Genome-wide identification and characterization of TCP transcription factors in peanut

doi:10.21203/rs.3.rs-2482268/v1

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Genome-wide identification and characterization of TCP transcription factors in peanut

https://doi.org/10.21203/rs.3.rs-2482268/v1

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The plant-specific TCP transcription factors (TFs) play important roles in multiple processes of plant development and environmental stress response. However, the characteristics of TCP TFs in peanut are still unclear. In this study, we characterized the peanut TCPs including the numbers, classification, structures, and expression patterns based on the information from the available database. We identified 28, 9, and 8 TCPs in A. hypogaea cv. Tifrunner (AhTCP1-28), A. duranensis (AdTCP1-9), and A. ipaensis (AiTCP1-8), respectively. These genes can be divided into three groups based on the phylogenetic tree: PCF, CIN, and CYC/TB1. A total of 10 paralogous gene pairs in A. hypogaea cv. Tifrunner, five orthologous gene pairs between A. duranensis and A. hypogaea cv. Tifrunner and five orthologous gene pairs between A. ipaensis and A. hypogaea cv. Tifrunner, were identified respectively. Most duplicated gene pairs underwent purifying selection except AdTCP2-AhTCP15 and AiTCP8-AhTCP28 were subjected to positive selection. Hormone-related and abiotic stress response elements were abundant in several TCP gene promoters in A. hypogaea cv. Tifrunner. AhTCP12 and AhTCP25 contained four EREs (ethylene-response elements), and AhTCP15 included four TCA elements. Most of TCPs were expressed lowly in 22 tissues, however, several genes such as AhTCP25 and a homologous gene pair, AhTCP13 and AhTCP28, were expressed highly in peg tip and perianth, respectively. AhTCP duplicated gene pairs did not show significant functional differentiation. This study provided insight into the evolution and expression of AhTCPs and might assist peanut molecular breeding.

expression pattern

gene duplication event

peanut

plant development

TCP transcription factor

TCP, the plant-specific transcription factor (TF) family, is widely involved in plant growth and development, environmental stress response, hormone response regulation, and biological circadian rhythm regulation (Martín-Trillo and Cubas 2010). The TCP was named (Cubas et al. 1999) after the identification of transcription factors TEOSINTE BRANCHED1 (TB1) in maize (Zea mays), CYCLOIDEA (CYC) in snapdragon (Antirrhinum majus), and PROLIFERATING CELL FACTORS 1 and 2 (PCF1 and PCF2) in rice (Oryza sativa) (Doebley et al. 1997, Luo et al. 1996, Ohashi 1997) in the late 1990s. At the N-terminus, TCPs contained a highly conserved 59-amino acid non-canonical basic-helix-loop-helix (bHLH) motif which is associated with DNA binding, protein-protein interaction, and nuclear localization (Aggarwal et al. 2010, Kosugi and Ohashi 2002, Namrata et al. 2017). According to multiple sequence alignment, the TCPs were mainly classified into 2 classes (Kosugi and Ohashi 2002, Navaud et al. 2007). However, the second class can be further divided into 2 subclasses (Crawford et al. 2004, Howarth and Donoghue 2006). Therefore, TCP TFs currently were known as 3 classes: PCF, CIN, and CYC/TB1 (Ma et al. 2016, Wang et al. 2018, Liu et al. 2018, Xu et al. 2014, Wei et al. 2016).

Since the TCP gene was first identified in maize (Zea mays), more TCPs were reported, and the characteristics of TCPs were uncovered in different plants. TCPs were highly conserved in their structures and functions, most TCPs from the same group showed similar exon and intron numbers and motif structures (Ma et al. 2014, Yan et al. 2018). The upstream non-coding region usually contains cis-acting regulatory elements, such as promoters, enhancers, repressors, etc. These regulatory elements play critical roles in regulating the responsive events of plants during growth and development by controlling the function of downstream genes. The upstream cis-acting regulatory elements of TCPs have been reported in soybean (Glycine max), based on the binding sequence prediction, the regulatory elements of GmTCPs can be classified into three types: hormone-related, abiotic stress-related, and tissue-specific expression elements (Feng et al. 2018). Gene duplication played important role in gene family expansion and evolution, however, the selection pressure forced the duplicated gene to drift toward fixation. A study of TCPs in maize by Chai and colleagues (Chai et al. 2017) revealed that eight duplicated pairs of ZmTCPs undergoing purifying selection might largely be responsible for maintaining the functions of maize TCPs. Although the gene redundance and selection secured the function reliability, TCPs displayed expression varieties. The expression of TCPs varied in tissues and was regulated by phytohormones at different development stages. In Medicago truncatula, 21 MtTCPs were detected as highly expressed in the leaf, and most of the CYC/TB1 group members were detected as highly expressed in the flower (Wang et al. 2018). The expression level of MtTCP3/4/10A/10B was significantly reduced when overexpressing MtmiR319A (Wang et al. 2018). The expression levels of several PeTCPs were significantly upregulated under abscisic acid, methyl jasmonate, and salicylic acid treatments in Phyllostachys edulis (Liu et al. 2018). In Solanum lycopersicum, SlTCPs act together with RIN (RIPENING INHIBITOR), CNR (COLORLESS NON-RIPENING), and SlAP2a (APETALA2a) proteins, which are transcription factors with key roles in ripening (Parapunova et al. 2014).

Moreover, the important roles that TCPs participate in plants were verified in recent reports. BRC1 (AtTCP12) represented a key point at which signals controlling branching are integrated within axillary buds (Aguilar-Martínez et al. 2007). BRC1 is expressed in developing buds, where it arrests bud development and BRC1 downregulation leads to branch outgrowth (Aguilar-Martínez et al. 2007). Transposon-tagged lines for AtTCP14 showed delayed germination (Tatematsu et al. 2008). In addition, the germination of 14 attcp mutants exhibited hypersensitivity to exogenously applied abscisic acid and gibberellin biosynthesis inhibitor paclobutrazol (Tatematsu et al. 2008). Yeast two-hybrid assay showed the interaction between GmTCP8 and an abscisic acid receptor (GmPYL10) in Glycine max (Feng et al. 2018). The evolution and expression of TCPs revealed StTCP23 might participate in plant defense in Solanum tuberosum (Bao et al. 2019). AtTCP4, AtTCP10, and AtTCP24 were AtWRIl-interacting partners (Kong et al. 2020). WRINKLED I (WRIl) is a key TF governing oil biosynthesis in plants and co-expression of AtTCP4 with AtWRIl reduced AtWRIl-mediated oil biosynthesis in leaves of Nicotiana benthamiana (Kong et al. 2020). This provides theoretical support for the utilization of TCPs in peanut oil metabolism. AtTCP21 was discovered to bind to the CCA1 promoter region (GGTCCCAC) in Arabidopsis (Pruneda-Paz et al. 2009). CCA1 is a key gene participating in the circadian regulation of A. thaliana (Alabadi 2001).

Cultivated peanut (Arachis hypogaea) is a major oilseed crop grown mainly in tropical, subtropical, and warm temperate climates regions (Cuc et al. 2008). Cultivated peanut is an allotetraploid (AABB; 2n = 4x = 40), A. duranensis and A. ipaensis are the donors of the A and B sub-genomes respectively (2n = 2x = 20) (Halward et al. 1992, Kochert et al. 1996, Moretzsohn et al. 2013). In recent years, the whole genome sequences of A. duranensis, A. ipaensis, and A. hypogaea have been released (Bertioli et al. 2016, Bertioli et al. 2019, Chen et al. 2019, Yin et al. 2018a, Zhuang et al. 2019). In addition, more RNA-seq datasets were released from whole genome or transcriptome level studies (Gong et al. 2020, Karmakar et al. 2019, Liu et al. 2020, Zhang et al. 2020a, Zhang et al. 2020b, Zhao et al. 2018b), an integrated bioinformatics platform is provided for peanut study. Various important gene families including LOX, NAC, WRKY, MST, and GRF were identified and analyzed (Song et al. 2016a, Song et al. 2016b, Wan et al. 2020, Yuan et al. 2020, Zhao et al. 2019). However, the TCPs are not well-studied in peanut. In this study, 28 A. hypogaea cv. Tifrunner, 9 A. duranensis, and 8 A. ipaensis TCPs were identified, as well as sequence features, evolution, and expression pattern were analyzed.

Twenty-eight TCPs were identified in A. hypogaea cv. Tifrunner genome

To investigate the TCP family in peanut, we searched the genome database of A. hypogaea (Tifrunner) and we identified 28 full-length TCPs (AhTCP), naming AhTCP1- AhTCP28 according to their position on the chromosomes (Table 1). The fundamental characteristics including the size of the gene and translated protein, the isoelectric point (pI), the aliphatic index, and the hydropathicity of 28 TCPs based on their genomic and amino acid sequences were analyzed and summarized (Table 1). These AhTCPs are distributed on 14 chromosomes out of 20. The lengths of AhTCP amino acids ranged from 131 (AhTCP15) to 658 (AhTCP16). The molecular weight ranged from 14.16 kDa (AhTCP15) to 72.66 kDa (AhTCP16). The lowest value of the theoretical isoelectric point was 5.22 (AhTCP22), while the highest value of the theoretical isoelectric point was 9.72 (AhTCP25). The aliphatic index value ranged from 51.67 (AhTCP4) to 77.75 (AhTCP3) meant rich aliphatic amino acids in AhTCP proteins. All GRAVY of AhTCP proteins was less than zero, verifying that AhTCPs were hydrophilic (Table 1).

Table 1

The information of 28 AhTCPs
Sequencing ID	Name	Chromosomes	Groups	CDS length(bp)	Protein length (aa)	Molecular weight(kD)	Isoelectric points (pI)	Aliphatic index	Grand average of hydropathicity (GRAVY)
arahy.5LBE6G	AhTCP1	Arahy.01	CYC/TB1	1434	477	53.17	7.03	53	-0.988
arahy.6V3SWS	AhTCP2	Arahy.01	PCF	1317	438	47.45	7.15	57.76	-0.82
arahy.NAZY29	AhTCP3	Arahy.03	PCF	417	138	14.93	9.51	77.75	-0.54
arahy.7QL5S6	AhTCP4	Arahy.03	CIN	1938	645	70.81	6.78	51.97	-0.959
arahy.UL2QPJ	AhTCP5	Arahy.03	CIN	1752	583	64.74	8.44	69.69	-0.685
arahy.ABQ1RQ	AhTCP6	Arahy.04	PCF	1491	496	51.39	6.8	55.67	-0.61
arahy.47Q1NW	AhTCP7	Arahy.04	CYC/TB1	1371	456	50.57	8.47	54.41	-0.905
arahy.XF5UIG	AhTCP8	Arahy.06	PCF	1182	393	42.70	6.35	69.34	-0.57
arahy.D15RR9	AhTCP9	Arahy.08	CIN	1059	352	38.53	6.14	53.84	-0.84
arahy.CX086V	AhTCP10	Arahy.09	PCF	1347	448	47.75	8.84	61.94	-0.559
arahy.DG8AQJ	AhTCP11	Arahy.09	CYC/TB1	1305	434	48.81	6.47	61.13	-0.838
arahy.U9727D	AhTCP12	Arahy.10	CIN	1299	432	47.82	9.33	56.92	-0.697
arahy.18WYEQ	AhTCP13	Arahy.10	CYC/TB1	1287	428	47.03	9.26	55.98	-0.865
arahy.Q9YRAB	AhTCP14	Arahy.11	PCF	1320	439	47.57	7.15	57.63	-0.826
arahy.M1TALC	AhTCP15	Arahy.13	PCF	396	131	14.16	9.51	70.76	-0.727
arahy.T0E7SC	AhTCP16	Arahy.13	CIN	1977	658	72.66	6.23	54.48	-0.876
arahy.QI2904	AhTCP17	Arahy.13	CYC/TB1	1410	469	52.33	7.79	54.14	-0.979
arahy.JF9204	AhTCP18	Arahy.13	CIN	1416	471	52.11	8.22	53.35	-0.962
arahy.22FU97	AhTCP19	Arahy.14	PCF	1488	495	51.46	6.83	56.95	-0.603
arahy.Q1RL33	AhTCP20	Arahy.14	CYC/TB1	1416	471	51.66	8.47	52.23	-0.924
arahy.KNN8RN	AhTCP21	Arahy.16	PCF	1092	363	39.32	6.11	64.57	-0.668
arahy.Q2VML3	AhTCP22	Arahy.18	PCF	507	168	17.96	5.22	65.77	-0.537
arahy.6TI82F	AhTCP23	Arahy.18	CIN	1059	352	38.52	6.14	53.84	-0.84
arahy.I9FULJ	AhTCP24	Arahy.18	CYC/TB1	456	151	16.61	8.95	64.11	-0.868
arahy.W24P9U	AhTCP25	Arahy.19	PCF	801	266	27.83	9.72	73.8	-0.406
arahy.TU0X8W	AhTCP26	Arahy.19	CYC/TB1	1233	410	45.77	6.46	59.73	-0.789
arahy.5684ZR	AhTCP27	Arahy.19	PCF	1140	379	39.72	5.82	60.08	-0.5
arahy.46V6T7	AhTCP28	Arahy.20	CYC/TB1	1287	428	46.99	9.2	55.98	-0.868

Classification Of Ahtcps Based On The Phylogenetic Tree

The amino acid sequence analysis of TCPs indicated that the TCPs contained a highly conserved 59-amino acid non-canonical basic-helix-loop-helix (bHLH) domain, apart from this domain, TCP protein sequences are, in general, highly variable. According to this feature, TCPs in Arabidopsis thaliana, Medicago truncatula, and Glycine max have been identified and reported (Selahattin et al. 2013, Wang et al. 2018, Feng et al. 2018). In this study, besides the A. hypogaea, we also identified A. duranensis and A. ipaensis TCPs using the abovementioned method (Table S1, Table S2).

Multiple sequence alignment of AhTCPs showed that the basic domain of bHLH structure in all AhTCP family amino acid sequences has a highly conserved DRH-K-G-R-P polypeptide. A four amino acids deficiency was found in bHLH region of the TCP domain between class I and class II (Fig S1). A total of 145 TCP TFs, including 24 AtTCPs,22 MtTCPs, 54 GmTCPs, 9 AdTCPs, 8AiTCPs, and 28AhTCPs (Table S3), were calculated to construct the phylogenetic tree using the maximum-likelihood method with 1000 bootstrap replicates by IQ-tree program. All TCPs displayed clear classifications. Then we focused on the cultivated peanut, and it showed that 28 AhTCPs were classified into two classes, that is, 12 were included in class I (PCF) and 16 were class II. In addition, 7 CIN and 9 CYC/TB1 were further separated within class II based on the phylogenetic tree (Fig. 1). Arahis hypogaea cv. Tifrunner had a nearly 1 to 1 ratio of class I and class II, and this ratio was also observed in Arabidopsis thaliana, Medicago truncatula, and Glycine max. However, the wildtype peanut A. duranensis and A. ipaensis had a 1 to 3 ratio of class I/Class II group (Table S4).

Similar Gene Structure And Potential Function In Each Group Of The Phylogenetic Tree

To reveal AhTCPs' structure, we executed exon/intron structure and protein motif analysis. AhTCP genes have similar exon/intron gene structures in the same group in the phylogenetic tree (Fig. 2A). Among 28 AhTCP genes, 9 genes only contained exon, and the other 19 genes at least contained intron. AhTCP4 had the longest exon (1851bp), and AhTCP5 had the longest intron (3150bp). The motif results showed that Motif 1 and Motif 2 were identified as the conserved regions of AhTCPs, and Motif 1 was detected in all AhTCP proteins (Fig. 2B).

To investigate the functions of AhTCPs, cis-acting elements within 1500 bp upstream of the start codon of TCP genes were analyzed. Hormone-related, abiotic stress response and tissue-specific expression elements were found in the cis-acting sequence. Hormone-related and abiotic stress response elements were abundant in most TCP gene promoters. Four EREs (ethylene-responsive elements) were identified in AhTCP12 and AhTCP25, four TCA-elements(salicylic acid responsiveness) were identified in AhTCP15, three ABRE (abscisic acid responsiveness) in AhTCP6 and AhTCP19, three CGTCA-motif (MeJA-responsiveness) in AhTCP21, three LTRS in AhTCP11 and AhTCP26, and three MBS in AhTCP22(Fig. 3).

Asymmetric Tcps Between Cultivated And Wild Peanut, And Purifying Selection Action On Tcp Duplicates

Asymmetric TCPs between cultivated and wild peanut, and purifying selection action on TCP duplicates

Cultivated peanut is an allotetraploid (AABB) containing both chromosomes from A. duranensis (AA) and A. ipaensis (BB). The cultivated peanut chromosomes 1–10 were contributed by A. duranensis (A01-A10) and the chromosomes 11–20 were contributed by A. ipaensis (B01-B10). According to the previous investigation, the AhTCPs were distributed on 14 chromosomes: four on chromosome 13, three on chromosomes 3, 18, and 19, two on chromosomes 1, 4, 9, 10, and 14, and one on chromosomes 6, 8, 11, 16, and 20. Most of the AhTCPs was located at the apex of both ends of the chromosomes (Fig S2). The TCPs distribution of cultivated peanut was not exactly corresponding to two wild ancestral peanuts. AhTCPs were distributed on chromosomes 6, 9, 11, 16, and 19, but no corresponding chromosomes of the wild ancestral peanut contained TCPs. One AdTCP on chromosome A01 corresponded to two AhTCPs on A. hypogaea cv. Tifrunner chromosome 1. In addition, one AiTCP on chromosome B08 corresponded to three AhTCPs on A. hypogaea cv. Tifrunner chromosome 18 (Table S5).

To explore the collinearity relationship of the TCP family between wild ancestral peanut and cultivated peanut, the gene duplication event of the TCP family was investigated using synteny analysis based on the BLAST-based homolog identification method (Table S6). Ten paralogous gene pairs were found in A. hypogaea cv. Tifrunner. Five orthologous gene pairs were found between A. duranensis and A. hypogaea cv. Tifrunner, and five orthologous gene pairs were found between A. dipaensis and A. hypogaea cv. Tifrunner (Fig. 4).

To further analyze the selective pressure of the TCP family in peanut, the Ka (non-synonymous), Ks (synonymous) values, and Ka/Ks ratio of the duplicated gene pairs were calculated. The Ka value ranged from 0 to 0.0465, and the Ks value ranged from 0 to 0.0893. The Ka/Ks ratio of the duplicated gene pairs ranged from 0 to 1.1356 with an average of 0.4116(Table 2). Twenty-two out of 24 gene pairs were subjected to purification selection, except for AdTCP2-AhTCP15 (1.1356 of Ka/Ks value) and AiTCP8-AhTCP28 (1.0635 of Ka/Ks value) duplicated gene pairs. These two duplicated gene pairs were subjected to positive selection.

Table 2

Ka/Ks calculation of each duplicated TCP gene pairs in peanut
Duplicated gene pairs	Ka	Ks	Ka/Ks	Effective Len	Average S-sites	Average N-sites	Selection pressure
AiTCP4-AhTCP19	0	0	NaN	1365	332.50	1032.50	Purify selection
AdTCP4-AiTCP4	0.0098	0.0499	0.1962	1359	331.33	1027.67	Purify selection
AdTCP4-AhTCP6	0.0010	0.0121	0.0802	1365	333.50	1031.50	Purify selection
AiTCP6-AhTCP23	0	0	NaN	936	217.33	718.67	Purify selection
AdTCP7-AhTCP9	0	0.0088	0	996	228.00	768.00	Purify selection
AiTCP6-AdTCP7	0.0014	0.0186	0.0747	936	217.33	718.67	Purify selection
AiTCP1-AhTCP15	0.0089	0.0149	0.5945	393	90.33	302.67	Purify selection
AdTCP2-AhTCP3	0.0031	0	NaN	414	95.17	318.83	Purify selection
AdTCP2-AhTCP15	0.0300	0.0265	1.1356	384	89.75	294.25	Positive selection
AiTCP8-AhTCP28	0.0219	0.0206	1.0635	1269	279.33	989.67	Positive selection
AdTCP9-AhTCP13	0.0030	0.0214	0.1410	1278	283.83	994.17	Purify selection
AiTCP8-AdTCP9	0.0303	0.0893	0.3391	1269	281.00	988.00	Purify selection
AiTCP5-AhTCP20	0	0	NaN	1164	245.17	918.83	Purify selection
AiTCP5-AdTCP6	0.0465	0.0787	0.5913	1131	236.58	894.42	Purify selection
AhTCP2-AhTCP14	0	0.0265	0	1314	307.25	1006.75	Purify selection
AhTCP8-AhTCP21	0.0263	0.0478	0.5500	1086	241.08	844.92	Purify selection
AhTCP4-AhTCP16	0.0151	0.0234	0.6473	1842	427.67	1414.33	Purify selection
AhTCP11-AhTCP26	0.0383	0.0768	0.4992	1221	262.50	958.50	Purify selection
AhTCP10-AhTCP27	0.0071	0.0178	0.4006	1128	283.83	844.17	Purify selection
AhTCP1-AhTCP17	0.0204	0.0381	0.5351	1389	295.92	1093.08	Purify selection
AhTCP6-AhTCP19	0.0099	0.0494	0.1993	1479	355.33	1123.67	Purify selection
AhTCP9-AhTCP23	0.0012	0.0252	0.0488	1056	242.17	813.83	Purify selection
AhTCP3-AhTCP15	0.0265	0.0266	0.9973	384	89.42	294.58	Purify selection
AhTCP13-AhTCP28	0.0081	0.0587	0.1380	1275	283.17	991.83	Purify selection

Member Of Each Group Has Similar Expression Pattern Under Normal Growth Condition

The expression patterns of 28 AhTCPs in 22 different tissues were obtained from PeanutBase. All CYC/TB1 groups presented low expression, except two homologous genes AhTCP13 and AhTCP28 were both highly expressed in the flower, suggesting that this homologous gene pair might affect flower growth and development. Most of the CIN group presented higher expression levels in leaf, shoot and seed development stages than the other tissues. In the PCF group, AhTCP25 was highly expressed in the leaf, shoot, root, and flower, especially in the peg tip, suggesting it might play a significant role in the growth and development of A. hypogaea cv. Tifrunner. Four similar phylogenetic relationship genes (AhTCP6, AhTCP19, AhTCP2, and AhTCP14) presented similar high expression levels in fruit Pat.1 stage. In addition, AhTCP6 and AhTCP19 also presented similar high expression levels in shoot tissues (Fig. 5).

AhTCP duplicated gene pairs have the same expression pattern

To investigate whether the AhTCP duplicated gene pairs had gene expression divergence, the expression level ratio of 10 AhTCP duplicated gene pairs in 22 different tissues was calculated and analyzed. AhTCP1-AhTCP17, AhTCP2-AhTCP14, and AhTCP11-AhTCP26 represented a high expression level ratio in leaf, flower, peg tip, and pericarp, respectively (Fig. 6). However, further analysis revealed that the original reads of these expression levels were very low, suggesting these results might be false positives. In general, 10 AhTCP duplicated gene pairs did not show significant expression divergence.

Expression patterns of AhTCP genes in different seed developmental stages

Seed is the most important economic part of peanut, and the TCP gene family is widely involved in the seed developmental stages. Therefore, the expression pattern of AhTCP genes during different seed developmental stages was analyzed. The CYC/TB1 group TCP genes presented low expression and The CIN group TCP genes presented a higher expression level than CYC/TB1 group TCP genes in most of the seed developmental stages. PCF group gene (AhTCP25) expression level was significantly high at the early stage of seed development (Fig. 7A). Notably, the homologous gene pair (AhTCP2 and AhTCP14) presented a gradual increase in expression level with the seed development process (Fig. 7B). This discovery was also verified in soybean. The homologous genes (GmTCP8 and GmTCP43) of AhTCP2 and AhTCP14 in soybean were identified and the expression level was analyzed, and the GmTCP8 and GmTCP43 also presented a gradual increase in expression level with different seed developmental stages (Fig. 7C). The result suggested that AhTCP2 and AhTCP14 pair might play a significant regulatory role in seed development.

TCP gene family is an important transcript factor family in most plants. Since it was named in 1999 (Cubas et al. 1999), the study of TCP genes were reported continuously. TCP family members in plants including Arabidopsis, tomato, poplar, cotton, cabbage, soybean, wheat, and grape were identified in recent years (Feng et al. 2018, Leng et al. 2019, Li et al. 2017, Ma et al. 2014, Yan et al. 2018, Yin et al. 2018b, Zhao et al. 2018a, Ma et al. 2016, Parapunova et al. 2014, Selahattin et al. 2013). Unfortunately, the report of TCP gene family in the important oil crop peanut is yet blank. Benefit from the release of whole genome sequences of wild ancestral peanut and cultivated peanut (Bertioli et al. 2016, Bertioli et al. 2019, Chen et al. 2019, Yin et al. 2018a, Zhuang et al. 2019), Omics analysis and group analysis research become feasible. Here, we identified 17 TCP genes from wild ancestral Peanut (9 AdTCPs and 8 AiTCPs) and 28 TCP genes from cultivated peanut (AhTCPs) (Table 1, Table S1, Table S2). Sequence alignment, homology analysis, gene structure, protein motifs, TCP promoter analysis, chromosomal location, gene duplication event, and expression pattern analysis were conducted to explore the evolution and function of peanut TCP family.

A total of 45 TCP genes were identified in all three peanut species. We found 16 TCP genes (14 AhTCPs, 2 AdTCPs, and 2 AiTCPs) in the PCF group, 15 TCP genes (7 AhTCPs, 4 AdTCPs, and 4 AiTCPs) in the CIN group, and 14 TCP genes (9 AhTCPs, 3 AdTCPs, and 2 AiTCPs) in CYC/TB1 group (Fig. 1). The AhTCP genes of cultivated peanut possessed an expanded family, with approximately three-fold size compared with A. duranensis or A. ipaensis. The result indicated that during the evolution of peanut from wild ancestral peanut to cultivated peanut, the TCP gene family not only had chromosome doubling but also had gene duplication and differentiation (Fig. 4). Notably, the number of TCP in cultivated peanut was not the simple addition of two wild-type peanuts. Interestingly, the class I TCP (PCF) in cultivated peanut was tripled during the formation of the allotetraploid genome, however, the class II (CIN) showed high conservation in the numbers and class III (CYC/TB1) was doubled compared with the total number of CYC/TB1 from two wildtype peanuts (Table S4). When comparing the class I TCPs ratio in Arabidopsis thaliana, Glycine max, Medicago truncatula, and cultivated peanut, the class I TCPs showed a similar ratio that is around 50% of total TCPs (Table S4). However, the class I TCPs in wild ancestral peanuts had only 25% of total TCPs. It suggested that class I might play more important roles during the evolution of cultivated peanut since the duplication of PCF genes occurred frequently while the class II and III TCPs were more conserved. Though the number of TCPs varied in plants, gene structure and protein motifs analysis demonstrated that the same group had similar exon-intron structure and conserved motif. Being identified as a TCP region, Motif 2 was discovered only in CYC/TB1 and CIN group and not found in the PCF group (Fig. 2).

TCP genes are widely involved in plant growth and development, environmental stress, hormone response regulation, and biological circadian rhythm (Martín-Trillo and Cubas 2010). In Arabidopsis, the TCP genes were extensively studied. Five CIN group TCP genes (AtTCP2, 3, 4, 10, and 24) were targeted by miRNA319 and were reported to be involved in regulating leaf development (Koyama et al. 2017, Efroni et al. 2008). The TCP transcription factor BRC1 (AtTCP18) was reported to be responsible for controlling the branching of axillary buds. BRC1 arrests bud development however the downregulation of BRC1 leads to branch outgrowth (Aguilar-Martínez et al. 2007, Finlayson 2007). A novel protein named TCP10 (AtTCP13) binding to the subset of AHPs suggested a possible regulatory function of TCP genes in regulating the His-to-Asp phosphorelay systems and plant growth (Alabadi 2001). It is worth mentioning that a recent study discovered coexpression of TCP4 with AtWRI1 reduced AtWRI1-mediated oil biosynthesis in Nicotiana benthamiana leaves (Kong et al. 2020). The result demonstrated TCP genes are involved in fatty acid biosynthesis and provided a novel idea for utilizing TCP genes in peanut fatty acid regulation. However, in peanut, few reports regarding TCP function are available currently.

Seed development is a critical process in plants, especially for plants that production relied on seeds such as peanuts. In this study, we found that the homologous gene pair AhTCP2 and AhTCP14 are homologous with Arabidopsis thaliana TCP14 (AtTCP14). AtTCP14 was highly expressed at the general seed Coat pre-globular stage, embryo proper linear- Cotyledon stage, and embryo proper Mature Green stage during the whole seed development, suggesting that AtTCP14 plays an important function in arabidopsis seed germination (Tatematsu et al. 2008). It further supported our inference about the function of the AhTCP2 and AhTCP14 homologous gene pair. After 24 hours of imbibition in the water of arabidopsis seeds, the expression level of AtTCP14 was significantly increased by nearly 3 times than the control group, suggesting that AtTCP14 also plays a key role in seed germination (Nakabayashi et al. 2005). Since the TCP genes are highly conserved, AhTCP2 and AhTCP14 were highly likely to play important regulatory roles in peanut seed germination and development. Furthermore, the function related to the circadian rhythm which was considered to affect germination was predicted in peanut TCPs as well. AhTCP25 is homologous with AtTCP21, which was discovered to bind to the CCA1 promoter region (GGTCCCAC) in Arabidopsis (Pruneda-Paz et al. 2009). CCA1 is a key gene participating the circadian regulation (Alabadi 2001). AhTCP25 was predicted to play a similar function to AtTCP21, thus regulating peanut circadian rhythm.

TCP genes were extensively studied in Arabidopsis and reported in other plants such as soybean and corn. Unfortunately, we are unable to dig out corresponding studies of TCPs in peanut at the molecular level, however, in this report, we predicted the functions of peanut TCPs based on studies on other species at the bioinformatic level. In addition, we summarized the characteristics of peanut TCPs that we believe this work will facilitate the study of TCP genes in peanut.

Identification of TCPs in A. duranensis, A. ipaensis, and A. hypogaea cv. Tifrunner genomes

A. duranensis, A. ipaensis, and A. hypogaea cv. Tifrunner genome sequences were downloaded from PeanutBase (https://peanutbase.org). To identify TCPs, the Hidden Markov Model (HMM) profile of the TCP domain (PF03634) was downloaded from the pfam database (http://pfam.xfam.org). HMMER program was used to identify TCPs (Finn et al. 2011). To further eliminate the problematic result, all protein sequences were checked in the pfam database. The physical and chemical properties including molecular weights, isoelectric points, aliphatic index, and grand average of hydropathicity (GRAVY) were estimated by the ExPASy website (https://web.expasy.org/protparam).

Sequence Alignment And Phylogenetic Analysis

DNAMAN was used to identify the characteristics of the conserved domain of AhTCPs. To verify the homology of Arachis TCP, A. thaliana (AtTCP), M. truncatula (MtTCP), and G. max (GmTCP) were obtained from the previous studies (Selahattin et al. 2013, Feng et al. 2018, Wang et al. 2018). The AtTCP, MtTCP, GmTCP, and AhTCP domains were extracted based on pfam database. Multiple sequence alignment was executed by MAFFT 7.0 program (Katoh and Standley 2013). Best-fit models of protein evolution were estimated by ProtTest 3 (Darriba et al. 2011). The phylogenetic trees were constructed by IQ-tree program using the maximum-likelihood method with 1000 bootstrap replicates (Nguyen et al. 2015).

Gene Structure, Protein Motifs, And Tcp Promoter Analysis

The exon-intron region of AhTCPs was downloaded in cultivated peanut genomic sequences from PeanutBase. The diagrams of exon–intron structures were generated by gene structure display server 2.0 (GSDS 2.0, http://gsds.cbi.pku.edu.cn/). The protein motif analysis was executed by submitting the sequences to the MEME website (http://meme-suite.org/) with parameters: motif number 20, minimum motif width 6, maximum motif width 50, and zoops model. The 1500 bp upstream promoter sequences of AhTCPs were retrieved from the genomic sequence. The putative cis-acting element was found by PlantCARE online program (http://bioinformatics.psb.ugent.be/webtools/plantcare/html) (Lescot et al. 2002).

Chromosomal Location, Gene Duplication Event Of Tcp Genes

The chromosomal location of AdTCPs, AiTCPs, and AhTCPs was obtained from PeanutBase (https://peanutbase.org). Orthologous and paralogous genes of wild and cultivated peanut were analyzed using the BLAST-based homolog identification method (Bertioli et al. 2019, Chen et al. 2019, Zhuang et al. 2019). The method included the following three parameters: ≥80% sequence length coverage of two sequences, ≥ 80% nucleotide identity of two sequences, and E-value ≤ 1E − 10. The collinearity analysis and non-synonymous (K_a) and synonymous (K_s) substitution of each duplicated gene were estimated by TBtools (Chen et al. 2020).

Tissue Expression Patterns Of Tcps And Gene Expression Divergence Analysis

Arachis hypogaea cv. Tifrunner expression datasets (Clevenger et al. 2016) were obtained from PeanutBase. AhTCP gene expression patterns were analyzed in 22 different tissues, including seedling leaf 10 days post-emergence (leaf 1), main stem leaf (leaf 2), lateral stem leaf (leaf 3), vegetative shoot tip from the main stem (veg shoot), reproductive shoot tip from first lateral (repr shoot), 10-day roots (root), 25-day nodules (nodule), perianth, stamen, pistil, aerial gynophore tip (peg tip 1), subterranean peg tip (peg tip 2), Pattee 1 stalk (peg tip Pat. 1), Pattee 1 pod (fruit Pat. 1), Pattee 3 pod (fruit Pat. 3), Pattee 5 pericarp (pericarp Pat. 5), Pattee 6 pericarp (pericarp Pat. 6), Pattee 5 seed (seed Pat. 5), Pattee 6 seed (seed Pat. 6), Pattee 7 seed (seed Pat. 7), Pattee 8 seed (seed Pat. 8), Pattee 10 seed (seed Pat. 10).

The expression level ratio of 10 AhTCP duplicated gene pairs in 22 different tissues was calculated using the high expression gene in duplicated gene pairs divided by the low expression one. The ratio was used to indicate the expression divergence between two homologous genes.

bHLH

Basic helix-loop-helix

BRC1

Branched1

CCA1

Circadian clock associated 1

CYC

Cycloidea

GRAVY

Grand average of hydropathicity

GRF

Growth-Regulating Factor

HMM

Hidden Markov Model

LOX

Lipoxygenase

MEME

Multiple Em for Motif Elicitation

MST

Monosaccharide transporter

Molecular weight

PCF

Proliferating cell factor

Isoelectric points

TB1

Teosinte branched1

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The work was financially supported by the grants from the SAAS programs for science and technology development (1+9KJGG005), the Key research and development program of Sichuan (2021YFYZ0018), and the Frontier research program of the Sichuan Academy of Agricultural Sciences (2019QYXK018). We would like to thank Dr. Hui Song from Qingdao Agricultural University for assistance in bioinformatics analysis.

Author contributions

Shuang Li, Xiaojun Zhang, and Xunlu Zhu contributed to the study's conception and design. Data collection, analysis, and artwork were performed by Shuang Li, Rui Hou, Yongju Xu, and Xiaohong Zhang. The manuscript was written by Shuang Li and Xunlu Zhu. All authors reviewed the manuscript.

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No competing interests reported.

FigS1.jpg
Multiple sequence alignment of AhTCP proteins. Dark blue represented absolute conservation. Light blue and pink represented high conservation. The number on the right represented the number of amino acids in each row.
FigS2.jpg
Chromosome location and homologous analysis of AhTCPs. White rectangles represented 20 chromosome of A. hypogaea. Red lines linked homologous genes of AhTCPs. The scale bar represented 30,000,000 bp.
TableS1.xlsx
HMM result of TCP family in A. duranensis.
TableS2.xlsx
HMM result of TCP family in A. ipaensis.
TableS3.xlsx
Information about TCP members in A. thaliana, M. truncatula, and G. max.
TableS4.xlsx
The group TCP gene numbers of 6 species participating phylogenetic tree.
TableS5.xlsx
TCP gene position comparison of A. hypogaea cv. Tifrunner, A. duranensis, and A. ipaensis.
TableS6.xlsx
The BLAST-based homolog identification result.

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Genome-wide identification and characterization of TCP transcription factors in peanut

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Abstract

Figures

Introduction

Results

Classification Of Ahtcps Based On The Phylogenetic Tree

Similar Gene Structure And Potential Function In Each Group Of The Phylogenetic Tree

Asymmetric Tcps Between Cultivated And Wild Peanut, And Purifying Selection Action On Tcp Duplicates

Member Of Each Group Has Similar Expression Pattern Under Normal Growth Condition

Discussion

Materials And Methods

Sequence Alignment And Phylogenetic Analysis

Gene Structure, Protein Motifs, And Tcp Promoter Analysis

Chromosomal Location, Gene Duplication Event Of Tcp Genes

Tissue Expression Patterns Of Tcps And Gene Expression Divergence Analysis

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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