for a Y1H assay. The yeast with FcPG12 promoter pABAi-P2 grew on the SD/-Ura/+AbA agar plate, indicating that the P2 segment has strong self-activation. Therefore, only P1-positive yeast colonies were selected for the following cotransformation test. The yeast cotransformed with pABAi-P1 and AD-ERF5 plasmids grew on an agar plate containing SD/-Leu/+AbA, and the yeast colony became blue after adding X-α-Gal, whereas the control yeast could not grow on SD/-Leu agar plates, indicating that FcERF5 binds to the first motif fragment of the FcPG12 promoter (Fig. 8B).

Dual-luciferase reporter assay was performed by transfecting the leaves of N. benthamiana with 62sk-FcERF5 plasmid and ProFcPG12: LUC (FcPG12 promoter) plasmid to measure the LUC activity and detect the luminescence signal (Fig. 8C). The leaf cells of N. benthamiana with FcERF5 and FcPG12 promoter exhibited higher LUC activity than those with 62-sk control and FcPG12 promoter (Fig. 8D). A strong luminescence signal was detected in the region where FcERF5 and the FcPG12 promoter were co-injected, but not in the negative controls (Fig. 8D), demonstrating that FcERF5 activates FcPG12 expression via binding to its promoter. In addition, we determined that FcERF22 (FCD_00010137), from the same cluster as FcERF5, does not bind to the FcPG12 promoter (Supplemental Fig. S3), suggesting that FcERF5 binding to the FcPG12 promoter is specific.

Transient overexpression FcERF5 in fig fruit

The constructed 62sk-FcERF5 plasmid was transiently overexpressed in one half of a fig fruit, and the other half served as a control. The firmness of the fruit half with transient overexpression of FcERF5 was lower than that of the fruit half with empty vector (Fig. 9A). Based on qRT-PCR analysis, FcERF5 was upregulated in the fruit tissues of the half with FcERF5 overexpression (Fig. 9B), verifying the effectiveness of the overexpression at the transcriptional level. The activity of PG enzyme in the receptacle with transient overexpression of FcERF5 was higher than that of the control (Fig. 9C). The expression of FcPG12 was upregulated the fruit half with transient overexpression of FcERF5 (Fig. 9D). Taken together, the results indicate that FcERF5 activates the expression of FcPG12, thereby promoting fig fruit softening.

Fig. 9
figure 9

Effect of transient overexpression of FcERF5 in fig fruit. (A) Fruit tissue firmness. Quantitative RT-PCR analysis of (B) FcERF5 and (C) FcPG12 expression. (D) PG enzyme activity

Discussion

FcPG gene family

A total of 43 PG genes were identified in F. carica, less than the 62 and 54 PG genes found in the genomes of F. microcarpa and F. hispida (Supplemental Table S2), respectively. There were two FcPG tandem repeat regions (FcPG9, 10, 11, 12, 13 and FcPG17, 18, 19, 20, 21, 22, 23 gene clusters) on the fig chromosome (Fig. 1A), located on clades C and D of the phylogenetic tree, respectively (Fig. 1B). This is similar to the 28 tomato PG genes present in clusters on the chromosome, including five tandem repeat gene clusters [14]. In addition, there were four and three PG gene clusters of tandem repeats in F. microcarpa and F. hispida, respectively (Supplemental Figs. S4 and S5). A comparative collinearity map with F. pumila and F. hispida was constructed according to the FcPG9, 10, 11, 12, 13 gene cluster. Three members in F. pumila and four members in F. hispida were homologous to the FcPGs of this gene cluster, with more dispersed distribution, indicating that tandem duplication occurred on this chromosome segment in fig (Supplemental Fig. S6).

The sequences of FcPG9, 10, 11, 12, 13 and FcPG1 were clustered with the PG genes related to fruit softening in apricot (PaPG) [30], peach (PpPG) [12], Prunus domestica (PdPG1) [31], pear (PcPG3) [32] and papaya [16], suggesting that these FcPG genes are related to fruit softening (Fig. 1B). Grape VvPG1 [33], kiwifruit CkPG [34], citrus CitPG [35] and apple MdPG1 [36, 37], with identified roles in fruit softening, were clustered on clade B. FcPG4, 31 and 37 were also clustered on clade B. However, only FcPG4 expression was detected in fig fruit (Fig. 1B). FcPG4 had opposite expression patterns in fig flesh and peel during fruit development, suggesting its tissue-specific expression (Fig. 4).

Most of the FcPGs were not found to be expressed in fig fruit. Approximately one-third of the FcPGs were transcribed during fig fruit development and ripening, similar to peach fruit, where only 16 of the 45 PG family members were detected [12]. Most of the FcPGs which were not expressed in fig fruit, were also not expressed in the profile of the vegetative tissues, i.e., root, leaf and shoot, and they could therefore be pseudogenes, like some of the Arabidopsis PGs [6]. The transcript abundance of FcPGs which were expressed in fig fruit showed differential patterns during fruit development, and as can be inferred from analysis of the FcPG promoter, some PG expression may be induced by phytohormones such as ABA or ethylene, together with various stress conditions.

FcPG12 is a major player in fig fruit texture change during ripening

Among the FcPGs expressed in the fruit, the transcript c43883_g4 demonstrated unique expression characteristics. There was almost no expression of c43883_g4 in young fruit, but it increased rapidly and continuously from the start of ripening, when its level became much higher than that of the other FcPGs (Fig. 4). Moreover, c43883_g4 was induced by ethephon (Fig. 6). Transcript c43883_g4 was annotated as the gene cluster FcPG9, 10, 11, 12, 13 and phylogenetically clustered as FcPG1. In general, only one gene in the tandem repeat region shows relatively high expression, whereas the other genes have either low expression or no transcript product at all [6]. In our study, FcPG12 was most similar to the transcript c43883_g4 (Fig. 7A). In addition, only FcPG12 transcripts were identified when five specific primers were respectively used to detect the transcripts of the five PG genes (FcPG913). Pure FcPG12 without the SNP (single-nucleotide polymorphism) base of the amplified transcripts was validated by multiple clone sequencing, and the presence of only single-peak melting curves in qRT-PCR. Taken together, that FcPG12 is the major member transcribed during fruit development in this gene cluster.

Blast alignment analysis indicated that FcPG12 and FcPG39 in this study correspond to the sequences of FcPG1 and FcPG2, respectively, that were previously reported and published in GenBank [28]. In the phylogenetic tree, FcPG12 is clustered on clade C, with strong homology to the key PGs of other species on this clade. However, it has a distant relationship with clade B (FcPG39 is in this clade), which also carries many key fruit softening-related PGs (Fig. 1B). It therefore seems that the key PGs involved in fruit softening in different species are on different phylogenetic clades. FcPG12, with a relative molecular mass of 4.18 kDa and pI of 8.73 is similar to the key PG proteins of other species, such as PpPG, PaPG and PdPG1, collected in this paper (Fig. 1B). Sequence similarity analysis showed that the PG domains were highly conserved, but the PG protein sequences varied greatly among the different species (Supplemental Fig. S7). To obtain evidence that FcPG12 affects fruit softening, we performed transient overexpression of FcPG12 in fig. Increased PG enzyme activity, together with decreased firmness of the receptacle revealed that FcPG12 plays an important role in fruit softening by degrading pectin (Fig. 7). This is similar to the results of AaPG18 overexpression in kiwifruit [15].

Identification of key PG genes is the basis for improving fruit storage and extending shelf life. CRISPR/Cas9 technology was used to obtain 4-bp and 10-bp deletions and a 1-bp insertion in the SlPG gene in tomato. Frameshift mutation of SlPG led to early termination of amino acid translation, thus delaying fruit softening [38]. In another study, virus-induced gene silencing technology was used to interfere with the expression of the peach β-galactosidase gene, resulting in decreased expression of PpPG21 and PpPME3 and their corresponding enzyme activities, which inhibited the softening of peach fruit [39]. Interference of MdPG1 in ‘Royal Gala’ apple resulted in decreased pectin-degradation rate and increased fruit firmness [40]. As the key glycoside hydrolase in figs, modulating the expression of FcPG12 may be an important way to slow down the rapid fruit softening and improve its texture.

The expression regulation of FcPG12 by transcription factors

Understanding FcPG12 expression regulation is of great significance for analyzing the mechanism of softening during fig ripening. The expression pattern of FcERF5 during fruit softening was similar to that of FcPG12, increasing when the fig fruit started to ripen (Supplemental Fig. S2). FcERF5 is closely clustered with AtERF5. According to similarity prediction in the SWISS-PROT protein database, FcERF5 is a transcription factor that can bind to the GCC-box and may be a transcription activator involved in signal-transduction pathways regulating gene expression. Through Y1H and dual luciferase reporter assays, we verified that FcERF5 functions as an activator of FcPG12 by binding to the FcPG12 promoter (Fig. 8). This is in agreement with a recent report that PpERF/ABR1 promotes PpPG expression in peach by binding to the PpPG promoter [41]. Subcellular localization of FcERF5 supported occurrence of the regulation process in the nucleus (Fig. 8A). Fruit firmness and PG enzyme activity after overexpression of FcERF5 were similar to that following FcPG12 overexpression, providing further evidence that FcERF5 upregulates FcPG12 and promotes fruit softening (Fig. 9).

AP2/ERF is a large family that includes many members related to fruit ripening and softening in Fig. [29]. FcERF62 also had expression characteristics similar to those of FcPG12. FcERF62 (FCD_00028202) belongs to FcERF IV subfamily. The expression of FcERF62 gradually increased with fruit development. At fruit ripening, it reached its highest abundance in the fruit flesh (FPKM of 100), while its expression level was lower in the peel (highest FPKM was 12). The expression of FcERF62 increased in fruit treated with ethephon. However, direct regulation of FcPG12 by FcERF62 requires further verification.

In addition to these FcERFs, weighted gene coexpression network analysis (WGCNA) showed that FcMYB108, FcMYB4, FcbHLH137, FcbHLH149 and FcNAC29 in coexpression module ‘MEcyan’ and ‘MEblack’ had expression characteristics similar to those of FcPG12 (Supplemental Fig. S8). RhMYB108, which is highly homologous to FcMYB108, has been reported to be involved in ethylene- and jasmonic acid-induced petal senescence in roses [42]. NAC gene family members play an important role in ethylene and ABA signal-transduction pathways. They are involved in the fine regulation of cell wall modification genes and play a role in regulating texture changes during fruit ripening and softening [1]. AtNAP, which is closely related to FcNAC29, may function in the transition between active cell division and cell expansion [43]. MYB and NAC transcription factor binding sites were predicted on the FcPG12 promoter. However, more experimental evidence is required to reveal the panorama of FcPG12-expression regulation.

Conclusion

In this study, 43 FcPGs were identified in the fig genome for the first time. According to their gene structure, motif and cis-acting element characteristics, they were assigned to seven groups (A–G). Based on RNA-Seq data analysis, gene cloning and sequence comparison, FcPG12 was isolated as a key PG related to fruit softening. The function of FcPG12 in promoting fruit softening was confirmed by transient overexpression and qRT-PCR analyses. FcERF5 binds to the FcPG12 promoter in the nucleus and promotes its expression, resulting in fruit softening, as proven by subcellular localization, Y1H, dual luciferase and transient expression experiments. Our results provide a basis for exploring the molecular mechanism of fig softening, and an understanding of how ERF regulates cell wall-modifying PG gene transcription during fruit softening.

Materials and methods

Plant materials

Five-year-old common figs (Ficus carica var. 108B) were used for gene isolation and transient genetic transformation. The figs were grown at Shangzhuang Experimental Station of China Agricultural University, Beijing, with 3 × 3 m spacing and a vertical trellis system. The development of fig fruit was divided into six stages. Fruits of similar maturity, uniform size and clean surface were selected. For each sample collection, the flesh, peel and receptacle of fruits were separated to freeze in liquid nitrogen and store at -80 °C separately for further use.

Identification of FcPG gene family members

F. carica genome data were downloaded from NCBI (DDBJ/EMBL/GenBank access code: VYVB01000000) [44]. Fig PG genes were obtained by homologous blast of Arabidopsis PG protein sequences and HMM search of the PG conserved domain in the Pfam database (http://pfam.xfam.org/). Arabidopsis PG protein sequences were downloaded from TAIR (https://www.arabidopsis.org). Fig PGs were searched using BlastP (e-value threshold ≤ 1e-5) with Tbtools and AtPG protein sequences as queries [45]. The Hidden Markov Model (HMM) file of the PG protein domain (PF00295) was download from the Pfam database. Sequences containing PG protein domains were retrieved from the fig genome database using HMMER 3.0 [46]. Combining the results of the two screening methods, protein sequences without PG conserved motifs and short proteins (< 50 amino acids) were eliminated. The retained protein sequences were verified to contain PG domains by NCBI Batch-CD. The relative molecular weights and isoelectric points (pIs) of the predicted PGs were calculated by ExPASy (https://www.expasy.org/). Prediction of signal peptides was carried out by SignalP (https://services.healthtech.dtu.dk/service.php?SignalP-5.0).

Chromosomal location, gene duplication, phylogenetic tree construction and PG sequence analysis

Gene structure visualization of the GFF3 file of the fig genome was conducted using TBtools [45]. All PG protein sequences of fig and Arabidopsis thaliana were aligned with Clustal W [47], and a phylogenetic tree was constructed by MEGA 6.0 software using the maximum likelihood (ML) method with 1000 replicates [48]. Multiple sequence alignment was done with Clustal X software and ESPript online website (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). Conserved motifs were analyzed using MEME (https://meme-suite.org/meme/tools/meme). The FcPG promoter (2000-bp region upstream of the start codon of each gene) was extracted from the fig genome and submitted to the PlantCare database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to determine putative cis-elements. Transcription factor binding sites were predicted by JASPAR (https://jaspar.genereg.net/). Corresponding coding regions were aligned using Clustal W, and the number of non-synonymous substitutions per non-synonymous site (Ka) and the number of synonymous substitutions per synonymous site (Ks) were calculated by KaKs Calculator 2.0 [49]. The time of gene duplication was determined by the formula T = Ks/2r, where r is the divergence rate of nuclear genes. In A. thaliana, r was considered to be a synonymous substitution of 7 × 10− 9 per site per year [50]. Collinear analysis of Ficus carica, Ficus hispida, and Ficus pumila was performed using the multicollinear scanning toolkit (MCScanX) [51].

FcPG expression during fruit ripening

The expression pattern of FcPGs was analyzed by re-mining two RNA-Seq libraries of fig fruit established in our laboratory. The first library consisted of data on ‘Purple Peel’ fig fruit development (SRA login: PRJNA723733) [52]. The RNA-Seq data were generated and the gene-expression counts were normalized to fragments per kilobase of transcript per million mapped reads (FPKM). The second library contained data on the flesh and receptacle of figs after ethephon treatment [53]. Briefly, ‘Brown Turkey’ fig fruit before ripening were injected with 1 mL of 250 mg L− 1 ethephon, and the flesh and receptacle of the fruit were collected 2, 4, and 6 d after treatment for transcriptome sequencing. The gene-expression counts were normalized to transcripts per million (TPM).

Quantitative reverse transcription (qRT)-PCR validation of gene expression

Total RNA was extracted from the receptacle of fig fruit by cetyltrimethylammonium bromide (CTAB) [54], and RNA was reverse-transcribed to cDNA using a reverse transcription kit (TaKaRa Biotechnology, Dalian, China). The primer pairs were designed by Primer3 (https://bioinfo.ut.ee/primer3/). The qRT-PCR was performed on an Applied Biosystems QuantStudio 6 Flex system, using TB Green® Premix Ex Taq (RR420Q, TaKaRa). Each sample was run in three technical repeats. The reaction conditions were as follows: 40 cycles of 95 °C for 30 s, 95 °C for 5 min, and 60 °C for 34 s. The FcActin gene was used as an internal control [55], and gene relative expression was determined by the 2−ΔΔCT method [56]. All primers are listed in Supplemental Table S1.

Subcellular localization

Subcellular localization was predicted using the online tool Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/). The gene primers were designed by SnapGene 4.2.4 software. The full-length gene’s coding sequence (CDS) was cloned from cDNA and inserted into the binary vector pBI121 with enhanced green fluorescent label (EGFP). The 35 S:FcPG12-EGFP, 35 S:FcERF5-EGFP and control vectors were introduced into Agrobacterium tumefaciens strain GV3101 by heat shock. Agrobacterium cells were cultured on Luria broth (LB) agar plates containing rifampicin (25 µg mL− 1) and kanamycin (50 µg mL− 1) at 28 °C for 2–3 d. For each transformation experiment, a single colony was cultured in 5 mL LB containing antibiotics at 28 °C for 16 h. The culture was centrifuged at 6000 g and the pellet was resuspended in 25 mL medium (10 mM MES, pH 5.7, 100 µM Acetosyringone (AS), 10 mM MgCl2) to OD600 of 0.6–0.8. After standing at 28 °C for 2 h, the suspension was infiltrated into 4-week-old Nicotiana benthamiana leaves [57]. Fluorescent signal was observed 48–72 h later using a confocal laser-scanning microscope (Olympus FV3000, Tokyo, Japan).

Transient genetic transformation in fig fruit

The full-length CDSs of FcPG12 and FcERF5 were cloned into vector pGreenII 62-sk [58] and introduced into Agrobacterium strain GV3101 for transient overexpression in fig fruit. Using a transient genetic transformation method for Fig. [59], the fruits before ripening (stage 4 in Song et al., 2021) were cut into two equal parts and one half was injected with Agrobacterium with the recombinant plasmids. The other half served as a control. After 3 d, fruit hardness was measured and the fruit receptacle tissue was collected. Around 60 fruits were injected for each gene.

Fruit firmness and PG enzyme measurements

Fruit firmness was measured with AICE digital fruit hardness tester GY-4, and each measurement was repeated three times. PG enzyme activity was measured using PG enzyme activity kit G0701F (Grace Biotechnology, Suzhou, China).

Yeast one hybrid (Y1H) assay

The full-length CDS of FcERF5 was inserted into vector pGBKT7-AD (AD-TF vector, Clontech, Mountain View, CA, USA), and the FcPG12 promoter fragment with the GCC-box domain was inserted into vector pABAi. The constructed pABAi-ProFcPG12 plasmid was transformed into the Y1H Gold yeast strain by PEG/LiAC method and grown on an agar plate with SD growth medium without uracil (SD/-Ura). After 3 d, the positive yeast colony was transferred to an SD/-Ura agar plate and screened to determine the minimum inhibitory concentration of aureobasidin A (AbA) (50–500 µg L− 1) for yeast growth. Then the constructed pAD-FcERF5 and pABAi-ProFcPG12 plasmids were co-transformed into yeast strains, and these were inoculated on SD plates without leucine (SD/-Leu) containing X-α-Gal to observe color changes.

Dual-luciferase reporter assay

The FcPG12 promoter fragment (1500 bp region upstream of the start codon) was cloned and inserted into the pGreenII 0800-LUC vector [58], and introduced into Agrobacterium strain GV3101. The Agrobacterium with constructed 62sk-FcERF5 and 0800-ProFcPG12 plasmids (1:9) was co-transformed into N. benthamiana leaves by infiltration. After 48 h, 1 mM fluorescein was sprayed on the leaves, and luciferase imaging was performed using LUCK2019 (Vilber Lourmat, Paris, France). Luciferase activities (LUC and REN) were measured using a GloMax® -20/20 signal tube luminescence meter (Promega, Madison, WI, USA), and the ratio of the two was used as the calculated result.

Weighted gene coexpression network analysis (WGCNA)

WGCNA of the RNA-Seq data (Sect. 2.4) was performed using R package ‘WGCNA’ [60]. The RNA-Seq data covering the top 75% of the variance were screened, and the recommended threshold was automatically calculated. Genes with expression pattern similarity higher than 90% were selected.

Statistical analysis

Statistical analyses, consisting of one-way ANOVA followed by Duncan’s new multiple range test and Student’s t-test, were performed with IBM SPSS Statistics Version 20.0.

Data Availability

All data and supporting materials can be found within the manuscript. Accession numbers of sequence data from this article can be found in the GenBank data libraries: FcPG12 (OQ982383), FcERF5 (OQ982384), CkPG (L12019), CmPG1 (AF062465), CmPG2 (AF062466), CitPG (EF185420), CpPG1 (FJ007644), CpPG4 (GQ479796), MaPG3 (AY603339), MdPG1 (L27743), PaPG (ADT82706), PcPG1 (AB084461), PcPG3 (BAF42034), PdPG1 (DQ375247), PpPG (EF568784), PpPG1 (BAH56488), PpPG2 (CAA54448), TAPG1 (NM001246866), TAPG2 (U70480), TAPG4 (U70481), VvPG1 (AY043233).

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Acknowledgements

Thanks to Dr. Song Miao, Dr. Wang Zhe and Dr. Gu Xiaojiao from the college of Horticulture, China Agricultural University, for providing experiment and technical guidance.

Funding

This work was financially supported by the key research project for fig development of Weiyuan County, China.

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Yuan Wang conceived and designed the studies, performed most of the experiments, analyzed the data, and participated in writing and revising the manuscript. Zhiyi Fan performed most of the experiments, analyzed the data, and participated in writing and revising the manuscript. Yanlei Zhai and Hantang Huang performed some of the experiments. Alexander Vainstein participated in writing and revising the manuscript. Huiqin Ma conceived and designed the studies, and participated in writing and revising the manuscript. All of the authors have read and approved the final version of the manuscript.

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Correspondence to Huiqin Ma.

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Wang, Y., Fan, Z., Zhai, Y. et al. Polygalacturonase gene family analysis identifies FcPG12 as a key player in fig (Ficus carica L.) fruit softening. BMC Plant Biol 23, 320 (2023). https://doi.org/10.1186/s12870-023-04315-7

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