Comparative and Expression Analysis of Ubiquitin Conjugating Domain-Containing Genes in Two Pyrus Species.

Ripening affects the nutritional contents and quality of fleshy fruits, and it plays an important role during the process of fruit development. Studies have demonstrated that ubiquitin-conjugating (UBC or E2) genes can regulate fruit ripening, but the characterization of UBCs in pear is not well documented. The recently published genome-wide sequences of Pyrus bretschneideri and Pyrus communis have allowed a comprehensive analysis of this important gene family in pear. Using bioinformatics approaches, we identified 83 (PbrUBCs) and 84 (PcpUBCs) genes from P. bretschneideri and P. communis, respectively, which were divided into 13 subfamilies. In total, 198 PbrUBC paralogous, 215 PcpUBC paralogous, and 129 orthologous gene pairs were detected. Some paralogous gene pairs were found to be distributed on the same chromosome, suggesting that these paralogs may be caused by tandem duplications. The expression patterns of most UBC genes were divergent between Pyrus bretschneideri and Pyrus communis during pear fruit development. Remarkably, the transcriptome data showed that UBC genes might play a more important role in fruit ripening for further study. This is the first report on the systematic analysis of two Pyrus UBC gene families, and these data will help further study the role of UBC genes in fruit development and ripening, as well as contribute to the functional verification of UBC genes in pear.

1. Introduction

Ubiquitination is an essential cellular process for eukaryotes [1]. The ubiquitin proteasome pathway involves many aspects of eukaryotic cell regulation because of its ability to degrade intracellular proteins [2,3]. Ubiquitin conjugation is a multistep reaction mediated by the action of three enzymes, including E1s (ubiquitin-activating enzymes), E2s (ubiquitin-conjugating enzymes), and E3s (ubiquitin ligases). E2s act in the middle step of the protein ubiquitination pathway [4]. Previous reports suggested that the E2 family members have a certain degree of expansion during evolution, for example, more ancestral eukaryotes such as algae have fewer E2 enzymes (< or =20) than certain plants and animals (>40) [5]. The Saccharomyces cerevisiae genome encodes 13 UBC proteins [6]; 19, 18, and 12 UBC proteins are identified in the algae Chlamydomonas reinhardtii, Micromonas sp. RCC299, and Ostreococcus, respectively; 20 in Caenorhabditis elegans [7]; and 75, 74, 52, 48, 34, and 37 UBC proteins in Zea mays, Musa nana, Solanum lycopersicum, Oryza sativa, Carica papaya, and A. thaliana, respectively [1,8,9,10,11,12].

Researchers have studied UBC genes in some plants, indicating that these genes are involved in tolerance against biotic and abiotic stresses, and plant growth and development [1,8,9,10,11,12]. For example, the A. thaliana UBC1 and AtUBC2 participate in the activation of the flowering suppressor FLC gene and inhibit flowering [13]. Overexpression of the AtUBC32 gene in A. thaliana reduced the sensitivity of plants to salt stress [14]. Overexpression of Vigna radiata UBC1 (VrUBC1), Arachis hypogaea UBC2 (AhUBC2), or Glycine max UBC2 (GmUBC2) in A. thaliana can enhance plant drought resistance [15,16,17]. The A. thaliana UBC13 (AtUBC13) has been implicated in iron deficiency responses and epidermal cell differentiation [18,19]. Additionally, AtUBC21 (AtPEX4) has been shown to be specific for ubiquitination in peroxisome maintenance [20]. In Z. mays, 16 and 48 ZmUBCs were significantly up-regulated in response to drought and salt stress [10], respectively. Similarly, in O. sativa, 14 OsUBC genes were differentially expressed under salt and drought stress [21].

Pear (Pyrus spp.) is one of the leading cultivated fruit trees which are widely grown in temperate regions, and its fleshy fruits play an important role in human health and nutrition [22,23]. The pear is the third largest temperate fruit tree after apple (Malus domestica) and grape (Vitis vinifera). Previously published manuscripts have carried out some studies on the mechanisms related to fruit ripening, as the fruit ripening is an important and complex process. The UBC genes play an important role in the fruit ripening process. In S. lycopersicum, Wang et al. (2014) found that SlUBC32 was down-regulated in the S. lycopersicum rin mutant and up-regulated during fruit ripening, indicating that this gene plays a key role in the regulation of fruit ripening [11]. In M. nana, Dong et al. (2016) identified that the expressions of 32 MaUBCs were increased or decreased during different ripening stages [9]. In C. papaya, Jue et al. (2017) suggested that 13 and two CpUBCs were up-regulated and down-regulated during one and two ripening stages, respectively [12]. However, the UBC genes involved in fruit ripening in two Pyrus species (P. bretschneideri and P. communis) have not yet been identified. To further understand the function of UBC genes, we carried out a systematic analysis of two Pyrus species, including phylogenetic relationships, sequence characteristics, chromosomal locations, and the expression differences during fruit development. These results highlight the role of UBC genes in pear fruit development and provide important information for further exploring the functional differences between two Pyrus species.

2. Materials and Methods

2.1. Sequence Retrieval and Identification of UBC Genes

To identify the UBC proteins in two Pyrus species (i.e., P. bretschneideri and P. communis), we used HMMER v3.1b2 obtained from the HMMER website (http://www.hmmer.org/download.html) [24]. The HMM profile of the UBC domain (PF00179) was downloaded from Pfam 31.0 (http://pfam.xfam.org/) [25]. Using HMMER v3.1b2 software, an HMM search was carried out for P. bretschneideri [23] and P. communis [26] genomes with a significance e-value of 0.001. SMART [27], Pfam [25], and INTERPRO [28] were used to confirm the presence of the UBC domain. Information on PbrUBCs and PcpUBCs, including intron and exon numbers, chromosomal locations, and coding sequences (CDS), was obtained from the GigdDB (http://gigadb.org/) and GDR databases (https://www.rosaceae.org/) [29], respectively.

2.2. Phylogenetic Analysis and Gene Duplication

The MUSCLE program was used to perform the alignments of all UBC amino acid sequences using default parameters [30]. ModelFinder was used to detect the best substitution model of these alignment sequences. The phylogenetic tree was generated using full-length sequences by the Maximum Likelihood (ML) method with 1000 bootstrap replications and the VT + G4 model implemented in IQ-TREE software obtained from IQ-TREE (http://www.iqtree.org/) [31]. The FigTree software (http://tree.bio.ed.ac.uk/software/figtree/) was used to visualize the ML tree. We have utilized the MCscanX software for the analysis of gene duplication events [32], and the “add_ka_and_ks_to_collinearity.pl” script for the analysis of the non-synonymous (Ka)/synonymous (Ks) substitution ratio.

2.3. Gene Structure and Motif Analysis

The GFF files of P. bretschneideri and P. communis were downloaded from GigdDB (http://gigadb.org/) and GDR databases (https://www.rosaceae.org/), respectively. The TBtools software was used to plot the map of the exon-intron structure [33]. The MEME online tool was used to search for the conservative motif of both PbrUBC and PcpUBC proteins, with the maximum width of 200 amino acids and a limit of 20 motifs, and other default parameters [34].

2.4. Expression Analysis

To further understand the expression of UBC genes in both P. bretschneideri and P. communis, we downloaded the RNA-Seq data from the public NCBI database. The sample details and accession numbers for the above data are presented in the availability of data and materials section. The FASTX-toolkit was used to remove the low-quality base-calls (Q < 20) of raw reads [35]. The TopHat2 software was used to map the clean reads to the reference genome with default parameters [36], and the Cufflinks software was used to assemble and calculate the expression FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) values [37]. The R software was used to plot the heatmap of these UBC genes.

2.5. Expression Correlation of Orthologous UBC Genes in Two Pyrus Species

Using RNA-Seq data, we obtained the expression profiles of orthologous UBC genes. Then, we estimated the similarity between the expression patterns of the orthologous gene pair by using Pearson’s correlation coefficient (r). The degree of expression diversity was confirmed by significant values (r) based on previous studies [38,39]. In general, r > 0.5, 0.3 < r < 0.5, and r < 0.3 suggest non-divergence, ongoing divergent, and divergent, respectively [38,39].

2.6. Availability of Data and Materials

P. bretschneideri fruit developmental stage 1 (Fruit_stage1: 15DAB), Accession: SRX1595645; P. bretschneideri Fruit_stage2 (30DAB), Accession: SRX1595646; P. bretschneideri Fruit_stage3 (55DAB), Accession: SRX1595647; P. bretschneideri Fruit_stage4 (85 DAB), Accession: SRX1595648; P. bretschneideri Fruit_stage5 (115 DAB), Accession: SRX1595650; P. bretschneideri Fruit_stage6 (mature stage), Accession: SRX1595651; P. bretschneideri Fruit_stage7 (fruit senescence stage), Accession: SRX1595652; P. communis Fruit_stage1 (15DAB), Accession: SRX1595636; P. communis Fruit_stage2 (30DAB), Accession: SRX1595637; P. communis Fruit_stage3 (55DAB), Accession: SRX1595638; P. communis Fruit_stage4 (85 DAB), Accession: SRX1595639; P. communis Fruit_stage5 (115 DAB), Accession: SRX1595640; P. communis Fruit_stage6 (mature stage), Accession: SRX1595641; P. communis Fruit_stage7 (fruit senescence stage), Accession: SRX1595644. P. bretschneideri drought-tolerant for 0 h, Accession: SRX4110141; P. bretschneideri drought-tolerant for 1 h, Accession: SRX4110140; P. bretschneideri drought-tolerant for 3 h, Accession: SRX4110143; P. bretschneideri drought-tolerant for 6 h, Accession: SRX4110142; P. bretschneideri recovery for 24 h, Accession: SRX4110139.

3. Results

3.1. Identification of UBC Genes in Two Pyrus Species

The protein of P. bretschneideri and P. communis was downloaded from the GigdDB (http://gigadb.org/) and GDR database (https://www.rosaceae.org/), respectively. To identify the potential UBC protein family in two Pyrus species, we obtained the UBC domain from the Pfam database and generated the HMM profile in the HMMER 3.0 package. A total of 85 and 94 putative UBC proteins were identified by searching the generated HMM profile with the E-value of 0.001 against the P. bretschneideri and P. communis protein sequence database, respectively. Further scanning of these UBC proteins for the UBC domain was conducted by a motif scan using the INTERPRO, SMART, and Pfam database, and found some UBC proteins not contained in the UBC domain. Finally, we identified 83 and 84 putative UBC proteins in the P. bretschneideri and P. communis genome, named PbrUBC01-83 and PcpUBC01-84 according to their order on the chromosomes, respectively. The information of these PbrUBC and PcpUBC genes, such as chromosome location, gene identifier, and protein length (aa), is shown in Table 1.

Table 1

The detailed information of UBC family members in both P. bretschneideri and P. communis.

Gene Name	Gene Identifier	Chromosme	5′ End	3′ End	Protein Size (aa)
PcpUBC01	PCP011547.1	chr2	908379	915288	378
PcpUBC02	PCP008190.1	chr2	2303235	2304455	406
PcpUBC03	PCP026226.1	chr2	9163625	9165622	161
PcpUBC04	PCP025062.1	chr2	11398030	11400462	160
PcpUBC05	PCP029820.1	chr2	11983749	11985588	154
PcpUBC06.1	PCP020092.1	chr3	547762	556678	986
PcpUBC06.2	PCP041697.1	chr3	551276	556678	387
PcpUBC07	PCP000885.1	chr3	3236140	3247151	841
PcpUBC08	PCP013747.1	chr3	4266095	4267481	148
PcpUBC09	PCP031109.1	chr3	6092388	6096394	192
PcpUBC10	PCP029016.1	chr3	9480497	9483572	146
PcpUBC11	PCP029037.1	chr3	11156660	11161769	769
PcpUBC12	PCP008664.1	chr3	16102272	16105426	277
PcpUBC13.1	PCP006596.1	chr4	3944944	3952328	304
PcpUBC13.2	PCP033076.1	chr4	3944944	3946278	148
PcpUBC14	PCP032557.1	chr4	13065481	13065759	92
PcpUBC15	PCP004584.1	chr5	4531710	4535205	920
PcpUBC16	PCP000343.1	chr5	6602829	6603942	120
PcpUBC17	PCP000325.1	chr5	6785831	6791593	1148
PcpUBC18	PCP013561.1	chr6	7061585	7063890	183
PcpUBC19.1	PCP003994.1	chr6	9051182	9054610	490
PcpUBC19.2	PCP038680.1	chr6	9053326	9054610	152
PcpUBC20	PCP006677.1	chr6	10001882	10012807	890
PcpUBC21	PCP025848.1	chr7	1250213	1253437	172
PcpUBC22	PCP025852.1	chr7	1277835	1279055	189
PcpUBC23	PCP027463.1	chr7	2394123	2396438	148
PcpUBC24	PCP039252.1	chr7	5395929	5399115	181
PcpUBC25	PCP041398.1	chr7	9425098	9426267	78
PcpUBC26	PCP023269.1	chr7	14434633	14435892	419
PcpUBC27	PCP018282.1	chr8	526845	534958	439
PcpUBC28	PCP024977.1	chr8	2320747	2321922	189
PcpUBC29	PCP014622.1	chr8	5359408	5371794	989
PcpUBC30	PCP013024.1	chr8	12182295	12185615	188
PcpUBC31.1	PCP025013.1	chr9	5181126	5187890	423
PcpUBC31.2	PCP042586.1	chr9	5181126	5183531	195
PcpUBC32.1	PCP029211.1	chr9	5697502	5704326	356
PcpUBC32.2	PCP037499.1	chr9	5697502	5700526	237
PcpUBC33	PCP022975.1	chr10	4876099	4877045	137
PcpUBC34	PCP006043.1	chr10	13347113	13347565	150
PcpUBC35	PCP019803.1	chr10	17163601	17167148	921
PcpUBC36	PCP037747.1	chr11	5179080	5182742	153
PcpUBC37	PCP003934.1	chr11	5615653	5620782	686
PcpUBC38	PCP014945.1	chr11	12048835	12052442	227
PcpUBC39	PCP007359.1	chr11	13280818	13282231	148
PcpUBC40	PCP041407.1	chr12	2420398	2421551	148
PcpUBC41	PCP043134.1	chr12	15435377	15437609	146
PcpUBC42	PCP003304.1	chr13	3769096	3771160	152
PcpUBC43	PCP028885.1	chr13	7446155	7448170	373
PcpUBC44	PCP010847.1	chr14	1973506	1975549	195
PcpUBC45	PCP000493.1	chr14	3899314	3905194	754
PcpUBC46	PCP006717.1	chr14	4144989	4159895	1373
PcpUBC47	PCP033096.1	chr14	4155864	4157211	152
PcpUBC48	PCP017973.1	chr14	6488805	6490257	144
PcpUBC49	PCP031605.1	chr14	8511513	8512945	265
PcpUBC50.1	PCP001680.1	chr14	12856983	12862739	444
PcpUBC50.2	PCP032125.1	chr14	12860593	12862739	146
PcpUBC51	PCP022610.1	chr15	2571489	2577399	467
PcpUBC52	PCP032902.1	chr15	3995850	3996286	78
PcpUBC53	PCP013613.1	chr15	5801442	5811234	828
PcpUBC54	PCP005846.1	chr15	15546784	15549513	160
PcpUBC55	PCP020991.1	chr15	18866853	18872463	319
PcpUBC56	PCP014540.1	chr15	21362811	21364686	161
PcpUBC57	PCP006806.1	chr16	1486195	1494553	341
PcpUBC58	PCP026158.1	chr16	4290275	4292359	152
PcpUBC59	PCP021281.1	chr16	5293770	5294891	373
PcpUBC60	PCP021299.1	chr16	5419315	5421158	307
PcpUBC61	PCP010786.1	chr17	6829215	6832764	267
PcpUBC62	PCP042470.1	chr17	15393404	15402997	596
PcpUBC63	PCP012277.1	chr17	17062013	17063062	349
PcpUBC64	PCP007143.1	chr17	17728444	17733945	621
PcpUBC65	PCP018399.1	scaffold00612	73468	83219	651
PcpUBC66	PCP021641.1	scaffold00634	76924	79227	148
PcpUBC67	PCP007398.1	scaffold00805	133398	134716	174
PcpUBC68	PCP018521.1	scaffold00852	13601	20410	278
PcpUBC69	PCP004264.1	scaffold00983	16646	19316	148
PcpUBC70	PCP021954.1	scaffold01394	34467	36817	191
PcpUBC71	PCP007629.1	scaffold01465	20581	22904	291
PcpUBC72	PCP002761.1	scaffold01522	35381	36867	160
PcpUBC73	PCP020361.1	scaffold01593	68663	70923	191
PcpUBC74	PCP045045.1	scaffold01774	29298	33191	754
PcpUBC75	PCP001255.1	scaffold01881	39525	44855	1147
PcpUBC76	PCP004531.1	scaffold01923	15838	21766	337
PcpUBC77	PCP028469.1	scaffold02358	32352	36077	463
PcpUBC78	PCP022164.1	scaffold02454	7209	11865	389
PcpUBC79	PCP012568.1	scaffold02548	27443	29342	178
PcpUBC80	PCP043259.1	scaffold04878	3046	6910	134
PcpUBC81	PCP001444.1	scaffold05041	9798	11719	183
PcpUBC82	PCP022346.1	scaffold17014	829	2135	180
PcpUBC83	PCP040042.1	scaffold23907	470	1254	125
PcpUBC84	PCP011188.1	scaffold27287	92	1717	232
PbrUBC01	Pbr021045.1	Chr1	3279060	3282148	149
PbrUBC02	Pbr022046.1	Chr1	5355491	5357926	192
PbrUBC03	Pbr018716.1	Chr1	9129424	9132884	149
PbrUBC04	Pbr013632.1	Chr1	9364933	9367901	149
PbrUBC05	Pbr029889.2	Chr2	11205065	11207039	195
PbrUBC06	Pbr025178.1	Chr2	13123810	13126801	161
PbrUBC07	Pbr022865.1	Chr2	15059120	15062337	184
PbrUBC08	Pbr022866.1	Chr2	15070696	15073441	182
PbrUBC09	Pbr040498.1	Chr2	15603251	15605586	162
PbrUBC10	Pbr024232.2	Chr3	7098302	7101956	160
PbrUBC11	Pbr027637.1	Chr3	9231735	9233562	181
PbrUBC12	Pbr023139.1	Chr3	17780615	17782004	149
PbrUBC13	Pbr000740.1	Chr3	19467052	19469759	170
PbrUBC14	Pbr013150.2	Chr3	22108425	22111475	149
PbrUBC15	Pbr034016.1	Chr3	25287937	25291916	168
PbrUBC16	Pbr030934.3	Chr4	12531057	12533684	161
PbrUBC17	Pbr027417.1	Chr5	12936909	12946872	201
PbrUBC18	Pbr027395.1	Chr5	13135775	13142057	1149
PbrUBC19	Pbr000361.1	Chr5	25903450	25906904	853
PbrUBC20	Pbr011471.1	Chr6	1539205	1541448	153
PbrUBC21	Pbr009129.1	Chr6	7334609	7336195	77
PbrUBC22	Pbr014124.1	Chr6	9284322	9290606	296
PbrUBC23	Pbr018194.1	Chr6	13523851	13526524	184
PbrUBC24	Pbr040529.1	Chr6	16795923	16797553	181
PbrUBC25	Pbr032353.1	Chr7	10867139	10869902	224
PbrUBC26	Pbr006183.1	Chr8	15645575	15647047	190
PbrUBC27	Pbr004154.1	Chr8	4690292	4691740	129
PbrUBC28	Pbr032653.1	Chr9	4250106	4253822	461
PbrUBC29	Pbr032645.1	Chr9	4153561	4154900	279
PbrUBC30	Pbr031810.1	Chr10	268749	273571	458
PbrUBC31	Pbr016259.1	Chr10	4489977	4494846	922
PbrUBC32	Pbr009080.1	Chr10	10152737	10153189	151
PbrUBC33	Pbr009081.1	Chr10	10158759	10159211	151
PbrUBC34	Pbr020740.1	Chr10	17324391	17325467	149
PbrUBC35	Pbr020719.1	Chr10	17573570	17574635	149
PbrUBC36	Pbr020703.1	Chr10	17785964	17790523	891
PbrUBC37	Pbr038220.3	Chr11	4285552	4289283	190
PbrUBC38	Pbr038323.1	Chr11	5463176	5468616	589
PbrUBC39	Pbr017901.1	Chr11	11843306	11844937	181
PbrUBC40	Pbr031559.1	Chr11	13000377	13002724	149
PbrUBC41	Pbr041320.1	Chr11	21287679	21289091	152
PbrUBC42	Pbr017298.1	Chr11	24728725	24731067	149
PbrUBC43	Pbr028474.1	Chr12	194166	195789	149
PbrUBC44	Pbr016440.1	Chr12	3192332	3192601	90
PbrUBC45	Pbr039044.1	Chr12	10209302	10211625	309
PbrUBC46	Pbr015391.1	Chr12	19750784	19753479	147
PbrUBC47	Pbr010810.1	Chr13	289004	291284	149
PbrUBC48	Pbr011958.2	Chr13	9713060	9715379	231
PbrUBC49	Pbr010372.1	Chr14	2425473	2427613	147
PbrUBC50	Pbr010424.1	Chr14	2933078	2935217	147
PbrUBC51	Pbr038166.1	Chr14	7236972	7240490	273
PbrUBC52	Pbr026720.1	Chr14	8739052	8742374	169
PbrUBC53	Pbr027115.1	Chr14	12937009	12939516	196
PbrUBC54	Pbr005908.1	Chr15	2962026	2964448	162
PbrUBC55	Pbr009224.1	Chr15	4281248	4283960	158
PbrUBC56	Pbr019673.1	Chr15	7696112	7697156	100
PbrUBC57	Pbr016945.1	Chr15	14705287	14708021	524
PbrUBC58	Pbr017248.1	Chr15	19933740	19935641	141
PbrUBC59	Pbr017249.1	Chr15	19937558	19939328	182
PbrUBC60	Pbr015294.2	Chr15	23696990	23705032	371
PbrUBC61	Pbr024308.1	Chr15	24667752	24669854	153
PbrUBC62	Pbr024286.1	Chr15	24961413	24963994	190
PbrUBC63	Pbr024279.1	Chr15	25164896	25167005	178
PbrUBC64	Pbr017425.1	Chr15	26387149	26392677	702
PbrUBC65	Pbr040652.1	Chr15	36951735	36952300	112
PbrUBC66	Pbr020836.2	Chr15	42195161	42198738	228
PbrUBC67	Pbr012108.1	Chr16	3304638	3306350	202
PbrUBC68	Pbr013690.1	Chr16	9644295	9646021	188
PbrUBC69	Pbr022472.1	Chr17	2772442	2779627	468
PbrUBC70	Pbr026816.1	Chr17	3678331	3681687	454
PbrUBC71	Pbr034051.1	Chr17	5335721	5339074	454
PbrUBC72	Pbr008641.1	Chr17	6164722	6165771	350
PbrUBC73	Pbr040232.1	Chr17	20655869	20656618	106
PbrUBC74	Pbr003941.1	scaffold1182.0	19987	21731	175
PbrUBC75	Pbr005003.1	scaffold1241.0	889	1709	133
PbrUBC76	Pbr005004.1	scaffold1241.0	11548	13331	182
PbrUBC77	Pbr006049.1	scaffold1301.0	22029	24744	149
PbrUBC78	Pbr009005.1	scaffold1564.0	5159	7986	149
PbrUBC79	Pbr028213.1	scaffold467.0	324186	326069	190
PbrUBC80	Pbr028219.1	scaffold467.0	348935	352574	172
PbrUBC81	Pbr032413.2	scaffold581.0.1	30262	34773	126
PbrUBC82	Pbr034367.1	scaffold640.0	26565	30347	154
PbrUBC83	Pbr042566.1	scaffold992.0	77372	83394	147

Note: Red logo represents tandem duplication.

3.2. Phylogenetic Analysis of UBC Genes in Two Pyrus Species

To gain insight into the evolutionary relationships of UBC genes in two Pyrus species, we built an ML tree with all PbrUBCs and PcpUBCs sequences using IQ-TREE software with the VT+G4 model, and investigated the gene structures of PbrUBC and PcpUBC genes based on the GFF3 annotation files. Phylogenetic analysis revealed that these PbrUBCs and PcpUBCs could be clustered into 13 subfamilies (Figure 1), using A. thaliana UBC genes as a template [1]. Subfamily H had 45 Pyrus UBC members and was the largest clade of all subfamilies, which represented 26.01% of the total Pyrus UBC genes. However, subfamily M and subfamily G only contained three and two Pyrus UBC members, respectively. We also found that the distribution of Pyrus UBC members was uneven in some subfamilies, suggesting that they had undergone dynamic changes from the common ancestor. Based on the phylogenetic analysis, we found that the UBC members from these Pyrus species presented a higher similarity with each other, which was consistent with their (i.e., P. bretschneideri and P. communis) evolutionary relationship. Additionally, we also detected the orthologous gene pairs between P. bretschneideri and P. communis. Finally, 129 orthologous gene pairs were found in these Pyrus species (Figure 2 and Table S1). This orthologous analysis supported the evolutionary relationships and the classification of subfamilies of UBC genes between the P. bretschneideri and P. communis genome.

Figure 1

Phylogenetic tree of UBC genes from P. bretschneideri, P. communis, and A. thaliana. The phylogenetic tree was built using IQ-TREE software, and HsUfc1 from Homo sapiens was used as the out-group. According to published articles, the ML tree could be divided into 13 subfamilies (A–M).

Figure 2

Microsynteny of UBC genes across P. bretschneideri and P. communis. The outermost scale represents the megabases (Mb). The P. communis and P. bretschneideri chromosomes are labeled Pcp and Pbr, and are represented by different color boxes, respectively. Blue, green, and red lines represent the P. bretschneideri paralogous gene pairs, P. communis paralogous gene pairs, and orthologous gene pairs, respectively.

Observation of the gene structure in these two Pyrus species UBC genes showed that the numbers of introns in the 83 PbrUBC and 84 PcpUBC genes varied from 0 (PbrUBC44, PcpUBC34, PbrUBC33, PcpUBC14, PbrUBC32, PbrUBC72, PcpUBC02, PcpUBC34, PcpUBC59, and PcpUBC63) to 16 (PcpUBC29) (Figure S1). Additionally, we found that most of the Pyrus UBC genes clustered in the same subfamily contained highly similar gene structure maps, including intron numbers and exon length. For instance, PbrUBC53 and PcpUBC44 in the subfamily C contained four introns, and PbrUBC10 and PbrUBC37 in the subfamily B had five introns. We also scanned the conserved motifs in these UBC genes, and found that motif 2, −3, and −15 encoded the UBC domain (Figure S2). To sum up, the gene structures and conserved motifs of UBC genes were basically consistent with the above evolutionary relationship.

In general, the different protein isoforms produced by alternative splicing may affect the diversity of transcriptomics and proteomics, ultimately affecting gene expression regulation and protein function. In our study, the occurrence of alternative splicing events was revealed in the UBC family during evolution, such as PcpUBC06, PcpUBC13, PcpUBC19, PcpUBC31, PcpUBC32, and PcpUBC50 (Table 1). The mRNAs of PcpUBC13.1/PcpUBC13.2, PcpUBC19.1/PcpUBC19.2, PcpUBC32.1/PcpUBC32.2, and PcpUBC50.1/PcpUBC50.2, which are produced by variable splicing, are different in the 3′-end. However, the mRNAs of PcpUBC06.1/PcpUBC06.2 and PcpUBC31.1/PcpUBC31.2, which are produced by variable splicing, are different in the 5′-end (Figure S3). These results suggested that changes in the transcript sequence of the UBC gene caused by alternative splicing events may have an effect on the interaction ability and function of the encoded proteins.

3.3. Chromosomal Distribution and Gene Duplication of UBC Genes in Two Pyrus Species

In the present study, 167 genes were identified as members of the UBC gene family, with 83 PbrUBC genes in P. bretschneideri and 84 PcpUBC genes in P. communis (Table 1). Then, we determined the chromosomal distribution of each UBC gene. As shown in Figure 3 and Table 1, the distribution of 167 UBC genes on the chromosome is random, and some of them were located on scaffolds. The genome maps of the UBC genes suggested that PbrUBC genes were dispersed across all chromosomes; however, PcpUBC genes were mainly found on 16 out of 17 chromosomes, except for chromosome 1. In the P. bretschneideri genome, chromosome 15 had the maximum number of PbrUBC genes (13), while chromosome 7 contained only one gene (PbrUBC15) gene. In the P. communis genome, both chromosome 3 and 14 contained the most PcpUBC genes, followed by chromosome 7 (6) and 15 (6) (Figure 3).

Figure 3

Localization and duplication of UBC genes in the P. bretschneideri (a) and P. communis (b) genome, respectively. The localizations of PbrUBCs and PcpUBCs mapped on the P. bretschneideri and P. communis genome, respectively, were obtained from Circos software [40]. The P. communis and P. bretschneideri chromosomes are labeled Pcp and Pbr, and are represented by different color boxes, respectively. Red regions indicate tandem duplication, and grey lines represent segment duplication. The outermost scale represents the megabases (Mb).

Gene duplication contributes to the expansion of gene family members and diversification of protein functions. In general, if two genes are collinear, they are considered to have evolved from a duplication event. In order to further investigate the expansion mechanism of UBC gene family members, the occurrence of segmental duplication and tandem duplication events were analyzed during the evolution of this gene family. Finally, 198 and 215 duplication events (Figure 3) of the P. bretschneideri and P. communis UBC genes were identified, respectively. Among these duplication gene pairs, four and one gene pairs were identified to have evolved from tandem duplications in P. bretschneideri and P. communis, respectively, and the remaining gene pairs were involved in segmental duplications. Additionally, a series of several-for-one duplication events in P. bretschneideri and P. communis UBC genes were observed, such as PbrUBC07/PbrUBC26, PbrUBC07/PbrUBC20, PcpUBC12/PcpUBC34, and PcpUBC12/PcpUBC48, and it is envisaged that these genes may contribute to the expansion of UBC gene family members during evolution. The pear genome shared two whole-genome duplication (WGD) events, the ancient WGD occurred in ~140 MYA (Millions of years ago) (Ks ~ 1.5–1.8) and the recent WGD occurred in 30–45 MYA (Ks ~ 0.15–0.3). Subsequently, 15 and 14 duplication gene pairs (Table S2) were identified as being derived from ancient WGDs and recent WGDs in the P. communis genome, respectively. In the P. bretschneideri genome, 17 duplication gene pairs were evolved from the recent WGDs, and 13 from the ancient WGDs. These results suggested that two WGDs contribute to the expansion of UBC gene family members in the Pyrus genome.

3.4. Evolutionary Patterns in Two Pyrus Species

To investigate the evolutionary divergence and patterns of the UBC genes in P. bretschneideri and P. communis, the selection pressures of 198 paralogous gene pairs in P. bretschneideri, 215 paralogous gene pairs in P. communis, and 129 orthologous gene pairs in P. bretschneideri and P. communis were analyzed. All gene pairs, including paralogous and orthologous, are listed in Table S1. To avoid the risk of saturation [41], we removed any Ks values >2.0 in our study. In P. bretschneideri, 99 paralogous pairs contained Ka/Ks ratios below one, while the remaining gene pairs had ratios greater than one (Table S2). In P. communis, 94 paralogous pairs had Ka/Ks ratios below one, while the remaining gene pairs had ratios greater than one. The maximum Ka/Ks value was 5.055 in P. bretschneideri (PbrUBC04-PbrUBC70) and 5.65 in P. communis (PcpUBC51-PcpUBC58) (Table S1 and Figure 4). Among these orthologous pairs, we found that the most of the gene pairs had Ka/Ks ratios that were below one, indicating that these genes (which evolved from a common ancestor) have undergone purify selection with slow evolution at the protein level. Remarkably, these genes might also evolve through positive selection (Ks = 0; Ka ≠ 0, such as PbrUBC32-PcpUBC32), negative selection (i.e., Ka = 0; Ks ≠ 0, such as PcpUBC42-PcpUBC58), and strongly negative selection (i.e., Ka = Ks = 0, such as PbrUBC09-PcpUBC03) due to these gene pairs being subject to strong constraints (Table S2).

Figure 4

The distribution of Ka (nonsynonymous), Ks (synonymous), and Ka/Ks values of paralogous and orthologous gene pairs. (a–c) represent Pbr-Pbr, Pcp-Pcp, and Pbr-Pcp gene pairs, respectively. The X- and Y-axes denote the synonymous distance and Ka/Ks ratio for each pair, respectively.

3.5. Expression Profiles of UBC Genes in Pyrus Fruit Development

The genome sequences of both P. bretschneideri and P. communis provided an excellent opportunity to further study gene expression. Previous studies have shown that UBC genes may play an important role during fruit development [11,12]. To further understand the potential roles of PbrUBC and PcpUBC genes during pear fruit development, we obtained the transcriptome data of these UBC genes and built a heat map. From the transcriptome data results, it was apparent that 50.6% (42/83) PbrUBCs and 25% (21/84) PcpUBCs were not detected in each fruit developmental stage, suggesting their activity in other organs, such as the flower, root, or leaf. In P. bretschneideri, 41 PbrUBC genes were expressed in one or more developmental stages (Figure 5 and Table S2). Among them, 17 PbrUBC genes were expressed in all P. bretschneideri fruit development stages, indicating that these genes might be very important for the development and maturation of fruit. In P. communis, 47 PcpUBC genes were expressed in all P. communis fruit development stages, implying that these have functional activity in all fruit development stages. Remarkably, we found that the different isoforms produced by alternative splicing were not expressed in the period of pear fruit development, suggesting that the alternative splicing events might not play a role during pear fruit development. Additionally, we found that some UBC genes continuously increased or reduced at one or several stages, such as PbrUBC24 and PbrUBC80, which were highly expressed in Fruit_stage3 (55 days after full blooming), and PcpUBC14, which was highly expressed at Fruit_stage5 (115 days after full blooming), implying that these genes might be very important for fruit-specific developmental stages.

Figure 5

Expression of UBC genes from P. bretschneideri and P. communis during fruit development and ripening, including Fruit_stage1 (15 days after full blooming (DAB)), Fruit_stage2 (30 DAB), Fruit_stage3 (55 DAB), Fruit_stage4 (85 DAB), Fruit_stage5 (115 DAB), Fruit_stage6 (mature stage), and Fruit_stage7 (fruit senescence stage). The color scale represents normalized log 2-transformed, where grey indicates a medium level, blue indicates a low level, and red indicates a high level. Circos software was used to visualize the heat map. The FPKM values of PbrUBCs and PcpUBCs are presented in Table S3. The outermost ring represents Fruit_stage7, followed by Fruit_stage6, Fruit_stage5, Fruit_stage4, Fruit_stage3, and Fruit_stage2, and the innermost ring represents Fruit_stage1.

3.6. Comparison of the Expression Patterns of UBC Genes in Two Pyrus Species

Pear is one of the leading cultivated fruit trees of temperate regions, and the fruit is the focus of this study due to its economic value. Homologous genes may have gene functional redundancy or divergence during evolution [42]. In the present study, to gain insight into the degree of expression diversity of UBC gene family members between P. bretschneideri and P. communis, their expression correlations were estimated using Pearson’s correlation coefficient (r). Remarkably, we only considered the homologous genes which were expressed in at least one pear fruit development stage (Table S4). Twenty-two orthologous gene pairs (such as PbrUBC36-PcpUBC17, PbrUBC20-PcpUBC42, and PbrUBC06-PcpUBC54) were found to be non-divergent, five orthologous gene pairs (such as PbrUBC18-PcpUBC17, PbrUBC01-PcpUBC69, PbrUBC31-PcpUBC35, PbrUBC07-PcpUBC55, and PbrUBC80-PcpUBC21) were ongoing divergent, and the remaining orthologous gene pairs (such as PbrUBC50-PcpUBC53, PbrUBC19-PcpUBC35, PbrUBC04-PcpUBC21, and PbrUBC20-PcpUBC58) were divergent (Table S3). These results suggested that most of the UBC orthologous gene pairs have undergone functional divergence.

3.7. Expression Profiles of PbrUBC Genes Respond to Drought Stress

As a major abiotic stress, drought can affect plant productivity, growth, and development. Previous studies have shown that plants can enhance their drought tolerance by regulating gene transcription [9,15,16]. To identify UBC genes with a potential role in the drought stress response of P. bretschneideri, we carried out the expression analysis for 83 PbrUBC genes under drought stress. From the transcriptome data results, we found that only 34.9% (28/83) PbrUBCs were expressed under drought stress (Figure 6). Under drought stress treatment, five PbrUBC genes (PbrUBC02, PbrUBC10, PbrUBC15, PbrUBC37, and PbrUBC74) were up-regulated at early time points; however, they were down-regulated after a long period of stress treatment, indicating the existence of a possible feedback regulatory mechanism. Two (PbrUBC49 and PbrUBC63) and five PbrUBC (PbrUBC03, PbrUBC07, PbrUBC13, PbrUBC32, and PbrUBC62) genes under drought stress treatment were up- and down-regulated, respectively (Figure 6). Our data indicated that these genes might be important for drought stress responses and will help to select candidate genes for functional analysis under drought stress.

Figure 6

Expression analysis of PbrUBC genes under drought stress treatment. The color scale represents normalized log 2-transformed, where grey indicates a medium level, blue indicates a low level, and red indicates a high level. The treatments were indicated at the bottom of each column, and the genes are located on the right.

4. Discussion

As a part of the ubiquitin proteasome system, ubiquitin-conjugating enzymes have been proved to play an important role in plant growth and development [1,11,21]. Although members of the UBC gene family have potential functional significance, they are relatively few in higher plants. Pear is widely cultivated in temperate regions due to its high nutritional and economic value. For pears, the fruit is the focus of this study. Previous studies have shown that the UBC gene family plays a very important role in fruit development and ripening [11,12], but is still excluded in two Pyrus species (P. communis and P. bretschneideri).

In our study, 83 PbrUBCs and 84 PcpUBCs genes were identified from the P. bretschneideri and P. communis genome, respectively. The number of PbrUBCs and PcpUBCs is much larger than 75, 74, 52, 48, 48, and 34 UBC genes previously reported from Zea mays, Musa nana, Solanum lycopersicum, Oryza sativa, Arabidopsis thaliana, and Carica papaya, respectively [1,8,9,10,11,12]. The genome sizes of Zea mays, Musa nana, Solanum lycopersicum, Oryza sativa, Arabidopsis thaliana, and Carica papaya are ~2300, ~523, ~900, ~466, ~125, and ~372 Mb, respectively. Then, we found that the genome sizes of S. lycopersicum and O. sativa are 7.2 and 3.7 times larger than that of A. thaliana, respectively; however, the genomes of these species have a similar number of UBCs, including 52, 48, and 48, respectively. In addition, the genome size of Z. mays is 4.67 times larger than that of Pyrus species (i.e., P. bretschneideri and P. communis), but the genomes of both P. bretschneideri (83) and P. communis (84) have a larger number of UBCs compared to Z. mays (75) and other studied species. Therefore, we speculate that the difference in the number of UBC genes is not related to the size of the genome.

Alternatively, gene duplication events, including segmental and tandem duplication, play a significant role in the expansion of gene family members in the genome. Two WGD events, including recent WGD [23] and ancient WGD [43], were shared by both the P. bretschneideri and P. communis genome during evolution. In order to understand the contribution of gene duplication events to the expansion of UBC family members in two Pyrus species, we analyzed the expansion mechanism of both the PbrUBC and PcpUBC gene family. In the P. bretschneideri genome, 192 PbrUBC gene pairs were determined to be involved in segmental duplication events and four gene pairs were identified that were involved in tandem duplication events. Similarly, 211 and one UBC gene pairs were involved in segmental duplication and tandem duplication events in the P. communis genome, respectively. These data indicate that the common expansion mechanism of the UBC gene family is mainly segmental duplication events, which is shared by both PbrUBCs and PcpUBCs. Therefore, we can infer that the expansion of UBC gene family members may not completely depend on independent duplications of individual sequences, and it may also be the result of rearrangement events and segmental chromosome duplication. A growing number of studies have shown that segmental duplications play a major role in the expansion of the pear gene family, such as the VQ, MYB, PRX, PHD, and WOX gene families [22,42,44,45,46].

UBC genes have been demonstrated to play an important role in plant growth and development, and physiological processes. For instance, OsUBC1 from O. sativa involves cellular responses to abiotic and biotic stresses [47], and the expression of five A. thaliana UBC genes (AtUBC13, AtUBC17, AtUBC20, AtUBC26, and AtUBC31) and three O. sativa UBC genes (OsUBC2, OsUBC5, and OsUBC18) is significantly down-regulated under drought and salt stress treatments; however, three OsUBC genes (OsUBC13, OsUBC15, and OsUBC45) are significantly up-regulated [21]. In the present study, we found that 34.9% (28/83) of PbrUBCs can respond to drought stress treatment at the transcriptional level, implying these genes play essential roles in responsive to drought stress in P. bretschneideri, such as PbrUBC02, PbrUBC10, PbrUBC15, PbrUBC37, and PbrUBC74, which were up-regulated at early time points. In the O. sativa and Z. mays, similar expression changes among UBC genes were also observed, including the expression of 34 ZmUBC genes that changed significantly and were up-regulated during early time points. These data indicated that these UBC genes might have important roles under drought stress treatment during P. bretschneideri development. The function of UBC genes in plant development and response stress has been well studied, but little is known about the role of protein ubiquitination in fruit development and ripening, except for M. nana, S. lycopersicum, and C. papaya. In M. nana, five UBC genes (MaUBC1, MaUBC9, MaUBC70, MaUBC68, and MaUBC71) presented about 10-fold to 40-fold higher expression levels at the fifth stage than at other stages of fruit ripening; however, seven other UBC genes (MaUBC8, MaUBC16, MaUBC17, MaUBC33, MaUBC34, MaUBC56, and MaUBC61) presented continuously increasing expression during all the fruit development stages [9]. In S. lycopersicum, six UBC genes (SlUBC6, SlUBC8, SlUBC24, SlUBC32, SlUBC41, and SlUBC42) were directly regulated by RIN, a fruit-ripening regulator [11]. In C. papaya, 13 (CpUBC4, CpUBC6, CpUBC7, CpUBC8, CpUBC9, CpUBC11, CpUBC12, CpUBC14, CpUBC16, CpUBC19, CpUBC20, CpUBC28, and CpUBC34) and two (CpUBC2 and CpUBC10) were up-regulated and down-regulated during C. papaya fruit ripening stages, respectively [12]. Our results suggested that PbrUBC82, PcpUBC10, and PcpUBC62, orthologs of MaUBC3 and MaUBC8 and AtUBC36, respectively (Figure S4), contained high expression levels during the fruit developmental period. S. lycopersicum SlUBC6 orthologs in two Pyrus species PbrUBC18 and PcpUBC17 were directly regulated by RIN (a fruit-ripening regulator), as reported in S. lycopersicum, and in two Pyrus species, were also highly expressed in fruits, suggesting a major role in fruit development. Additionally, we found that orthologs from different species exhibit different expression profiles. SlUBC32, MaUBC72, MaUBC47, PcpUBC44, PbrUBC53, PcpUBC31, and CpUBC5, which belong to the same subfamily (Figure S4), contained different expression profiles. For example, MaUBC72 was expressed during all M. nana fruit development, while its P. communis and S. lycopersicum orthologous genes, SlUBC32 and PcpUBC44/-31, were not expressed during fruit development. The contrary expression patterns suggested that these genes have different regulatory mechanisms in the development of plant fruits. Taken together, the present study indicated that some PbrUBCs and PcpUBCs might contribute to the regulation of fruit development and ripening processes. For UBC genes, the expression of most UBC orthologous gene pairs from P. bretschneideri and P. communis has undergone functional divergence, indicating functional redundancy evolved from a common ancestry for some orthologous gene pairs, and from neo-functionalization or sub-functionalization for others.