The Genome-Wide Identification, Characterization, and Expression Patterns of the Auxin-Responsive PbGH3 Gene Family Reveal Its Crucial Role in Organ Development

Abstract

The regulation of vital plant activities by hormones is governed by a family of macromolecular peptides referred to as GH3 genes. This work analyzed the expression patterns of GH3 family genes in pear tissues using transcriptome data and bioinformatics analysis. In the Bai Li pear genome, a total of 18 PbGH3 genes were identified. Comparative evolutionary studies have shown a strong association between PbGH3 and AtGH3 class I and class II proteins. The role of PbGH3 genes in growth activities and hormone regulation was revealed using gene ontology (GO) and promoter region analysis. In addition, although certain PbGH3 genes exhibited tissue-specific expression in sepals, the majority had a ubiquitous expression across all tissues. Bioinformatics and expression studies suggest that the GH3 gene family in pears may have a role in controlling the abscission of the fruit’s sepals. This work sheds light on the pear fruit sepal shedding process and may inspire further research.

1. Introduction

The pear (Pyrus L.), a member of the Rosaceae family, is the most extensively cultivated fruit tree globally, making it highly favored among consumers [1]. A premium pear cultivar, ‘Yuluxiangli’ has been all the rage recently thanks to its delicate skin, juicy meat, sweet flavor, long shelf life, and resilience to stress [2]. Nevertheless, the fruit’s quality, commodity value, and market competitiveness might be negatively impacted by persistent sepals [1]. Sepals constitute the outermost layer of the floral organ, comprising mesophyll cells, vascular bundles, and both the upper and lower epidermis. In summary, they avert mechanical damage from precipitation and moisture depletion while also protecting internal structures from additional elements [3]. As an example, ‘Yuluxiangli’ can have problems including delayed ripening, uneven surface, irregular form, and rustiness due to the sepals’ persistence. Multiple hormones interact in a complicated way to regulate organ abscission in plants, which is in turn regulated by both internal plant genetic variables and external environmental influences [4]. The process of organ shedding is regulated by nearly every plant hormone, including auxin (IAA) [5]. GH3 genes are key in the inactivation of IAA, which is vital for preventing shedding [5]. Our group utilized the ‘Korla fragrant pear’ and ‘Xuehuali’ hybrid F1 population to build a DNA-BSA library in the first stage of our research. Different pear cultivars exhibited variable expressions of these genes during sepal shedding, according to previous research [6].

Growth hormones are crucial for plant development as they influence cell division and biomass enhancement and are integral to phototropism and gravitropism [7,8,9]. The auxin early-response genes can be categorized into three primary groups: Aux/IAAs (auxin/indoleacetic acid), SAURs (small auxin up-RNAs), and GH3s (Gretchen Hagen 3), which facilitate the swift and elevated expression of specific genes [10,11]. GH3 genes are also transcriptionally regulated by stress-related hormones ABA (abscisic acid), JA (jasmonic acid), and SA (salicylic acid); growth-promoting hormones GA (gibberellins) and BRs (brassinosteroids); and ripening/senescence-associated hormone ETH (ethylene) [12]. The auxin-activated GH3 genes regulate several biological processes. Prior research has demonstrated that the GH3 gene is involved in various biological processes such as plant defense response, light signal transduction pathway, plant hormone signaling, cell wall disintegration, and fruit ripening and softening [13]. Additionally, GH3 genes participate in enhancing plant resistance against abiotic and biotic stresses [14]. It has been found that the GH3 gene promoter contains AuxRE, an auxin response element that can bind specifically to ARF and mediate auxin response genes. In particular, the GH3 protein is involved in the binding of IAA and amino acids and forms a negative feedback loop to regulate auxin isoenzymes [14]. Most group II members of GH3 are IAA amide synthetases, which can bind IAA to amino acids and change free IAA into the bound form, indicating that the GH3 gene participates in the signaling pathway of IAA [15]. The expression-based analysis of ‘Korla fragrant pear’ revealed that the GH3 gene was upregulated in the exfoliated sepals, indicating that GH3 may participate in the process of sepal exfoliation [6].

In this study, in silico and expression analysis of the auxin amide synthetase gene GH3 were carried out with ‘Yuluxiangli’ as the research material. In particular, we conducted an extensive bioinformatic analysis to understand the structural and functional characteristics of the GH3 gene family in pears. The expression analysis of PbGH3 genes displayed varied expression in different organs of the pear. The study’s overarching goal is to provide genetic resources for researchers interested in the heritability and shedding mechanisms of pear tissues, particularly sepals. Additionally, it will provide a reference for the study of the GH3 gene family in other Rosaceae species.

2. Materials and Methods

2.1. Plant Materials

The test materials for this experiment were ‘Yuluxiangli’ pears that were cultivated in the pear germplasm resource garden of the Fruit Tree Research Institute of Shanxi Agricultural University (112°30′26.454″ E and 37°21′4.980″ N). To support the growth of pear trees, a double-arm parallel scaffolding mode was used in the current study.

2.2. Treatments

Three ‘Yuluxiangli’ branches with a nine-year-old tree were chosen for biological replication under the same field management. At the initial stage of full bloom in spring, gibberellin acid 3 (GA₃, calyx retention agent), Paclobutrazol (a gibberellin inhibitor, PP₃₃₃), and distilled water (CK) were sprayed, respectively, until the entire flower was fully wet and the solution dripped. Subsequently, two weeks after spraying, at the end of the full bloom period, the calyx abscission zone tissues of pear fruits, the peel, pulp, and calyx tube tissue of the persistent fruit, as well as the single tissue of the petals, sepals, leaves, and leaf buds before the shedding of the young fruit were collected for IAA and RNA extraction. The experiment was designed using a random block design, and the single plant plot was repeated three times.

2.3. Statistics of Calyx Shedding Rate of Pear Fruits

Three weeks after the application of the treatment (during the first physiological fruit drop), the calyx shedding rate was investigated and calculated for a total of 100 fruits, with three repetitions per individual plant plot. The calyx shedding rate was defined as the ratio of the number of fruits with calyx shedding to the total number of fruits set.

2.4. Measurement of Endogenous IAA Content in the Calyx Abscission Zone

The enzyme-linked immunosorbent assay (ELISA) method was employed to determine the endogenous IAA content [16,17]. Specifically, the endogenous auxin (IAA) content was measured in the calyx collected at the late full bloom stage.

2.5. Identification of GH3 Genes in Pear Genome

The GH3 family members were identified in the Chinese white pear genome and European pear genome (http://peargenome.njau.edu.cn/, accessed on 7 September 2022) using blastp and hmmsearch methods [18,19]. Firstly, a Hidden Markov Model search (HMM search) was conducted to examine the presence of the GH3 domain in the protein sequences [20]. This was achieved by using Pfam (http://pfam.xfam.org/, accessed on 7 September 2022), smart (http://smart.embl-heidelberg.de/, accessed on 7 September 2022), and the InterProScan tool (http://www.ebi.ac.uk/Tools/pfa/iprscan/, accessed on 7 September 2022).

2.6. Chemical Properties and Chromosomal Location of Pear GH3 Protein Family

The amino acid sequence length, molecular weight, aliphatic amino acid index, isoelectric point, and hydrophilicity of the pear GH3 protein family were analyzed using the online tool ProtParam (https://web.expasy.org/protparam/, accessed on 7th September 2022). The chromosome locations of pear GH3 genes were analyzed using BLAST software v2.2.25 (https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/, accessed on 7th September 2022). This involved aligning the GH3 sequences against the pear genome sequence and obtaining the chromosome positions of all GH3 genes [21].

2.7. Phylogenetic Analysis of Pear GH3 Protein

Protein sequences of the pear PbGH3, western pear, and Arabidopsis GH3 families were aligned using the ClustalW program in MEGA 7.0, and then manual correction was performed [22,23]. Next, we used the maximum likelihood technique to build an evolutionary model, keeping all other parameters at their default values and setting the BootStrap parameter to 1000.

2.8. Conserved Domain and Motif Analysis of Pear GH3 Protein

The conserved motif of the pear GH3 protein family was analyzed using the online software MEME (http://meme-suite.org/tools/meme, accessed on 9 September 2022) and visualized with TBtools-II (Toolbox for Biologists) v1.108.

2.9. Collinearity Analysis

The OrthoMCL algorithm was used to identify paralogous genes within the pear genome, and then the MCScan algorithm was applied to detect syntenic blocks containing pear GH3 genes [24]. The syntenic relationships between the pear genomes were visualized using Circos and TBtools-II (Toolbox for Biologists) v1.108 software.

2.10. Analysis of Cis-Acting Elements of Pear GH3 Gene Family Promoter

The 2 kb region, upstream of the start codon, was downloaded from the pear genome database (http://peargenome.njau.edu.cn/, accessed on 9 September 2022). The cis-elements were identified using the PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 9 September 2022) online database and visualized using TBtools-II (Toolbox for Biologists) v1.108 software.

2.11. Gene Ontology and Interactive Protein Network Analysis

The putative functions of the PbGH3 genes were predicted using BLAST2GO (2.2.31+). The predicted functions at biological, molecular, and cellular levels were investigated and presented. The STRING database (https://string-db.org/, accessed on 4 April 2024) was used to estimate the interaction network of GH3 proteins. The STRING database was searched for pear data using the PbGH3.1 proteins as the query. The network encompassed both physical and functional protein relationships, with a minimum interaction score of 0.400 being established for medium confidence.

2.12. RNA Extraction and qRT-PCR Analysis

Total RNA was extracted from the mixture of pear samples using the CTAB method [25,26]. cDNA was synthesized using TaKaRa’s PrimeScript RT reagent Kit. First-strand cDNA was synthesized using the EasyScript^® One-Step cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) following the manufacturer’s instructions. Primer pairs for qRT-PCR were designed using Primer Premier 6.0 (Premier Biosoft, San Francisco, CA, USA). qRT-PCR was performed on a Bio-Rad CFX96 thermal cycler (Bio-Rad Laboratories, CA, USA). The Actin gene (KT943411.1) was used as the standard internal control. The relative expression levels were calculated using the comparative 2^−ΔΔCt method.

2.13. The Source of Expression Analysis of PbGH3s

Transcriptomic RNA-seq data were collected from different developmental stages of pear tissues in Pyrus bretschneideri (BioProject: PRJNA309745) and fruit stages in five cultivars of Pyrus (PRJNA309745).

2.14. Statistical Analysis

The results of the experiment were expressed as the mean standard error. The data were subjected to a one-way analysis of variance (ANOVA) using SPSS 24.0 for analysis. Duncan’s multiple range test was used to evaluate statistically significant differences between WT and transgenic lines under different treatments, with a significance level set at p < 0.05.

3. Results

3.1. Effects of Different Chemical Treatments on the Calyx Removal Rate and Calyx IAA Content in‘Yuluxiangli’

Previous research has shown that gibberellin inhibits the calyx removal of ‘Yuluxiangli’, but Paclobutrazol promotes it. The purpose of studying ‘Yuluxiangli’ calyx detachment and IAA content was to examine the association between these two chemical treatments and IAA content [27,28]. While the calyx decaying rate rose after spraying paclobutrazol, the IAA content of ‘Yuluxiangli’ reduced dramatically compared to the control (Figure 1A). On the other hand, following GA spraying, there was an increase in IAA content and a drop in the calyx removal rate (Figure 1B). The results showed that the Calyx removal rate was proportional to the concentration of IAA in the calyx, with a lower declining rate observed for samples with larger concentrations of IAA (Figure 1A).

3.2. Identification and Sequence Analysis of the Pear GH3 Gene Family

The HMM model of the GH3 protein family was used to identify a total of eighteen candidate members in the white pear reference genome. Subsequently, a total of eighteen GH3 family genes in white pear were screened using Pfam database analysis (

Table 1. Genomic information and protein characteristics analysis of pear GH3 family gene members.

Gene Named	Gene ID	Chromosome Location	Genomic Location	CDS Length (bp)	Protein
					Length (bp)	PI	MW (kD)	Subcellular Localization
PbGH3.1	Pb021060	Chr1	3,422,119–3,427,546	1716	572	5.57	63.62	cytoplasm
PbGH3.2	Pb025212	Chr2	12,797,476–12,800,533	1800	600	5.97	67.51	cytoplasm
PbGH3.3	Pb007550	Chr5	9,944,262–9,946,297	1806	602	5.45	67.48	cytoplasm
PbGH3.4	Pb030571	Chr5	10,665,377–10,667,400	1806	602	5.80	67.51	cytoplasm
PbGH3.5	Pb030587	Chr5	10,922,247-10,924,270	1806	602	5.80	67.51	cytoplasm
PbGH3.6	Pb027334	Chr5	13,774,253–13,776,424	1845	615	6.62	69.28	plasma membrane
PbGH3.7	Pb030688	Chr9	20,040,241–20,043,789	1770	590	6.04	66.87	cytoplasm
PbGH3.8	Pb018629	Chr13	8,170,306–8,172,681	1824	608	5.28	69.21	cytoplasm
PbGH3.9	Pb021158	Chr15	9,092,247–9,094,625	1845	615	7.14	69.58	plasma membrane
PbGH3.10	Pb015325	Chr15	24,177,962–24,180,054	1800	600	5.32	67.34	cytoplasm
PbGH3.11	Pb037834	Chr16	5,069,847–5,072,238	1836	612	5.23	69.46	cytoplasm
PbGH3.12	Pb006240	scaffold1310.0	40,731–41,132	402	134	9.13	152.62	cytoplasm
PbGH3.13	Pb034084	scaffold633.0	110,010–112,450	1779	593	6.13	67.00	cytoplasm
PbGH3.14	Pb041132	scaffold921.0	133,377–135,548	1845	615	7.09	69.31	plasma membrane
PbGH3.15	Pb034085	scaffold633.0	149,274–150,901	1368	456	5.57	51.52	mitochondrial matrix space
PbGH3.16	Pb034086	scaffold633.0	151,196–151,716	435	145	8.81	16.30	mitochondrial matrix space
PbGH3.17	Pb026872	scaffold435.0.1	214,889–216,981	1800	600	5.32	67.34	cytoplasm
PbGH3.18	Pb026873	scaffold435.0.1	270,065–272,157	1800	600	5.32	67.34	cytoplasm

). Isoelectric point (PI) values ranged between 5.23 and 9.13, with the lowest value being PbGH3.11 and the highest value being PbGH3.12. The molecular weight (MW) of GH3 protein ranged from 15.26 to 69.58 (KD). PbGH3.12 had the lowest MW among the GH3 members, while PbGH3.9 had the highest MW. Further analysis indicated that PbGH3.9 had the highest number of amino acids (615), whereas PbGH3.12 had the lowest. For chromosome mapping, GH3 family genes were distributed on several chromosomes of Bai Li, with chromosome 5 having the highest number of PbGH3 genes (PbGH3.3, PbGH3.4, PbGH3.5, and PbGH3.6). The PbGH3.12, PbGH3.17, PbGH3.18, PbGH3.13, PbGH3.15, and PbGH3.16 were not integrated into the assembled chromosomes.

3.3. Chromosomal Mapping

Chromosomal mapping research was carried out to visualize the physical location of the PbGH3 gene family (Figure 2). Out of all the chromosomes, chromosome 5 has the most genes, with four different ones: PbGH3.3, PbGH3.4, PbGH3.5, and PbGH3.6. Specifically, scaffold663.0 has three genes: PbGH3.13, PbGH3.15, and PbGH3.16. Along with that, Scaffold435.0.1 possesses two genes, specifically PbGH3.17 and PbGH3.18. In order to keep mitotic chromosomes structurally intact, scaffold chromosomes play a crucial role. Scaffold chromosomes are composed of nonhistone proteins present in low abundance, which further define the fate of chromosome shape.

3.4. Phylogenetic Analysis of Pear GH3 Family Proteins

The results of the evolutionary relationship between the GH3 protein of white pear and Arabidopsis thaliana showed that they could be divided into two groups (Group I and Group II) (Figure 3). with 3 and 15 members, respectively. Proteins from the A. thaliana Group III family did not cluster with any PbGH3-related proteins. The proteins PbGH3.1, PbGH3.7, and PbGH3.12 showed high homology with Arabidopsis GH3 class I protein, while the other protein members displayed high homology with Arabidopsis GH3 class II protein. The white pear PbGH3.3 and PbGH3.4 proteins showed the strongest similarity to the AtGH3.1 protein from A. thaliana, while the AtGH3.10 protein showed the strongest distance. PbGH3.3 and PbGH3.4 were the closest relatives to PcGH3.5 and PcGH3.6 from Western pear, respectively, and the most distant relatives to PcGH3.9.

3.5. Synteny Analysis of the PbGH3 Gene Family

Gene duplication leads to the expansion of gene families through segmental and tandem duplications. We speculated about the exact mechanism that had driven the expansion of the GH3 gene family in pears. We analyzed gene duplication events in the PbGH3 gene family. As shown in Figure S1, a total of 18 PbGH3 genes were unevenly mapped onto chromosomes 1, 2, 5, 9, 13, 15, 16, and 4 scaffold chromosomes. Scaffold chromosomes, along with chromosome 5, contain the highest number of duplicated genes. Interestingly, Chr5 contains five PbGH3 genes, in which PbGH3.6 duplicated with PbGH3.9 on Chr15. In addition, PbGH3.2 on Chr2 duplicated with PbGH3.10 on Chr15 and PbGH3.17 on Scaffold435.0.1. Furthermore, PbGH3.15 and PbGH3.18 duplicated on the unknown regions of Scafflold633.0 and Scaffold435.0.1, respectively. The gff3 file of the pear genome contains 17 chromosomes as well as numerous scaffolds that could not be placed. All the other chromosomes exhibit at least one duplicated gene.

3.6. Analysis of Conserved Motifs of Pear GH3 Protein Family

The conserved motifs of the pear GH3 protein family were analyzed, and the results are shown in (Figure 4 and Figure S2). Ten conserved motifs were named motif1-motif10, respectively, and out of ten, only nine motifs were presented in the amino acid sequence. Among the 18 protein members, PbGH3.13 lacks motif8, while PbGH3.15 lacks motif10 and motif8. Additionally, PbGH3.1 lacks motif8, while PbGH3.16 only has motif10 and motif5. Interestingly, PbGH3.12 only possessed motif4, the only motif observed in all PbGH3 proteins. Sequence analysis of the conserved motifs, as shown in Figure 4, showed that GH3 protein is highly conserved in pears.

3.7. Cis-Acting Element of the Promoter of Pear GH3 Family Gene

The cis-acting elements in the promoter region of the pear GH3 gene family were analyzed and are presented in Figure 5. The results highlighted numerous stresses, hormone regulation, and light-responsive cis-elements in the promoter region of the GH3 gene family. For example, a large number of elements responsive to light reaction, including G-Box, ACE, TGACG-motif, TCCC-motif, GATA-motif, G-box, WUN-motif, AT1-motif, CAAT-box, ABRE, and I-box were found in the promoter region of the GH3 gene family. All 18 GH3 family genes exhibited hormone-related cis-elements, such as the salicylic acid-responsive element SARE and TCA-element, as well as MeJA response elements CGTCA-motif and TGACG-motif. Among them,12 genes, including PbGH3.12, PbGH3.10, PbGH3.8, PbGH3.1, PbGH3.2, PbGH3.17, PbGH3.6, PbGH3.4, PbGH3.16, PbGH3.11, and PbGH3.14 have abscisic acid response elements (ABREs). Seven genes, namely PbGH3.8, PbGH3.1, PbGH3.9, PbGH3.13, PbGH3.5, PbGH3.16, and PbGH3.11 have gibberellin response elements such as TATC-box, GARE-motif, and P-box. PbGH3.3, PbGH3.10, PbGH3.8, PbGH3.9, PbGH3.2, PbGH3.17, and PbGH3.18 carry AuxRR-core and TGA-element, which are the major auxin responsive cis-elements. Some drought and low-temperature-responsive elements were also identified in the promoter region of the PbGH3 genes. It is suggested that the GH3 genes may be involved in the process of light signal transduction and plant hormone signal transduction, such as auxin signal transduction.

3.8. Gene Ontology and Protein–Protein Interaction Analysis

The gene ontology (GO) analysis was performed to investigate the predictive functions of PbGH3 genes. The GO analysis revealed that the PbGH3 gene family is involved in an array of biological, molecular, and cellular processes (Figure 6A). In the biological process category, the PbGH3 genes are mainly involved in the regulation of auxin homeostasis, hormonal response, response to light and chemicals, and other environmental stimuli. As stated in the molecular processes category, the PbGH3 genes are also responsible for the synthesis of IAA, modulation of ligase activity, and catalytic activity (Figure 6A). The STRING database was used for protein interaction to identify the potential interactive partners. The PbGH3.1 was used as a reference, and the analysis yielded several interactive partners. For instance, the XP_009372106 (Flowering time control protein FCA isoform X1), XP_009335003 (Probably inactive leucine-rich repeat receptor-like protein), XP_009354357 (LRR receptor-like serine/threonine-protein kinase GSO1), XP_009351080 (Two-component response regulator arr5-like), XP_009334310 (4-coumarate--CoA ligase-like 5), and XP_009356576 (Phytochrome-interacting factor 4; Transcription factor PIF4-like). These interactive proteins are involved in a variety of biological processes. The gene arr5, a cytokinin response regulator, is crucial in fine-tuning key developmental processes and is highly interactive with PbGH3.1 (Figure 6B).

3.9. Gene Expression in Calyx Tube and Calyx Retaining Pear Fruits

We examined the expression of eight GH3 genes to learn about the function of the PbGH3 gene family. After abscission, the calyx tube showed an increased expression of the GH3 gene family compared to the retained. The GH3 gene is a significant component of the early auxin signal response and could be involved in the induction of fruit calyx abscission via the auxin signaling pathway.

3.10. Expression Characteristics of GH3 Genes in Different Parts of Pear

The expression pattern of the PbGH3 gene family was analyzed in different tissues of ‘Yuluxiangli’ (Figure 7). All PbGH3 genes showed moderate expression in leaf tissue, with PbGH3.9 showing the highest expression and PbGH3.1 the lowest. Similarly, moderate to high mRNA levels were observed in pulp tissue for all the genes. The PbGH3.8, and PbGH3.10 genes showed more prominent expression patterns in pericarp (T) tissue than other genes. Almost every PbGH3 gene, with the exception of PbGH3.16, displayed increased expression in the pericarp (S). All PbGH3 genes showed high expressions in the calyx and calyx tubes. It is worth noting that flower tissue did not exhibit strong expression of all PbGH3 genes. The white pear bud tissue showed noticeably greater expression of all PbGH3 genes.

3.11. Expression Characteristics of GH3 Genes in Different Organs and Cultivars of Pear

In order to understand the role of the PbGH3 genes in growth processes, expression analysis was carried out in several pear organs and cultivars. For instance, the PbGH3.1 gene displayed prominent expression in the ovary, petals, S4F (flower), S4L (leaf), sepal, bud, and stem (Figure 8A). Similarly, high expression was also observed in the different cultivars of pears, such as Pbr, Pco, Ppy, Psi, and Pus (Figure 8B). The PbGH3.2, except for the ovary, did not exhibit expression in other organs, whereas it had mild expression in various cultivars of pear. The PbGH3.3 demonstrated higher expression in different pear cultivars, with the highest level recorded in Pbr. However, PbGH3.3 was only abundant in S4L and stem. The other members of PbGH3 exhibited inconsistent expression trends in the organs and cultivars. In this regard, PbGH3.4 showed moderate expression in the organs but not in cultivars. All the other genes showed no or moderate expression in the organs and cultivars. It can be inferred that PbGH3 genes, particularly PbGH3.1, PbGH3.2, and PbGH3.3, could play a crucial role in modulating the development of organs in various pear cultivars, owing to their uniform expression pattern.

3.12. Graphical Representation of PbGH3.1 in Pear Tissues

After finding that PbGH3.1 is highly expressed in all five species of pear, we visually represented this expression in pear tissues (Figure 8). Throughout the pear life cycle, the PbGH3.1 gene exhibited considerable expression levels, including the sepal, as demonstrated in the model (Figure 9).

4. Discussion

Auxin is one of the earliest discovered plant hormones. It regulates physiological responses, including phototropism, geotropism, vascular tissue formation, root development, and flower and fruit development [29,30]. It participates in stress responses, including drought stress, salt stress, and pathogen attack. Alteration of auxin levels in plants can induce the rapid expression of some genes. These genes, known as auxin early response genes (AERGs), include the Aux/IAA family, GH3 family, and SAUR family [29]. Herein, we studied the role of PbGH3 genes in pear organ shedding and abscission.

4.1. IAA Is Crucial in Controlling Pear Organ Shedding

Fruit abscission is a naturally occurring process that enables fruit trees to abandon no longer needed, infected, or damaged organs, which can be negative to plant survival [31]. Previous research showed that IAA is crucial in regulating organ shedding [31]. For instance, the direct application of IAA or its synergistic partner GA can significantly reduce the shedding [10]. Research has shown that organ abscission is hindered by high levels of IAA, but it can be enhanced by spraying an external chemical called PP₃₃₃, which lowers the levels of IAA [32]. The amount of IAA in the pear calyx was shown to be significantly correlated with the rate of calyx removal in this study. More precisely, a decrease in the concentration of IAA leads to an increase in the rate at which calyxes are removed. Here, we observed that the application of GA₃ induced the IAA contents, which eventually reduced calyx removal (Figure 1).

4.2. PbGH3 Are Widely Distributed in Pear Genome

The process of organ abscission is facilitated by auxin [32]. In the genome of ‘Dangshansuli’, 18 members of the GH3 gene family were identified. Predictions of subcellular localization placed the majority of the genes in the cytoplasm, plasma membrane, or mitochondrial matrix (Table 1). Nine substantially conserved motifs were found via homologous sequence comparison. All members of the GH3 gene family possess the motif4 (DNSIGPLEIRVVKNGTFEELMDYAISRGASINQYKTPRCVN), which suggests that these genes have been conserved throughout evolution at the amino acid level (Figure 4). Numerous plant species, notably rice, have been the subject of substantial research into the GH3 gene family in the past few years [33], grapes [34], and others. The GH3 gene family can be further subdivided into two or three groups based on their evolutionary relationships with one another (Figure 3). A. thaliana has 19 GH3 proteins, including an incomplete protein. Based on the sequence similarity of GH3 protein and their substrate specificity, the GH3 gene family can be divided into three different subfamilies. In this study, the sequence of the PbGH3 protein family was compared to the AtGH3 protein family, and a phylogenetic tree was constructed. The analysis showed that the evolutionary relationship of three pear PbGH3 protein members with Arabidopsis class I was similar, suggesting that these three members may have similar functions with class I proteins and may play a role in catalyzing the connection of JA and ethylene (ETH) synthesis precursor 1-amino cyclopropyl carboxylic acid (ACC). Furthermore, 15 genes exhibited a similar evolutionary relationship to Arabidopsis class II proteins, suggesting their potential involvement in catalyzing the adenylation of IAA or binding with amino acids and participating in the regulation of IAA balance in plants [35]. No members with a similar evolutionary relationship to Arabidopsis class III proteins were identified. Previous research has demonstrated that the conserved motif TGTCTC of AuxREs, a cis-acting auxin-responsive element, is necessary for auxin-responsive element regulation [1]. In this study, no AuxREs were identified at the upstream promoter region of pear PbGH3. However, functional elements containing phytohormones and stress responses, including TGA-element and AuxRR-core, with sequences of AACGAC and GGTCCAT, respectively, were identified at the upstream region of pear PbGH3 (Figure 5).

4.3. PbGH3 Regulate Sepal Shedding in Pear

Auxin is the master regulator of almost all the developmental events in plants and depends largely on auxin homeostasis [10,32,36]. GH3 genes are key components in plant growth and developmental activities via auxin homeostasis [37]. Regarding production biology, GH3 genes have previously been reported to be instrumental in regulating the organogenesis of reproductive organs. For instance, the gene AcGH3.1 is prominently expressed during the postharvest ripening of kiwi fruit [38]. Similarly, tomato SlGH3.1 and SlGH.3 sharply increase during ripening and could be vital in regulating auxin-mediated postharvest ripening in tomato fruit [32]. The DlGH3.2 displayed pericarp-specific expression in longan fruit [39]. The majority of the Capsicum chinense CcGH3 were differentially expressed in sepals, petals, and mature pericarp [40]. According to the research of [41], the number of stamens in the GH3.9 gene overexpressing plants is less than that of pistils, and the stamens are shorter, which is not conducive to the self-pollination of Arabidopsis and will further affect the formation of fruit.

The importance of GH3s has been observed in many A. thaliana GH3 mutants. For example, the ydk1-D (yadokari) mutant and dfl1-D (dwarf in light) mutant, isolated by the screening of activation-tagged lines, demonstrate upregulated gene expressions of GH3.2/YDK1 and GH3.6/DFL1, respectively [13]. The effect of GH3 gene expression on plant growth and development has also been studied in other plant species. For example, a gain-of-function mutation of rice OsGH3.1, OsGH3.2, OsGH3.8, or OsGH3.13/TLD1 (tld1-D mutant), which encode IAA-amino synthetases, results in dwarfed plant phenotypes often with reduced fertility [13,42]. Similarly, different tissues of the pear showed unique expression patterns for the 18 members of the GH3 gene family, according to the current study. PbGH3.10, PbGH3.15, PbGH3.16, PbGH3.2, PbGH3.17, PbGH3.18, and PbGH3.11 had the most significant levels of expression in sepals, indicating their role in the growth and development of sepals (Figure 6). On the other hand, PbGH3.3, PbGH3.14, and PbGH3.9 showed the highest expression, indicating their potential role in leaf buds (Figure 6). Additionally, PbGH3.13 exhibited the highest expression in the flesh of persistent calyx, suggesting its significance in the development of the fruit flesh of ‘Yuluxiangli’. Our data provided the latest information on the role of PbGH3 genes in calyx removal and could be used as potential biomolecules in future pear breeding programs.

5. Conclusions

The fruit’s shape and quality largely depend on the pear’s persistent calyx. We found that IAA homeostasis is instrumental during the calyx abscission process, and the GH3 gene family fine-tunes this process. We identified 18 GH3 family members of auxin amide synthetase genes in white pear, which showed typical structural characteristics of the GH3 family. Most of the GH3 members in pear were significantly upregulated in sepals, and under Paclobutrazol treatment, the gene expression of some GH3 members increased. Based on these findings, we speculate that members of the pear GH3 gene family may regulate the free IAA content and thus contribute to the development of pear tissues, particularly in sepals. However, it remains unclear whether the GH3 gene acts through the IAA pathway and how it affects the development of pear tissues, especially sepals.