Genome-wide identification of PbrbHLH family genes, and expression analysis in response to drought and cold stresses in pear (Pyrus bretschneideri)

Abstract

Background

The basic helix-loop-helix (bHLH) transcription factors play important roles in many processes in plant growth, metabolism and responses to abiotic stresses. Although, the sequence of Chinese white pear genome (cv. ‘Dangshansuli’) has already been reported, there is still a lack of clarity regarding the bHLH family genes and their evolutionary history.

Results

In this work, a genome-wide identification of the bHLH genes in Chinese white pear was performed, and we characterized the functional roles of these PbrbHLH genes in response to abiotic stresses. Based on the phylogenetic analysis and structural characteristics, 197 identified bHLH genes could be well classified into 21 groups. Expansion of PbrbHLH gene family was mainly driven by WGD and dispersed duplication with the purifying selection from the recent WGD. The functional annotation enrichment showed that the majority of PbrbHLHs were enriched in the GO terms and KEGG pathways involved in responds to stress conditions as TFs. Transcriptomic profiles and qRT-PCR revealed that PbrbHLH7, PbrbHLH8, PbrbHLH128, PbrbHLH160, PbrbHLH161 and PbrbHLH195 were significantly up-regulated under cold and drought treatments. In addition, PbrbHLH195-silenced pear seedlings display significant reduced cold tolerance, exhibiting reduced chlorophyll content, as well as increased electrolyte leakage and concentrations of malondialdehyde and H₂O₂.

Conclusion

For the first time, a comprehensive analysis identified the bHLH genes in Chinese white pear and demonstrated that PbrbHLH195 is involved in the production of ROS in response to cold stress, suggesting that members of the PbrbHLH family play an essential role in the stress tolerance of pear.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-021-02862-5.

Background

Transcription factors (TFs) are protein molecules with special structure and function of regulating gene expression, which plays many crucial roles in plant growth and development [1]. The basic helix-loop-helix (bHLH) transcription factor family is the second largest family in plants. The members of this family are designated by a highly conserved domain called the bHLH which are able to bind and form DNA dimers [2]. The conserved bHLH domain consists of about 60 amino acids and has two functional segments, the basic region and the HLH region. The N-terminal basic region, which contains 13–17 major basic amino acids, serves as the DNA binding domain to identify and specifically bind to DNA motifs in the promoter of the target gene [3–6]. The HLH region is located at the C-terminus of the bHLH domain, which consists of two parental α-helixes, mainly composed of hydrophobic residues, connected by a relatively dispersed (length and primary sequence) loop region. The HLH domain promotes protein-protein interactions and allows the formation of homo-dimer or hetero-dimer complexes [7]. bHLH transcription factors are involved in many process about plant growth and metabolism, such as stomata development [8], light signal transduction [9, 10], flowering regulation [11], anthocyanin and secondary metabolism [12–14]. There have been reported that bHLH genes are mainly involved in abiotic stress in plants, such as the responses to drought, low temperature, salt, ABA and mechanical damage [15, 16]. For example, AtbHLH006, AtbHLH17, AtbHLH32, AtbHLH92, AtbHLH122, AtbHLH128 and AtbHLH130 are directly or indirectly involved in ABA signaling pathway to improve drought resistance in Arabidopsis [17]. The over-expression of bHLH TF MYC-type ICE1, ICE2 and CBF enhanced the tolerance of Arabidopsis to low temperature stress [18]. In wheat, TabHLH1 is able to regulate ABA-mediated stress tolerance pathway to improve plant adaptability to drought and salt stresses [19]. The TabHLH39 gene is involved in regulating gene expression levels in stress responses, thereby increasing salt tolerance in over-expressing plants [20]. In rice, OsbHLH148 and OsbHLH006 (RERJ1) respond to drought stress through the jasmonic acid signaling pathway [21, 22]. The bHLH transcription factor RsICE1 can improve the cold tolerance of transgenic rice [23]. The expression of the PebHLH35 gene in populus increased during drought and ABA induction, and PebHLH35 had an active regulatory effect under drought stress, which mentioned plant tolerance [24]. Similarly, it was shown that VabHLH1 and VvbHLH1 are positive regulators of response to low temperature stress in Chinese wild Vitis amurensis and Vitis vinifera cv. Cabernet Sauvignon, and able to confer enhanced low temperature tolerance to transgenic plants by regulating the expression level of cold regulated (COR) genes [25].

To date, based on the rapid development of genome sequencing, a number of plant bHLH TF genes have been identified and characterized in many species. Although, there are 162, 167, 155, 124 and 188 bHLHs have been identified in Arabidopsis, rice, bean, potato and apple, respectively [26], there has been no report about the bHLH family in pear. Pear is an important cash crop and widely distributed in the world. However, pears were suffered from abiotic stresses such as drought, low temperature, and salt during the growth and development process, which not only restricts the cultivation area, but also affects their growth, development and yield. Therefore, investigating of pear bHLH transcription factors are necessary to elucidate the biological processes underlying pear stress responses.

In this study, we identified 197 pear bHLH (PbrbHLH) genes from the Chinese white pear genomic sequence and carried out phylogenetic analysis to determine the relationships among these genes. Analysis results of protein motifs and intron/exon structures support the classification of the bHLH family. At the same time, we identified duplication events that likely contributed to the expansion of the bHLH family. In addition, RNA-Seq data showed that the expression patterns of PbrbHLHs differed in response to drought and cold stresses. The data from this study will increase our understanding of PbrbHLH functions associated with stress responses. Meanwhile, our systematic analysis provided a foundation for further mechanisms of cold-tolerance and drought-tolerance for bHLH genes in pear, especially for aiming to identify candidate genes that may be involved in the cold- and drought-tolerance of pears.

Results

Identification, classification and function annotation of bHLH genes in Chinese white pear

To identify the PbrbHLH genes, we performed local HMM-search with the HMM file (PF00010) against Chinese white pear genome, with default parameters. 200 putative PbrbHLH protein sequences were identified. SMART and NCBI Batch CD-Search tools were used to confirm the existence of the conserved bHLH domain, and redundant sequences were removed. We finally obtained 197 sequences in pear bHLH family. According to the order of gene ID, these genes were named from PbrbHLH1 to PbrbHLH197 (Table 1 and Table S1). 168 PbrbHLH genes are randomly distributed on all 17 chromosomes ranging from 1 to 25 per chromosome, and the others were localized to 25 unanchored scaffolds (Table 1). Chromosome 15 has the most PbrbHLHs (25 genes), followed by chr 5 (21 genes) and chr 14 (15 genes).

Table 1.

Characteristics of identified PbrbHLH proteins

Gene name	Best hit in AT	Chr	start	end	ORF length	Extron num	MW (kDa)	PI	GRAVY
PbrbHLH1	AT5G54680.1	Chr5	27,094,335	27,096,404	720	5	26.13	6.29	−0.575
PbrbHLH2	AT4G21330.1	Chr5	27,016,336	27,016,952	441	3	16.28	4.24	−0.254
PbrbHLH3	AT1G61660.3	Chr5	26,993,507	27,001,224	1323	5	47.48	4.74	−0.502
PbrbHLH4	AT2G40200.1	Chr5	25,158,927	25,161,863	804	2	28.85	9.06	−0.296
PbrbHLH5	AT1G51140.1	Chr3	18,537,202	18,539,580	1254	6	45.25	6.24	−0.758
PbrbHLH6	AT1G35460.1	Chr15	40,505,769	40,508,649	657	4	23.32	5.67	−0.565
PbrbHLH7	AT5G57150.3	scaffold1040.0	66,902	68,814	774	5	29.31	8.72	−0.577
PbrbHLH8	AT5G65640.1	Chr8	6,468,092	6,469,857	1089	4	40.74	4.32	−0.569
PbrbHLH9	AT5G01305.1	Chr12	16,099,618	16,100,214	600	1	22.23	6.7	−0.57
PbrbHLH10	AT1G06690.1	Chr17	15,208,210	15,222,427	2412	15	88.59	9.32	−0.575
PbrbHLH11	AT4G29100.1	Chr17	16,366,147	16,368,813	1026	9	37.25	7.55	−0.702
PbrbHLH12	AT2G14760.1	scaffold1151.0	9562	11,543	1062	5	38.46	5.25	−0.539
PbrbHLH13	AT4G33880.1	scaffold1151.0	36,152	38,137	1104	5	40.15	4.94	− 0.688
PbrbHLH14	AT1G35460.1	Chr8	5,724,025	5,727,879	702	4	24.66	8.63	−0.676
PbrbHLH15	AT4G02590.2	Chr12	2,040,321	2,045,714	1014	7	35.47	6.24	−0.44
PbrbHLH16	AT5G43650.1	scaffold1203.0	77,475	79,190	546	3	21.27	10.1	−0.846
PbrbHLH17	AT5G65640.1	Chr15	20,949,598	20,951,150	1077	4	39.71	4.95	−0.481
PbrbHLH18	AT5G41315.1	scaffold1226.0	3	1454	690	3	25.9	5.7	−0.672
PbrbHLH19	AT5G65640.1	Chr15	35,299,620	35,301,589	1098	4	41.14	4.39	−0.585
PbrbHLH20	AT2G42280.3	Chr2	9,337,765	9,339,806	1278	6	47.26	6.38	−0.893
PbrbHLH21	AT5G41315.3	Chr14	10,423,756	10,458,851	2358	9	87.65	5.13	−0.457
PbrbHLH22	AT5G41315.3	Chr14	10,657,342	10,660,577	2052	8	76.58	5.91	−0.422
PbrbHLH23	AT1G27660.1	Chr15	3,038,629	3,044,314	1371	7	49.35	6.99	−0.467
PbrbHLH24	AT1G09530.5	Chr16	10,077,360	10,081,240	2154	7	76.57	6.55	−0.614
PbrbHLH25	AT3G06120.1	scaffold132.0.1	335,925	337,641	621	3	22.95	9.23	−0.194
PbrbHLH26	AT1G72210.1	scaffold132.0.1	415,469	417,397	981	3	36.18	6.51	−0.448
PbrbHLH27	AT1G72210.1	Chr4	632,585	633,725	783	3	29.21	5.83	−0.361
PbrbHLH28	AT1G09530.5	Chr4	2,380,472	2,383,676	2145	7	76.42	6.51	− 0.679
PbrbHLH29	AT5G08130.5	Chr14	14,953,088	14,955,645	1713	9	62.47	9.04	−0.704
PbrbHLH30	AT4G00050.1	Chr14	15,502,751	15,505,135	897	6	32.66	9.6	−0.811
PbrbHLH31	AT2G24260.2	Chr2	20,591,591	20,595,907	1410	7	48.56	6.66	−0.474
PbrbHLH32	AT3G22100.1	Chr17	16,734,318	16,735,664	1338	1	48.69	9.24	−0.584
PbrbHLH33	AT2G46810.3	Chr1	1,364,775	1,367,503	1302	4	48.52	6.03	−0.662
PbrbHLH34	AT2G41130.1	scaffold1497.0	3118	4544	726	2	27.16	8.62	−0.54
PbrbHLH35	AT2G41130.1	scaffold1497.0	33,505	34,931	726	2	27.16	8.62	−0.54
PbrbHLH36	AT1G49770.1	Chr5	9,948,267	9,951,160	849	3	30.42	9.21	−0.403
PbrbHLH37	AT3G21330.1	Chr9	9,245,860	9,247,209	1353	1	50.11	6.82	−0.71
PbrbHLH38	AT4G16430.1	Chr10	9,856,487	9,857,979	1425	2	52.65	7.24	−0.482
PbrbHLH39	AT4G16430.1	Chr10	9,876,054	9,878,083	1512	1	55.69	6.16	−0.454
PbrbHLH40	AT4G02590.2	Chr10	10,112,702	10,116,115	894	6	31.43	6.41	−0.368
PbrbHLH41	AT5G41315.3	Chr6	7,815,415	7,818,898	1878	6	70.05	6.01	−0.466
PbrbHLH42	AT2G40200.1	Chr1	7,131,102	7,132,823	831	2	29.84	9.37	−0.358
PbrbHLH43	AT2G43650.1	scaffold162.0	146,901	178,353	2154	19	80.5	4.75	−0.807
PbrbHLH44	AT3G26744.4	Chr14	12,125,142	12,127,506	1605	4	57.59	5.52	−0.497
PbrbHLH45	AT5G50915.1	Chr15	13,329,386	13,332,068	1068	7	39.35	7.02	−0.775
PbrbHLH46	AT3G26744.4	Chr15	13,269,686	13,272,442	1644	4	59.07	5.68	−0.52
PbrbHLH47	AT5G53210.1	Chr13	217,187	219,497	1260	3	44.87	5.92	−0.355
PbrbHLH48	AT2G40200.1	Chr7	11,517,886	11,519,641	858	2	31.01	8.33	−0.285
PbrbHLH49	AT2G31280.1	Chr10	8,959,774	8,964,350	2223	11	81.76	6.18	−0.364
PbrbHLH50	AT3G47640.1	Chr6	1,652,351	1,654,266	744	4	27.31	7.55	−0.745
PbrbHLH51	AT3G28857.1	Chr6	1,630,526	1,630,925	330	2	12.16	6.96	−0.443
PbrbHLH52	AT3G47710.1	Chr6	1,573,126	1,574,101	282	2	10.38	9.4	−0.685
PbrbHLH53	AT5G62610.2	Chr6	1,235,430	1,238,083	870	6	30.55	5.08	−0.712
PbrbHLH54	AT3G14270.1	Chr2	17,666,963	17,682,155	7410	18	274.01	5.39	−0.488
PbrbHLH55	AT3G19500.1	Chr10	15,027,760	15,030,379	783	5	27.99	8.59	−0.544
PbrbHLH56	AT1G69010.1	Chr3	21,842,280	21,845,477	1008	7	36.78	5.88	−0.869
PbrbHLH57	AT1G68920.3	Chr3	21,774,132	21,778,787	2283	11	83	7.4	−0.5
PbrbHLH58	AT1G68810.1	Chr3	21,442,039	21,445,012	1059	2	39.09	6.76	−0.628
PbrbHLH59	AT1G25330.1	Chr3	20,976,835	20,978,516	747	6	27.71	7.42	−0.593
PbrbHLH60	AT2G20180.7	Chr3	20,788,185	20,790,941	1113	6	40.55	8.21	−0.433
PbrbHLH61	AT5G53210.1	Chr1	9,416,218	9,418,685	1203	3	43.2	4.99	−0.386
PbrbHLH62	AT4G17880.1	Chr16	9,798,223	9,800,129	1563	2	58.32	6.68	−0.529
PbrbHLH63	AT3G07340.1	scaffold202.0.1	171,486	173,848	1632	7	58.63	7.43	−0.613
PbrbHLH64	AT1G22490.1	Chr6	19,415,337	19,417,849	969	3	35.91	9.29	−0.526
PbrbHLH65	AT2G41130.1	Chr17	10,057,378	10,058,791	726	2	27.1	8.01	−0.536
PbrbHLH66	AT1G66470.1	Chr9	6,731,103	6,744,452	1836	9	66.09	8.63	−0.445
PbrbHLH67	AT3G07340.1	Chr10	12,766,002	12,768,825	1713	8	61.84	7.14	−0.755
PbrbHLH68	AT4G29100.1	Chr15	15,967,308	15,972,064	1176	9	42.2	7.3	−0.576
PbrbHLH69	AT5G56960.2	Chr15	14,814,368	14,817,543	1761	7	65.78	8.39	−0.418
PbrbHLH70	AT1G68810.1	Chr10	3,695,569	3,699,378	810	2	29.05	8.08	−0.275
PbrbHLH71	AT3G07340.1	Chr4	11,760,522	11,763,609	1587	8	56.76	8.19	−0.6
PbrbHLH72	AT1G69010.1	Chr17	17,757,708	17,762,671	1221	7	43.88	8.35	− 0.53
PbrbHLH73	AT2G31210.1	Chr2	10,054,241	10,056,179	1542	3	56.71	5.83	−0.586
PbrbHLH74	AT2G31210.1	Chr2	10,074,575	10,076,304	1395	3	51.69	6.47	−0.646
PbrbHLH75	AT2G31210.1	Chr2	10,457,137	10,458,866	1395	3	51.69	6.47	−0.646
PbrbHLH76	AT2G31210.1	Chr2	10,477,477	10,479,415	1542	3	56.71	5.83	−0.586
PbrbHLH77	AT1G72210.1	Chr12	721,524	723,015	975	3	36.53	5	−0.442
PbrbHLH78	AT4G09820.1	Chr15	25,886,991	25,893,792	2013	7	74.31	4.9	−0.561
PbrbHLH79	AT3G07340.2	Chr13	6,771,090	6,772,369	543	6	19.93	8.49	−0.388
PbrbHLH80	AT4G17880.1	Chr1	4,498,172	4,499,991	1494	1	55.23	5.61	−0.508
PbrbHLH81	AT5G53210.1	Chr1	9,180,049	9,182,549	1203	3	43.18	4.99	−0.393
PbrbHLH82	AT5G01305.1	Chr4	3,361,294	3,361,890	600	1	22.23	6.7	−0.57
PbrbHLH83	AT3G50330.1	Chr15	7,854,064	7,854,920	756	1	27.88	9.35	−0.562
PbrbHLH84	AT1G49770.1	Chr15	7,767,619	7,768,847	705	3	25.85	6.73	−0.308
PbrbHLH85	AT4G36930.1	Chr15	7,762,918	7,765,404	1056	6	38.16	5.96	−0.572
PbrbHLH86	AT2G14760.1	Chr15	7,261,485	7,263,160	1077	4	39.97	5.3	−0.758
PbrbHLH87	AT1G10120.2	Chr15	6,913,207	6,915,155	1281	7	45.8	6.29	−0.661
PbrbHLH88	AT5G08130.7	Chr6	4,286,812	4,291,392	1773	11	65.08	8.94	−0.788
PbrbHLH89	AT1G73830.1	Chr6	3,710,462	3,711,993	795	6	29.47	5.21	−0.644
PbrbHLH90	AT3G20640.1	Chr15	9,143,054	9,146,117	1368	7	49.92	6.44	−0.702
PbrbHLH91	AT2G34820.1	Chr10	2,735,718	2,736,795	930	2	35.01	4.61	−0.492
PbrbHLH92	AT3G26744.4	Chr17	23,941,072	23,943,930	1536	4	55.71	4.83	−0.496
PbrbHLH93	AT2G14760.1	Chr8	12,990,009	12,991,896	1086	4	40.29	4.99	−0.785
PbrbHLH94	AT1G69010.1	Chr9	18,756,829	18,761,640	1134	7	40.78	6.44	−0.647
PbrbHLH95	AT4G34530.1	Chr10	21,887,740	21,890,367	1320	7	48.14	5.75	−0.584
PbrbHLH96	AT4G34530.1	Chr10	21,977,209	21,979,827	1320	7	48.19	5.75	−0.599
PbrbHLH97	AT4G37850.1	Chr5	7,214,750	7,216,451	1029	4	38.08	7.61	−0.353
PbrbHLH98	AT4G37850.1	Chr5	7,248,643	7,250,344	1029	4	38.08	7.61	−0.353
PbrbHLH99	AT4G14410.2	scaffold351.0.1	189,513	191,335	675	4	24.94	5.45	−0.665
PbrbHLH100	AT1G51140.1	Chr11	23,652,606	23,656,188	1305	6	47.29	6.74	−0.775
PbrbHLH101	AT3G19860.1	Chr2	16,242,907	16,244,464	975	5	35.96	7.99	−0.951
PbrbHLH102	AT4G17880.1	Chr11	28,888,209	28,890,472	1386	2	50.93	6.99	−0.374
PbrbHLH103	AT2G42280.1	Chr7	453,441	455,564	1308	6	48.48	7.48	−0.905
PbrbHLH104	AT5G50915.1	Chr12	13,203,431	13,205,370	1071	7	39.45	8.36	−0.654
PbrbHLH105	AT4G37850.1	Chr15	25,516,823	25,518,252	1062	3	39.06	6.42	−0.49
PbrbHLH106	AT4G37850.1	Chr15	25,435,077	25,436,506	1062	3	39.06	6.42	−0.49
PbrbHLH107	AT5G37800.1	Chr17	7,619,610	7,621,431	792	6	29.3	7.97	−0.852
PbrbHLH108	AT4G34530.1	Chr5	7,750,598	7,753,931	1272	7	46.43	6.01	−0.602
PbrbHLH109	AT5G67060.2	Chr2	13,406,418	13,407,251	837	1	31.15	10.34	−0.595
PbrbHLH110	AT4G36930.1	Chr2	13,319,368	13,322,043	1122	8	40.81	6.09	−0.537
PbrbHLH111	AT4G02590.2	Chr5	19,146,494	19,150,070	909	6	31.91	6.05	−0.313
PbrbHLH112	AT4G16430.1	Chr5	18,934,846	18,936,967	1512	1	55.88	6.37	−0.442
PbrbHLH113	AT4G16430.1	Chr5	18,869,375	18,870,585	1128	2	41.25	7	−0.454
PbrbHLH114	AT2G31280.1	Chr5	18,633,552	18,637,955	2229	11	82.45	6.31	−0.373
PbrbHLH115	AT1G09530.5	Chr12	3,917,496	3,921,379	2154	7	76.57	6.55	−0.614
PbrbHLH116	AT2G28160.2	Chr3	14,840,221	14,842,265	1053	4	38.35	6.73	−0.396
PbrbHLH117	AT1G69550.1	Chr3	14,857,124	14,874,269	3528	9	134.38	7.71	−0.307
PbrbHLH118	AT2G28160.2	Chr3	14,886,366	14,888,189	1044	4	38.07	6.05	−0.405
PbrbHLH119	AT4G20970.1	Chr17	3,759,373	3,760,370	666	3	25.05	7.66	−0.583
PbrbHLH120	AT4G20970.1	Chr17	3,775,165	3,776,020	627	3	24.01	9.09	−0.656
PbrbHLH121	AT3G26744.4	Chr14	12,762,203	12,765,393	1605	4	57.59	5.52	−0.497
PbrbHLH122	AT5G50915.1	Chr14	12,855,296	12,858,003	1071	7	39.5	8.57	−0.669
PbrbHLH123	AT3G20640.1	Chr5	13,842,159	13,845,048	1371	7	49.94	6.36	−0.701
PbrbHLH124	AT5G54680.1	Chr5	13,525,331	13,527,890	693	5	25.14	9.13	−0.554
PbrbHLH125	AT2G16910.1	Chr5	13,127,432	13,130,656	1695	8	64.04	5.43	−0.692
PbrbHLH126	AT2G16910.1	Chr5	13,122,299	13,123,691	786	6	29.37	7.86	−0.181
PbrbHLH127	AT2G16910.1	Chr5	13,117,288	13,120,346	1818	8	67.88	4.9	−0.707
PbrbHLH128	AT1G01260.2	Chr1	3,882,499	3,884,328	1833	1	67.36	6.64	−0.512
PbrbHLH129	AT1G68810.1	Chr13	2,460,589	2,463,318	1053	2	38.75	6.83	−0.656
PbrbHLH130	AT4G29100.1	Chr2	19,901,470	19,905,690	1140	9	40.95	6.9	−0.604
PbrbHLH131	AT2G31730.2	scaffold467.0	196,436	199,525	870	9	32.43	7.61	−0.638
PbrbHLH132	AT5G01305.1	Chr12	5,216,197	5,216,784	591	1	21.8	8.46	−0.663
PbrbHLH133	AT3G21330.1	Chr17	9,031,563	9,032,906	1347	1	49.3	6.9	−0.605
PbrbHLH134	AT1G25330.1	Chr13	1,982,008	1,983,683	717	6	26.63	5.98	−0.684
PbrbHLH135	AT2G43010.2	Chr9	13,138,658	13,140,883	1674	7	61.03	6.95	−0.725
PbrbHLH136	AT1G69010.1	Chr13	4,199,817	4,203,034	1005	7	36.54	6.59	−0.843
PbrbHLH137	AT1G68920.3	Chr13	4,107,727	4,111,792	1653	8	58.78	5.06	−0.582
PbrbHLH138	AT4G00050.1	Chr6	4,887,155	4,889,553	1131	5	41.25	7.74	−0.666
PbrbHLH139	AT3G56970.1	scaffold526.0	20,152	21,309	759	3	28.92	8.42	−0.695
PbrbHLH140	AT3G24140.2	Chr4	12,461,187	12,462,781	1365	3	50.78	4.9	−0.598
PbrbHLH141	AT1G05805.1	Chr4	11,676,123	11,681,214	1086	6	38.44	8.86	−0.638
PbrbHLH142	AT5G53210.1	Chr11	12,922,232	12,924,549	1260	3	44.84	5.43	−0.354
PbrbHLH143	AT2G31730.2	Chr7	10,672,016	10,674,734	861	9	32.14	9.07	−0.581
PbrbHLH144	AT2G20180.6	Chr5	17,244,166	17,248,840	1464	9	53.02	9.9	−0.765
PbrbHLH145	AT3G06120.1	scaffold639.0.1	60,715	62,445	621	3	22.95	9.23	−0.194
PbrbHLH146	AT2G24260.1	Chr15	11,765,124	11,768,866	1413	7	48.48	6.56	−0.485
PbrbHLH147	AT5G50010.1	scaffold655.0	182,209	184,247	1083	1	39.75	4.43	−0.736
PbrbHLH148	AT3G19860.1	Chr13	14,656,478	14,658,132	1059	5	38.54	7.33	−0.966
PbrbHLH149	AT2G27230.2	Chr6	10,899,693	10,903,475	2310	11	84.66	4.58	−0.365
PbrbHLH150	AT4G00870.1	Chr6	11,224,408	11,226,263	1542	2	57.19	7.2	−0.605
PbrbHLH151	AT5G67060.1	Chr15	23,381,295	23,382,023	732	1	26.85	10.62	−0.605
PbrbHLH152	AT4G36930.1	scaffold697.0	138,071	140,687	1167	8	42.28	5.39	−0.484
PbrbHLH153	AT3G53690.1	Chr2	16,659,400	16,666,799	2487	9	92.52	4.9	−0.496
PbrbHLH154	AT5G61270.1	scaffold745.0.1	145,406	150,762	1320	6	47.07	7.27	−0.482
PbrbHLH155	AT4G17880.1	Chr14	19,188,604	19,190,693	1449	2	53.93	6.75	−0.515
PbrbHLH156	AT4G17880.1	Chr14	19,223,454	19,225,368	1566	2	58.53	6.46	−0.578
PbrbHLH157	AT2G27230.2	Chr14	19,633,839	19,638,140	2385	10	87.47	4.82	−0.343
PbrbHLH158	AT3G53690.1	Chr15	17,135,114	17,139,758	1623	7	60.85	7.13	−0.471
PbrbHLH159	AT3G21330.1	scaffold763.0.1	64,350	65,696	1350	1	49.94	6.93	−0.709
PbrbHLH160	AT5G57150.1	Chr16	15,621,565	15,625,703	735	5	27.77	5	−0.444
PbrbHLH161	AT1G32640.1	scaffold773.0	23,504	24,874	1374	1	50.16	6.44	−0.657
PbrbHLH162	AT5G65640.1	scaffold775.0	130,165	131,652	1074	4	39.49	4.51	−0.461
PbrbHLH163	AT1G68810.1	Chr15	38,735,037	38,735,835	708	2	25.8	9.79	−0.708
PbrbHLH164	AT5G10530.1	Chr11	4,864,156	4,884,598	2229	4	82.36	5.41	−0.253
PbrbHLH165	AT3G19500.1	scaffold809.0	153,215	155,872	783	5	27.77	8.3	−0.491
PbrbHLH166	AT4G00870.1	Chr6	6,218,911	6,220,570	1410	2	52.11	6.03	−0.393
PbrbHLH167	AT5G46830.1	Chr6	6,266,723	6,268,150	1137	2	41.84	6.79	−0.353
PbrbHLH168	AT2G42280.1	Chr14	20,106,973	20,109,719	951	6	34.74	9.18	−0.707
PbrbHLH169	AT3G57800.2	Chr14	20,181,881	20,186,582	1110	8	40.67	5.6	−0.443
PbrbHLH170	AT1G73830.2	Chr14	16,070,706	16,072,305	798	6	29.51	5.21	−0.549
PbrbHLH171	AT1G59640.1	Chr13	3,371,476	3,373,746	891	6	32.07	6.88	−0.747
PbrbHLH172	AT1G29950.2	Chr10	6,704,729	6,706,250	837	2	30.51	5.21	−0.485
PbrbHLH173	AT4G38070.1	Chr15	26,803,608	26,804,342	636	2	23.13	10.48	−0.321
PbrbHLH174	AT5G67060.2	Chr15	23,542,843	23,543,574	735	1	26.97	10.62	−0.584
PbrbHLH175	AT4G29100.1	Chr17	8,804,002	8,806,668	1026	9	37.25	7.55	−0.702
PbrbHLH176	AT2G28160.1	Chr3	14,083,787	14,085,285	972	4	35.39	4.46	−0.457
PbrbHLH177	AT1G72210.1	Chr15	37,042,350	37,044,909	936	3	34.87	7.34	−0.613
PbrbHLH178	AT5G50010.1	Chr15	37,570,541	37,573,248	1083	1	39.82	4.48	−0.755
PbrbHLH179	AT2G31220.1	Chr7	3,923,753	3,925,356	870	4	32.13	6.95	−0.564
PbrbHLH180	AT2G31210.1	Chr7	3,929,075	3,931,367	1545	3	56.99	5.97	−0.643
PbrbHLH181	AT4G37850.1	scaffold911.0	70,566	72,288	1044	4	38.53	7.87	−0.403
PbrbHLH182	AT4G37850.1	scaffold911.0	148,794	150,516	1044	4	38.53	7.87	−0.403
PbrbHLH183	AT5G43650.1	scaffold914.0	94,761	97,214	756	3	29.2	8.99	−0.766
PbrbHLH184	AT1G30670.1	Chr5	20,425,616	20,427,088	924	2	34.42	5.16	−0.385
PbrbHLH185	AT3G19860.1	Chr5	10,201,972	10,206,005	1029	5	38.34	6.43	−0.934
PbrbHLH186	AT5G62610.2	scaffold927.0	32,483	35,092	870	6	30.54	5.24	−0.69
PbrbHLH187	AT4G20970.1	scaffold930.0	119,533	120,307	507	3	18.18	5.28	−0.21
PbrbHLH188	AT5G41315.3	Chr6	13,870,216	13,873,451	1986	8	73.87	5.34	−0.472
PbrbHLH189	AT3G19500.1	scaffold939.0	83,047	86,537	804	5	28.76	8.86	−0.513
PbrbHLH190	AT5G56960.2	Chr2	18,541,013	18,544,243	1791	8	67.08	8.89	−0.375
PbrbHLH191	AT4G37850.1	Chr16	18,387,503	18,388,918	1056	3	38.73	7.31	−0.474
PbrbHLH192	AT4G37850.1	Chr16	18,414,150	18,415,660	1062	3	38.85	6.51	−0.484
PbrbHLH193	AT2G14760.1	Chr10	16,254,387	16,256,363	1062	5	38.62	6.31	−0.526
PbrbHLH194	AT2G34820.1	Chr5	23,493,559	23,495,765	1134	2	42.55	5.64	−0.085
PbrbHLH195	AT1G32640.1	scaffold984.0	37,062	39,652	2067	1	75.5	5.8	−0.61
PbrbHLH196	AT2G42280.1	Chr14	4,887,436	4,890,120	999	5	36.51	9.64	−0.612
PbrbHLH197	AT3G57800.2	Chr14	4,960,408	4,965,046	1050	6	38.32	6.01	−0.581

The exact number of subgroup classifications for plant bHLH proteins is unknown, but is thought to be 15–32 [7, 8, 27]. To classify these genes and investigate their evolutionary relationships, phylogenetic tree was built applying the NJ method (Fig. 1, Fig. 2a and Fig. S1). The unrooted tree revealed that PbrbHLH gene family could be separated into 21 clades with the subfamily names A to U, which is the same number as those found in tomato [28] and Phaseolus vulgaris [29]. Unlike other clades, clade P and Q contained a single bHLH protein, respectively, meaning that PbrbHLH32 and PbrbHLH184 are unique. Furthermore, the NJ-tree built with these two PbrbHLHs and 167 AtbHLHs protein sequences indicated that the correlation between PbrbHLH132 and PbrbHLH184 and other bHLH proteins were relatively low (Fig. S1), which is consistent with the un-rooted tree. Except clade P and Q, the gene numbers of each clade varied wildly from 3 (clade L and M) to 22 (clade U). The results of gene structure analysis also showed that the PbrbHLH gene family have a broad range of exon numbers as well the gene structural diversity (Fig. 2c), such as the fact that there is no characteristic distribution pattern of exons and UTRs within most of certain subfamilies. However, the distribution patterns of exons were relatively conserved in clade D, F, G, H, J, K and U, and genes in these clades have a high similarity in exons number, exon pattern and the length of each exon, such as PbrbHLH73, PbrbHLH74, PbrbHLH75, PbrbHLH76 and PbrbHLH180 in clade F and PbrbHLH47 to PbrbHLH77 in clade H.

Fig. 1.

Un-rooted phylogenetic tree of PbrbHLH proteins.MEGA 7 was used to construct the phylogenetic tree based on the protein sequences. iTOL was used to annotate and review the phylogenic tree. The proteins were clustered into 21 groups. Different background colors indicate the different group of the PbrbHLH proteins

Fig. 2.

Schematics of the gene structure and conserved motifs in the PbrbHLH family. a Subgroup classification. Phylogenetic tree was generated using 197 PbrbHLH genes with MEGA7. The subgroup names were labeled accordingly. b Conserved motif analysis. Twenty distinct motifs were identified with MEME suite and each motif was represented with different color. c. Gene structural analysis

The characteristics of the PbrbHLH family and their coding genes are shown in Table 1 and Table S1. The protein molecular weights of bHLHs were from 10.38 to 274.01 kD. Protein isoelectric points (pI) ranged from 4.24 to 10.62, and 120 of them were lower than 7 (Table 1). The grand average of hydropathy (GRAVY) for all bHLH proteins in pear was positive, suggesting that all PbrbHLHs were likely soluble proteins which are consistent with their potential functions as TFs.

The annotation information from GO and KEGG databases were able to depict potential function of these genes. To predict the functions of identified PbrbHLH genes, the functional enrichment analysis of PbrbHLHs and a blastp analysis against the protein sequences of reported AtbHLH genes were all performed. As shown in Fig. S2a and Table S1, PbrbHLHs were mainly enriched in the terms of binding, cell part, cellular process, metabolic process and some regulation function, and all of these functions and processes are closely related to TFs. In addition, the KEGG enrichment result showed that these genes were largely enriched in circadian rhythm, MAPK signaling and plant hormone signal transduction pathways (Fig. S2b), all of which are the main mechanisms by which bHLH family TFs regulate the expression of downstream genes. Furthermore, the blastp result also showed that the crucial TF of these pathway were detected, including AtbHLH004 (the orthologous ofPbrbHLH62, PbrbHLH80, PbrbHLH102, PbrbHLH155, PbrbHLH156 and PbrbHLH162) and AtbHLH003 (the orthologous of PbrbHLH38, PbrbHLH39, PbrbHLH112 and PbrbHLH113), thepositive and negative regulator of jasmonate responses, respectively; AtbHLH008 (the orthologous of PbrbHLH24, PbrbHLH28, PbrbHLH115 and PbrbHLH135), a negative regulator of phyB signalling; and AtbHLH098 (the orthologous of PbrbHLH47, PbrbHLH61, PbrbHLH81 and PbrbHLH142), a substrate of kinases MPK3 and MPK6.

Synteny analysis of PbrbHLHs

The gene duplication events, such as WGD/segmental duplication, tandem duplication and transposition events, are the main causes for gene family expansion and affect the evolution of protein-coding gene families [30]. By using the MCScanX package, we detected the duplication events of bHLH gene family, and each of genes was assigned to one of five different duplication types: singleton, WGD/segmental, tandem, proximal or dispersed. Five duplication types were all detected driving the expansion of the PbrbHLH genes (Table 2 and Table S2). The results showed that 58.9% of bHLH genes in Chinese white pear were duplicated and retained from WGD/segmental events, and almost one quarter (23.9%) of PbrbHLHs was belonged to dispersed type.

Table 2.

Numbers of bHLH genes from different origins in Chinese white pear

Duplication type	Singleton	Dispersed	Proximal	Tandem	WGD/segmental
No. of bHLH genes from different origins (percentage)	3 (1.5)	47 (23.9)	11 (5.6)	20 (10.1)	116 (58.9)

To explore the evolutionary process behind the PbrbHLH genes, we performed intragenomic synteny analysis to identify conservation chromosome blocks within Chinese white pear. The landscape of ortholog PbrbHLH genes pairs were shown in Fig. 3 and their chromosomal distribution was random. The evolutionary date of WGD/segmental duplication events could be estimated by the Ks value (synonymous substitutions per site) [31]. As the previous reports, based on Ks values, the genome of pear had undergone two genome-wide duplication events: the ancient WGD (Ks ~ 1.5–1.8) that took place ~ 140 MYA [32] and the recent WGD (Ks ~ 0.15–0.3) occurred at 30–45 MYA [33] in pear. Therefore, we used Ks values to estimate the evolutionary date of the gene duplication events among the PbrbHLH gene family. The Ks values implied that most PbrbHLH genes were duplicated from a date around the recent WGD event, and some of others were originated from the ancient WGD (Table 3). The selection intensity and direction could be represented by Ka/Ks ratio, Ka/Ks value of one indicates neutral evolution, positive selection was indicated by a Ka/Ks value greater than one, and purifying selection was indicated by a Ka/Ks value less than one [34]. The Ka/Ks ratios of almost all homologous PbrbHLH genes were less than one (except the gene pair PbrbHLH110-PbrbHLH152), which implying that PbrbHLHs mainly evolved under purifying selection (Table 3).

Fig. 3.

Distribution and collinearity of the PbrbHLHs. Red lines along the circumference of the circle mark the positions of genes on chromosomes. The lines in different colors inside the circle indicate collinearity relationships among PbrbHLH genes

Table 3.

The duplicate mode and estimation of absolute date for large-scale duplication events for PbrbHLHs

Colinearity gene pairs		Duplication type		Method	Ka	Ks	Ka/Ks	MYA	P-Value (Fisher)
Gene1	Gene2	Gene1	Gene2
PbrbHLH6	PbrbHLH14	WGD	WGD	NG	0.14	0.29	0.47	97.86	0.000549
PbrbHLH10	PbrbHLH135	WGD	WGD	NG	0.04	0.13	0.30	44.85	6.55E-08
PbrbHLH20	PbrbHLH103	WGD	WGD	NG	0.08	0.21	0.38	70.19	1.28E-06
PbrbHLH21	PbrbHLH22	WGD	WGD	NG	NA	NA	NA	NA	NA
PbrbHLH24	PbrbHLH28	WGD	WGD	NG	0.06	0.16	0.39	53.45	1.34E-08
PbrbHLH25	PbrbHLH145	WGD	WGD	NG	NA	0.01	0.00	2.39	NA
PbrbHLH29	PbrbHLH72	WGD	WGD	NG	0.59	2.37	0.25	790.30	9.99E-18
PbrbHLH29	PbrbHLH88	WGD	WGD	NG	0.06	0.22	0.28	73.35	5.03E-14
PbrbHLH30	PbrbHLH138	WGD	WGD	NG	0.07	0.16	0.46	53.44	0.0025151
PbrbHLH38	PbrbHLH112	WGD	proximal	NG	0.08	0.25	0.31	82.40	6.76E-11
PbrbHLH40	PbrbHLH15	WGD	WGD	NG	0.25	1.73	0.15	577.13	1.60E-33
PbrbHLH40	PbrbHLH111	WGD	WGD	NG	0.03	0.20	0.13	67.82	1.39E-13
PbrbHLH42	PbrbHLH48	WGD	WGD	NG	0.09	0.24	0.35	80.89	4.09E-06
PbrbHLH44	PbrbHLH46	WGD	WGD	NG	0.03	0.16	0.18	53.81	2.18E-15
PbrbHLH49	PbrbHLH114	WGD	WGD	NG	0.07	0.16	0.41	54.11	2.60E-08
PbrbHLH53	PbrbHLH186	WGD	WGD	NG	0.01	0.01	0.59	3.39	0.416491
PbrbHLH61	PbrbHLH47	WGD	WGD	NG	0.09	0.17	0.50	57.55	0.000638
PbrbHLH61	PbrbHLH142	WGD	WGD	NG	0.09	0.17	0.52	57.55	0.0010697
PbrbHLH68	PbrbHLH130	WGD	WGD	NG	0.03	0.11	0.30	37.23	3.36E-05
PbrbHLH69	PbrbHLH190	WGD	WGD	NG	0.07	0.15	0.47	49.46	3.18E-05
PbrbHLH70	PbrbHLH4	WGD	WGD	NG	0.06	0.20	0.30	66.52	2.02E-06
PbrbHLH71	PbrbHLH63	WGD	WGD	NG	0.01	0.04	0.38	13.25	0.0088433
PbrbHLH72	PbrbHLH94	WGD	WGD	NG	0.20	0.40	0.50	131.94	8.95E-06
PbrbHLH73	PbrbHLH76	WGD	proximal	NG	NA	NA	NA	NA	NA
PbrbHLH74	PbrbHLH75	proximal	WGD	NG	NA	NA	NA	NA	NA
PbrbHLH74	PbrbHLH179	proximal	WGD	NG	0.05	0.32	0.17	105.65	2.57E-15
PbrbHLH75	PbrbHLH179	WGD	WGD	NG	0.05	0.32	0.17	105.65	2.57E-15
PbrbHLH81	PbrbHLH47	WGD	WGD	NG	0.09	0.18	0.49	60.41	0.000206
PbrbHLH81	PbrbHLH61	WGD	WGD	NG	0.00	0.01	0.24	4.66	0.0623594
PbrbHLH81	PbrbHLH142	WGD	WGD	NG	0.09	0.18	0.50	60.41	0.0003516
PbrbHLH83	PbrbHLH151	WGD	WGD	NG	0.33	2.81	0.12	938.19	4.15E-23
PbrbHLH86	PbrbHLH93	WGD	WGD	NG	0.05	0.11	0.50	35.10	0.007392
PbrbHLH90	PbrbHLH123	WGD	WGD	NG	0.03	0.13	0.21	43.84	7.79E-10
PbrbHLH91	PbrbHLH194	WGD	WGD	NG	0.19	0.37	0.51	123.02	0.0001704
PbrbHLH97	PbrbHLH98	tandem	tandem	NG	NA	NA	NA	NA	NA
PbrbHLH100	PbrbHLH5	WGD	WGD	NG	0.05	0.17	0.28	56.22	1.41E-08
PbrbHLH104	PbrbHLH45	WGD	WGD	NG	0.07	0.10	0.70	32.29	0.185088
PbrbHLH104	PbrbHLH122	WGD	WGD	NG	0.00	NA	NA	NA	NA
PbrbHLH110	PbrbHLH152	WGD	WGD	NG	0.01	0.01	1.78	2.63	0.722276
PbrbHLH115	PbrbHLH24	WGD	WGD	NG	NA	0.00	0.00	1.38	NA
PbrbHLH115	PbrbHLH28	WGD	WGD	NG	0.06	0.16	0.39	53.45	1.34E-08
PbrbHLH121	PbrbHLH46	WGD	WGD	NG	0.03	0.16	0.18	53.81	2.18E-15
PbrbHLH122	PbrbHLH45	WGD	WGD	NG	0.07	0.10	0.69	32.39	0.140337
PbrbHLH129	PbrbHLH58	WGD	WGD	NG	0.03	0.22	0.11	74.60	6.27E-17
PbrbHLH132	PbrbHLH82	WGD	WGD	NG	0.10	0.23	0.43	75.83	0.0033683
PbrbHLH133	PbrbHLH37	WGD	WGD	NG	0.07	0.17	0.39	57.16	2.79E-06
PbrbHLH134	PbrbHLH59	WGD	WGD	NG	0.08	0.19	0.45	61.87	0.0024985
PbrbHLH134	PbrbHLH170	WGD	WGD	NG	0.44	1.98	0.22	661.66	2.55E-15
PbrbHLH136	PbrbHLH29	WGD	WGD	NG	0.36	1.49	0.24	497.24	6.84E-23
PbrbHLH136	PbrbHLH72	WGD	WGD	NG	0.52	1.81	0.29	603.96	1.79E-15
PbrbHLH142	PbrbHLH47	WGD	WGD	NG	0.00	0.01	0.16	4.40	0.0337635
PbrbHLH143	PbrbHLH131	WGD	WGD	NG	0.07	0.14	0.49	47.75	0.0087228
PbrbHLH146	PbrbHLH31	WGD	WGD	NG	0.04	0.22	0.18	73.41	5.22E-17
PbrbHLH151	PbrbHLH109	WGD	WGD	NG	0.05	0.15	0.32	48.85	0.0001836
PbrbHLH151	PbrbHLH174	WGD	WGD	NG	0.00	0.02	0.07	8.22	0.0100508
PbrbHLH153	PbrbHLH54	WGD	WGD	NG	0.16	0.21	0.73	71.01	0.0116334
PbrbHLH155	PbrbHLH150	WGD	WGD	NG	0.24	0.75	0.32	249.03	9.36E-21
PbrbHLH157	PbrbHLH149	WGD	WGD	NG	0.06	0.17	0.37	55.19	5.61E-10
PbrbHLH170	PbrbHLH89	WGD	WGD	NG	0.06	0.20	0.30	66.11	4.02E-06
PbrbHLH171	PbrbHLH53	WGD	WGD	NG	0.32	1.62	0.20	541.67	9.61E-24
PbrbHLH174	PbrbHLH109	WGD	WGD	NG	0.04	0.13	0.34	43.89	0.0005669
PbrbHLH177	PbrbHLH26	WGD	WGD	NG	0.34	2.62	0.13	874.92	4.32E-31
PbrbHLH178	PbrbHLH147	WGD	WGD	NG	0.01	0.00	1.40	1.42	0.772501
PbrbHLH196	PbrbHLH168	WGD	WGD	NG	0.06	0.10	0.59	31.88	0.0536712
PbrbHLH197	PbrbHLH169	WGD	WGD	NG	0.01	0.02	0.37	7.33	0.0941955

Conserved motif analysis of PbrbHLH gene family

The types and composition of inner motifs mainly determine the protein function, and the evolutionary relationships among these PbrbHLH proteins were also determined by analyzing their conserved motifs. To further identify motif constructions of the PbrbHLH proteins, the online MEME program was used in this study to detect motif patterns. As showed in Fig. 2b, 20 conserved motifs with low E-values were recognized. The details of motif patterns were shown in Table S3. These composition patterns were nearly consistent with the phylogenetic analysis results, which were similar within the same group, but varying greatly between groups. Among PbrbHLHs, although pattern [#1,2] was detected in all members as the conserved motif pattern for bHLH TF in Chinese white pear, some of the other motifs were present only in certain groups, including motif #8 in group B, I and K; motif #10 in group F and O; motif #11 in group N; motif #9 in group S and motif #19 in T. However, some unique motifs patterns also only could be detected in specific subfamilies. Such as the pattern [#13,12,10,1,2,6,3,14] in clade F, the pattern [#15,7,5,18,1,2,6,3] in clade K and the pattern [#1,2,6,3,20] in clade J. We found that many subfamilies had relatively certain motif composition and there were significant differences among each other. However, there were some groups that have more than one pattern, and no conserved pattern was detected in some other clades, indicating that PbrbHLHs in these groups were not conservative in the evolutionary process, and the division among groups might have occurred in an early period.

Expression profile and patterns of PbrbHLH genes in response to drought and cold stresses

Previous transcriptome data revealed the expression patterns of candidate PbrbHLH genes in response to drought stress and cold stress, respectively (Fig. 4) [35, 36]. Overall, the results indicated that although the background expression of some PbrbHLH genes was rarely detected, that of others was significantly different among these investigated time points. Several differentially expressed genes showed up-regulation trend under both two stress conditions, to varying degrees, such as PbrbHLH119 and PbrbHLH120 in clade E, PbrbHLH7 and PbrbHLH160 in clade G and PbrbHLH128 to PbrbHLH80 from clade K. This suggested that these genes may be involved in some close-related pathways in response to drought and cold stresses. Interestingly, compared to the expression of these genes in cold treatment, the peak expression of them under drought condition was showed at a relatively late time point. In contrast, some other PbrbHLHs showed different (or even opposite) expression patterns, indicating that their responses might vary according to the different stress conditions.

Fig. 4.

Expression profile of PbrbHLHs under drought and cold stresses. Expression analyses of PbrbHLHs using previous published transcriptome data under cold and drought stress conditions

To further verify the functions of these identified PbrbHLHs, eight differentially expressed PbrbHLH genes (PbrbHLH119 from clade E, PbrbHLH7,PbrbHLH8 and PbrbHLH160 from clade G, PbrbHLH80, PbrbHLH128, PbrbHLH161 and PbrbHLH195 from clade K) were selected to examine the expression in response to drought and cold stresses, respectively (Fig. 5). Comparing with the expression at 0 hpt (hours post treatment), except PbrbHLH8 and PbrbHLH80 in drought treatment as well PbrbHLH7 and PbrbHLH119 under cold stress (data not shown), expressions for all other genes were significantly altered in the early stage of drought or cold treatment. Their responses tended to be more rapid under drought conditions, usually changing within the first 12 h. Under cold stress, expression of PbrbHLH8, PbrbHLH128, PbrbHLH160 and PbrbHLH161 initially showed down-regulating trend before being up-regulated as well as the expression of PbrbHLH7 and PbrbHLH195 under drought stress. The opposite trends between cold and drought stresses were noted for PbrbHLH128 and PbrbHLH160. Under drought stress, both were up-regulated at first and then down-regulated, whereas, under cold stress, their expression initially decreased before increasing. These results indicated that PbrbHLH genes were indeed involved in the responses to drought and cold stresses, and the pathways they taken part in under these stresses condition seemed to be different.

Fig. 5.

Expression analysis of PbrbHLHs undercold and drought stresses. a Relative expression of PbrbHLH8, PbrbHLH80, PbrbHLH128, PbrbHLH160, PbrbHLH161 and PbrbHLH195 with cold treatment. b Relative expression of PbrbHLH7, PbrbHLH119, PbrbHLH128, PbrbHLH160, PbrbHLH161 and PbrbHLH195 with drought treatment. The pear Actin was used as internal reference for the normalization. The statistical analyses were performed using student’s t-test (* p < 0.05)

Silencing of PbrbHLH195 reduced cold tolerance of P. betulaefolia

To understand whether PbrbHLHs is required for cold tolerance in pear, the VIGS system was employed to silence PbrbHLH195, which is significantly up-regulated under cold condition, in P. betulaefolia. The transcript abundance of PbrbHLH195 in the positive plants was substantially reduced by 50–90%, compared with that of WT (Fig. 6j, k). The positive silent plants (p-TRV1, p-TRV2 and p-TRV3) and WT plants were morphologically indistinguishable under normal growing conditions (Fig. 6a, d). However, upon exposure to 0 °C for 8 days, the silent plants displayed more severe damage in comparison with WT (Fig. 6a). The electrolyte leakage (EL) and malondialdehyde (MDA) concentrations in silent plants were significantly higher than those in WT under cold stress (Fig. 6b, c). Meanwhile, when they were subjected to cold treatment, Chl fluorescence in silent plants were prominently repressed, accompanied by significantly lower Fv/Fm ratio and Chl content, in comparison with WT (Fig. 6d-g). In addition, compared with silent plants, WT had lower H₂O₂ content (Fig. 6h, i). In situ accumulation of H₂O₂ was histochemically stained with DAB. In the presence of low temperature, the staining became darker, but silent plants staining was deeper and stronger than that of WT (Fig. 6h, i), which was further confirmed by quantitative measurement (Fig. 6i), which means that silencing plants accumulate more reactive oxygen species than WT. These results indicated that silencing of PbrbHLH195 promotes cold susceptibility in P. betulaefolia.

Fig. 6.

Cold tolerance assay of PbrbHLH195-silenced Pyrus betulaefolia plants. a-c Phenotype of 1-month-old PbrbHLH195-silenced plants before and after cold treatment for 8 days (a). Electrolyte leakage (EL) (b) and malondialdehyde (MDA) concentrations (c) after cold treatment. d-g Chlorophyll fluorescence imaging of silenced plants and WT plants(d), Fv/Fm ratios (e), Chl content (f) and phenotype (g) of WT and pTRV-PbrbHLH195 silencing plants (pTRV-1, pTRV-2 and pTRV-3) at the end of the chilling stress. h-i In situ accumulation of H₂O₂ of WT and silencing plants, as revealed by histochemical staining with 3, 3-diaminobenzidine (DAB) (h) after cold treatment. Quantitative measurement of H₂O₂ (i) levels after cold treatment. The expression of PbrbHLH195 was detected by RT-PCR (j) and qRT-PCR (k) at 8d after cold treatment

Discussion

After the release of the Chinese white pear genome sequencing data, there were many TF genes at the whole-genome level have been identified and characterized, including NAC-TF, BAM-TF and WRKY-TF et.al [22, 37, 38].. bHLH transcription factors are involved in many pathways in plant growth and metabolism [12]. However, no such detailed studies have been done with the bHLH family, and only a few examinations have been made of PbrbHLHs in pear. Here, we identified 197 PbrbHLH genes in Chinese white pear. Results of the phylogenetic analysis, gene structure and protein conserved motif analysis enable us to classify these PbrbHLH proteins into 21 groups, which is the same number reported in tomato and apple [28, 39], even though those organisms have fewer SlbHLHs (159) and MdbHLHs (188) than the members of PbrbHLHs in pear. On the basis of phylogenetic analysis, the un-rooted tree showed that PbrbHLHs were well separated into 19 clades with the wildly varied gene numbers from 3 (clade L and M) to 22 (clade U) and two one-gene clade P and Q. The gene and protein structure analysis showed that PbrbHLH family also has a broad diversity in intron/exon organizations as well the protein motif patterns. Although, the distribution pattern of exons and UTRs in clade D, F, G, H, J, K and U were relatively conserved, there was a broad range of exon numbers and structural diversity in many other clades, which is similar to the results of protein motif pattern analysis. By using online MEME software, 20 conserved protein motifs were detected among PbrbHLHs with low E values, and pattern [#1,2] were existed in all bHLHs which was regarded as the characteristic pattern for PbrbHLH TF. Meanwhile, some other motifs were present only in certain groups, including the motif #8 in group B, I and K and motif #10 in group F and O. Furthermore, three unique motif patterns only could be detected in specific subfamilies, respectively, such as the pattern [#13,12,10,1,2,6,3,14] in clade F, the pattern [#15,7,5,18,1,2,6,3] in clade K and the pattern [#1,2,6,3,20] in clade J. These results suggested that the PbrbHLH gene family may play diverse roles in the adaptive evolution to environmental stresses, and the division among groups might have occurred in an early period.

Gene duplication analysis revealed that the main driving force for the expansion of PbrbHLH family was WGD/segmental events, which is same as the case in apple. For instance, by applying MCScanX, 58.9% of bHLH genes in Chinese white pear were categorized into WGD/segmental type. Although pear was undergone the recent WGD events, almost one quarter of bHLH genes were duplicated from dispersed events. This may be due to the high ratio of self-incompatibility and the domestication process of pear. These results showed that WGD/segmental and dispersed gene duplications play critical roles in the expansion of the bHLH gene family in Chinese white pear. Furthermore, Ks values analysis implied that almost all WGD type PbrbHLH genes were duplicated from a date around the recent WGD event, and the Ka/Ks ratios indicated that PbrbHLHs evolved mainly under purifying selection and they seem to be necessary for adaptation to the current environment in their evolutionary history.

The function enrichment analysis showed that PbrbHLH genes were mainly enriched in the functions and processes closely related to TF, and the pathways they classified in were the main mechanisms by which bHLH family TFs regulate the expression of downstream genes, such as circadian rhythm, MAPK signaling and plant hormone signal transduction pathways. For example, OsbHLH148 and OsbHLH006 (REJ1) can improve drought stress by jasmonic acid signaling pathway in rice. Under salt and drought stress, in grapes VvbHLH1 confers a dominant effect on salinity and drought tolerance thought increasing the accumulation of flavonoids and ABA signaling in transgenic Arabidopsis thaliana. In addition, bHLH protein is also involved in plant stress resistance. Arabidopsis AtbHLH112 gene improves drought tolerance by increasing osmotic substances, eliminating ROS content and reducing water diversion. The results indicated that PbrbHLHs might play roles as other bHLHs.

By analyzing previous transcriptome data, we revealed the expression patterns of PbrbHLHs under cold and drought stress conditions. The results showed that, except some genes, the expression of most PbrbHLHs was significantly altered. For example, under both two stresses, PbrbHLH genes including PbrbHLH7, PbrbHLH119, PbrbHLH120, PbrbHLH160 and PbrbHLH128 to PbrbHLH80 in clade K had an up-regulation trend, which suggested that these genes might play similar roles in some close-related pathways in response to drought and cold stresses. Comparing with cold treatment, the peak expression of them under drought condition was showed at a relatively late time point, indicating that the responses of PbrbHLHs varied according to the treatment applied. To verify whether PbrbHLHs were involved in the response to cold or drought stresses, we performed stress treatments and qRT-PCR analysis. The results showed that the expression of all tested genes was significantly altered in the early stage of drought or cold treatments, however, the responses of same gene between two treatmentes could be diverse. For instance, under cold treatment, expression of PbrbHLH7, PbrbHLH8, PbrbHLH161, PbrbHLH128, PbrbHLH160 and PbrbHLH195 showed down-regulating trend at first before being up-regulated, whereas, under drought stress, PbrbHLH128 and PbrbHLH160 were up-regulated at first and then down-regulated. Furthermore, as a high up-regulated gene induced in both cold and drought stress conditions, the interference of PbrbHLH195 in transcription level significantly reduced the cold tolerance of the RNAi pear seedlings. These results indicate that PbrbHLH genes were involved in the responses to drought and cold stresses in pear, and the pathways they involved in seemed to be different under various stress conditions.

Our works in this study highlight the importance of bHLH TF in the cold and drought tolerance of pear. This is the first study to identify the PbrbHLH genes and examine their expression patterns in pear. QRT-PCR analysis showed that PbrbHLH is involved in stress tolerance pathways and functional analysis showed that PbrbHLH195 plays an important role in pear abiotic stress tolerance. However, further investigation will be required to understand the roles of PbrbHLHs in the stress response pathways, and the characterization of key (even the marker) bHLH TFs under each stress condition was also crucial to the revealing of the functional mechanisms of bHLH in pear.

Conclusions

In this study, we identified 197 PbrbHLH genes from Chinese white pear and carried out phylogenetic analysis to determine the relationships among these genes. Based on the results of protein motifs and intron/exon characteristics and phylogenetic analysis, PbrbHLH family was classified into 21 groups. According to the analysis of collinearity, WGD and dispersed duplication might have a role in the evolution of the PbrbHLH family. In addition, RNA-seq data, qRT-PCR and VIGS results revealed that PbrbHLH genes might have important roles in response to abiotic stresses, and the expression patterns of them differed in response to drought and cold stresses. The underlined collected data from this study provided a foundation for advanced studies to evaluate the mechanisms of cold-tolerance and drought-tolerance for bHLH genes in pear.

Methods

Plant materials and bacterial strains

The pear seedlings were grown in the greenhouse under a 16 h/8 h light/dark photoperiod, 75% relative humidity and 25 °C. A. tumefaciens GV3101 was grown in LB media supplemented with kanamycin and Rif at 28 °C in an orbital shaker at 200 rpm and harvested during the log phase of growth for infiltration.

Identification and functional annotation of bHLH gene family in Chinese white pear

To identify the bHLH genes in Chinese white pear, we performed multiple database-based searches. We downloaded all needed sequences and annotation file of Chinese white pear from Pear Centre of Nanjing Agricultural University (http://peargenome.njau.edu.cn/) and the seed file of bHLH conserved domain (PF00010) was downloaded from Pfam (http://pfam.sanger.ac.uk/). HMMER (Hidden Markov Model, HMM) software was used to detect conserved Pfam domain with default parameters E-value < 0.05 [40]. Then we checked the predicted bHLH transcription factors by using the NCBI Batch CD-Search tools (Batch CD-Search: https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) based on CDD v3.18 and SMART v6.0 databases to verify the existence of bHLH domain (Table S1). The proteins with E-values greater than 1e^− 6 or without a bHLH domain were removed. The relevant gene ID of PbrbHLH genes were shown in Table 1. The annotation information for Chinese white pear was extracted from the GFF file, and the result was visualized by a R script. The BLASTP was performed against 167 reported AtbHLH protein sequences [5], and the protein sequences were downloaded from TAIR (The Arabidopsis Information Resource, https://www.arabidopsis.org/).

Structure and conserved motif analysis of the PbrbHLH genes

The Gene Structure Display Server (GSDS 2.0) (http://gsds.cbi.pku.edu.cn/) was used to analyze the structures of the bHLH genes by aligning the cDNA sequences with their corresponding genomic DNA sequences [41]. Conserved motif analysis of bHLH proteins was performed by online Multiple Expectation Maximization for Motif Elicitation (MEME) (http://meme.nbcr.net/meme/cgibin/meme.cgi) with default parameters, and maximum number parameter of motifs were set as 20 [42]..

Phylogenetic analysis

The phylogenetic tree was built with Neighbor-Joining (NJ) method and a bootstrap of 1000 in MEGA7.0 (http://www.megasoftware.net/) [43]. The p-distance was used and the optional parameters for pairwise deletion were considered.

Chromosomal localization and synteny analysis

The chromosomal localization information was extracted from the GFF file. The same procedure used in the PGDD (http://chibba.agtec.uga.edu/duplication/) was performed to analyze the synteny among the PbrbHLHs. Primarily, the local all-vs-all BLASTP searches among identified PbrbHLH genes were conducted (E < 1e^− 10). Afterward, MCScanX was employed for the determination of syntenic gene pairs with the BLASTP result and gene location information used as input files [44]. The downstream analysis tool (duplicate_gene_classifier) in the MCScanX package was employed for the identification of tandem, proximal dispersed, and segmental/whole-genome duplications (WGD) of PbrbHLH family genes. The results were visualized using circos-0.69 software [45]. The Ka and Ks values were analyzed via KaKs_Calculator 2.0 [46]. For the estimation of the date of segmental duplication events, the succeeding pairs of homologous genes within 100 Kb on all sides of the PbrbHLH genes, considered for the mean Ks calculation.

Expression analysis of PbrbHLH genes under drought and cold stress conditions

Published transcriptomic data (FPKM values) characterizing the total RNA of drought treatment samples (D0, D1, D3 and D6 indicating the samples harvested at 0 hpt (hour post treatment), 1 hpt, 3 hpt and 6 hpt under drought stress) were downloaded from Li et al. (2016) [35]; cold treatment samples (C0, C5, C12 and C24 indicating the samples harvested at 0 hpt, 5 hpt, 12 hpt and 24 hpt under cold stress) were downloaded from Yang and Huang (2018) [36]. We determined the expression patterns of PbrbHLH family genes under drought and cold stress conditions. The differentially expressed genes were identified with the threshold |log2^FC| > 1. TBtools v1.068 was used to visualize the results [47].

For the qRT-PCR analysis, 9-week-old pear seedlings were treated with drought and cold, respectively. The leaves were cryopreserved with liquid nitrogen at 0 hpt, 1 hpt, 3 hpt, 6 hpt, 12 hpt and 24 hpt after drought stress treatment as well the leaves with cold treatment at 0 hpt, 1 hpt, 3 hpt,9 hpt, 12 hpt and 24 hpt. Total RNA extraction and the synthesis of cDNA were according to the instructions of RNA kit (Tiangen, Beijing, China) and PrimeScript RT reagent Kit (Trans Gen). Specialized primers of the constitutive TUB and eight tested PbrbHLH genes were designed via NCBI online tool Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) with the Specificity Parameters Organism option set as Pyrus bretschneideri (taxid:225117) (Table S4). The qRT-PCR assays were conducted with three technical copies. QRT-PCR reactions (20 μl per hole) were performed as previously reported [48]. The expression was evaluated for each sample via the 2^−ΔΔCt method, and Duncan’s multiple range test was conducted. A p-value of less than 0.05 was the considerable variation and the differentially expressed genes were identified with |log2^FC| > 1.

Generation of silenced plants

Virus-induced gene silencing (VIGS)-mediated suppression of PbrbHLH195 was performed according to previous methods [49]. A 182 bp fragment of PbrbHLH195 open reading frame (ORF) was inserted into EcoR I and BamH I sites of tobacco rattle virus-based vector 2 (TRV2) to generate the pTRV2-PbrbHLH195 construct. The vectors pTRV1, pTRV2 and pTRV2-PbrbHLH195 were transformed into A. tumefaciens strain GV3101 by heat shock. The bacterial cells (OD600 = 1.0) containing pTRV1 were mixed with pTRV2-PbrbHLH195 or pTRV2 in a 1: 1 volume ratio in 2-(Nmorpholino) ethanesulfonic acid (MES) buffer (10 mM MgCl₂, 200 mM acetosyringone, and 10 mM MES, pH 5.6) and kept slowly shaking in dark for 4 h at room temperature. The bacterial mixtures were injected into the leaves of seedlings and rinsed with water, grown in soil pots and transferred to a controlled growth chamber. Two weeks later, un-injected leaves were collected from each plant and subjected to genomic PCR and qRT-PCR analyses, and the VIGS plants exhibiting similar magnitude of PbrbHLH195 suppression were used for further analyses.

Physiological analysis

EL was measured according to [50]. MDA and H₂O₂ were detected according to the instructions using the corresponding detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Chl fluorescence was measured by Imaging PAM CHL fluorometer, Fv/FM ratio was calculated by imaging Winge software (Walz, Germany). Chl was extracted and analyzed according to [51].

Abbreviations

bHLH

Basic helix-loop-helix

TF

Transcription factor

COR gene

Cold regulated gene

pI

Protein isoelectric points

GRAVY

Grand average of hydropathy

Ks

Synonymous substitutions per site

hpt

Hours post treatment

EL

Electrolyte leakage

MDA

Malondialdehyde

HMM

Hidden Markov Model

GSDS

Gene Structure Display Server

MEME

Multiple Expectation Maximization for Motif Elicitation

NJ

Neighbor-Joining

RPKM

Fragments per kilobase of exon model per million mapped reads

WGD

Whole-genome duplications

VIGS

Virus-induced gene silencing

ORF

Open reading frame

MES

2-(Nmorpholino) ethanesulfonic acid

Authors’ contributions

HZD, YQD and WJH designed and carried out the experiments and QMC carried out all bioinformatics analysis and wrote the manuscript. XSH and SLZ directed and revised the manuscript. All authors read, reviewed and approved the final manuscript.

Availability of data and materials

All needed genome sequences and genome annotation files of Chinese white pear were obtained from the Nanjing Agricultural University pear genome project website (http://peargenome.njau.edu.cn). All data generated or analysed during this study are included in this published article and its supplementary information files.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.