Identification, expression, and association analysis of calcineurin B-like protein-interacting protein kinase genes in peanut.

Plants usually respond to the external environment by initiating a series of signal transduction processes mediated by protein kinases, especially calcineurin B-like protein-interacting protein kinases (CIPKs). In this study, 54 CIPKs were identified in the peanut genome, of which 26 were from cultivated species (named AhCIPKs) and 28 from two diploid progenitors (Arachis duranensis-AdCIPKs and Arachis ipaensis-AiCIPKs). Evolution analysis revealed that the 54 CIPKs were composed of two different evolutionary branches. The CIPK members were unevenly distributed at different chromosomes. Synteny analysis strongly indicated that whole-genome duplication (allopolyploidization) contributed to the expansion of CIPK. Comparative genomics analysis showed that there was only one common collinear CIPK pairs among peanut, Arabidopsis, rice, grape, and soybean. The prediction results of cis-acting elements showed that AhCIPKs, AdCIPKs, and AiCIPKs contained different proportions of transcription factor binding motifs involved in regulating plant growth, abiotic stress, plant hormones, and light response elements. Spatial expression profiles revealed that almost all AhCIPKs had tissue-specific expression patterns. Furthermore, association analysis identified one polymorphic site in AdCIPK12 (AhCIPK11), which was significantly associated with pod length, seed length, hundred seed weight, and shoot root ratio. Our results provide valuable information of CIPKs in peanut and facilitate better understanding of their biological functions.

Introduction

Calcium is an important second messenger in plants (Feng et al., 2017), and the signal transduction pathway mediated by calcium plays an important role in plant growth, development, and stress (Mao et al., 2016). Calcium signals formed during plant growth are further transmitted by calcium sensor proteins. Common calcium sensor proteins include calmodulins (CaMs), calmodulin-like proteins (CMLs), calcium-dependent protein kinases (CDPK), calcineurin B-like proteins (CBL), and CBL-interacting protein kinase (CIPK) (Zhao et al., 2009). CIPK, the interacting protein kinase of CBL, is a kind of protein that interacts with activated CBL as a downstream protein and is also a kind of Ca2+-dependent serine/threonine protein kinase (Kim et al., 2000), which contains a conserved catalytic kinase domain at its N-terminal (Albrecht et al., 2001). The C-terminal is an NAF regulatory control domain composed of 24 amino acids, which is highly conserved and mediates the interaction between CBL and CIPK protein (Kolukisaoglu et al., 2004).

Up to now, 25, 30, 16, and 43 CIPK genes have been identified in Arabidopsis thaliana (Yu et al., 2007), Oryza sativa (Kolukisaoglu et al., 2004), Vitis vinifera (Lu et al., 2017), and Zea mays (Chen et al., 2011), respectively. CIPK genes were widely involved in stress response, growth, and development regulation of plants (Hu et al., 2015). Studies of Arabidopsis have shown that the protein encoded by AtCIPK23 played an important role in potassium metabolism (Wang and Wu, 2009). The combination of AtCBL1, AtCBL9, and AtCIPK23 activated the potassium transport channel (Cheong et al., 2007). AtCIPK15 interacted with PP2C phosphatase ABI2 to form a complex, which controlled the expression of ABA-related genes (Li et al., 2006). AtCIPK3 responded to various abiotic stresses through ABA-dependent or ABA-independent pathway (Xu et al., 2006). It was found that overexpression of OsCIPK3, OsCIPK12, and OsCIPK15 improved cold, drought, and salt stress tolerance of rice, respectively (Xiang et al., 2007). Overexpression of wheat CIPK24 increased the contents of Na+ and antioxidant protective enzymes (Hrabak et al., 2003) and further improved the tolerance of A. thaliana to high salt stress (Deng et al., 2013). Apple CIPK6 interacted with AtCBL4 protein after transferring A. thaliana AtCBL4 gene into apple and improved the resistance of apple seedlings to low temperature, drought, and high salt stress (Wang and Liu, 2018). With the completion of large-scale plant genome sequencing, the research on CIPK genes in soybean (Feng et al., 2015), poplar (Sun et al., 2015), and many other plants received considerable attention, but research on CIPK genes in peanut has not been reported yet.

Peanut is one of the major oil and cash crops in China (Pandey et al., 2020). Calcium is the second largest nutrient element in peanut (Rui, 2015). In recent years, frequent occurrence of land drought, cold damage, and soil salinization has been a serious impact on the increase of peanut yield and the improvement of peanut quality (Lu et al., 2017). Under the pressure of reducing production cost and protecting the environment, screening peanut varieties with low calcium tolerance and exploring multistress response proteins and stress resistance mechanisms in an adverse environment have become the top priority in studying the stress resistance breeding of peanut (Sanders et al., 2002). To deal with abiotic stresses such as drought, cold, and salt damage and improve the yield and quality of peanut, it is of great significance to explore peanut CIPK genes and reveal their role in the calcium signaling pathway. In this study, the CIPK genes of cultivated peanut and its two diploid progenitors were comprehensively analyzed. At last, a total of 54 CIPK genes were explicitly identified. Their basic protein information, exon–intron structure, phylogeny, and cis-acting elements were systematically analyzed, which provided valuable theoretical basis and genetic resources for the high-yield breeding of peanut.

Materials and methods

Genome-wide identification of the CIPK genes in peanut

To identify CIPK genes in peanut, the protein sequences of three peanut genomes were downloaded from PeanutBase (http://www.peanutbase.org/). The conserved domains of all proteins encoded by peanut genome were analyzed using the HMMER 3.0 software, and genes including both the Pkinase (PF00069.24) and NAF (PF03822.13) domains were selected as peanut CIPK candidates. The PROSTIE and SMART software were used to verify the 54 CIPKs as calcineurin B-like protein–interacting protein kinases. Candidates with PROTEIN_KINASE_DOM (PS50011) and NAF (PS50816) in PROSTIE and the S_TKc (SM00220) domain in SMART were selected as CIPKs. The physicochemical data such as gene number, coding sequence (CDS) length, amino acid number, isoelectric point, molecular weight, and EF hand structure number were obtained from PeanutBase or analyzed using the ExPASy Proteomics Sever online tool (Gasteiger et al., 2005).

Evolution and structure analysis of CIPK genes in peanut

The gene structure diagram was drawn using the GSDS 2.0 mapping software based on the genome annotation information of CIPK genes with the GFF format, which was downloaded from PeanutBase (http://www.peanutbase.org/). The phylogenetic tree using 179 CIPK proteins from peanut, rice, grape, Arabidopsis, and soybean was constructed using the MEGA 5.2 software with the neighbor-joining method (Bootstrap value 1,000, Poisson model, uniform rates, pairwise deletion). The analyses of the composition of conserved motifs were conducted using MEME (http://meme-suite.org/tools/meme) with the maximum number 20 (classic mode, zero or one occurrence per sequence).

Cis-acting elements analysis of peanut CIPKs

We defined the 2-kb upstream sequence of the initiation codon as the promoter of the peanut CIPKs and downloaded it from PeanutBase to search for cis-acting regulatory elements through PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Then, only the cis-acting elements related to adversity stress were screened out statistically.

Expression profiles of AhCIPK genes in different tissues and treatments

RNA-seq datasets of 22 peanut tissues were downloaded from PeanutBase (http://www.peanutbase.org/) and NCBI SRA (https://www.ncbi.nlm.nih.gov/sra/), and the expression levels of AhCIPK genes in different tissues were obtained (Clevenger et al., 2016) with all raw data deposited as BioSamples SAMN03944933–SAMN03944990. The expression data (FPKM value) of peanut CIPK genes were normalized and output using the TBtools software (Chen et al., 2020). Ralstonia solanacearum infection was carried out as described before according to (Zhang et al. 2017). Submergence treatment followed the method described by (Zeng et al. 2021).

Candidate gene association mapping

The genotype data of the CIPK genes used for association analyses were obtained from the transcriptome sequencing data of a peanut germplasm population with 146 accessions (unpublished data). The phenotypes were collected from five environments (Wuhan 2016, Wuhan 2017, Yangluo 2016, Yangluo 2017, Zhanjiang 2016). Three replicates were randomly planted in each environment, with 12 plants in each row.

Results

Genome-wide identification of the CIPK genes in cultivated peanut and its diploid progenitors

To systematically determine CIPK genes in peanut, genes containing both the conserved Pkinase (PF00069.24) and NAF (PF03822.13) domains were searched through the whole peanut genome. The SMART and PROSTIE software tools were used to verify the Pkinase domains. A total of 54 CIPK candidates were identified from the peanut genome of cultivated species Arachis hypogaea (26, namely AhCIPK1–AhCIPK26) and its two wild species Arachis duranensis (13, namely, AdCIPK1–AdCIPK13) and Arachis ipaensis (15, namely, AiCIPK1–AiCIPK15) (Table 1). Then, we determined their chromosome locations, mRNA length, number of amino acids (aa), MW, theoretical pI, and transmembrane domain (TMD) (Table 1). AhCIPK genes were distributed on chromosomes 1, 2, 3, 7, 8, 9, and 10 (A genome) and 11, 12, 13, 17, 19, and 20 (B genome). Two wild species–specific CIPK genes (AdCIPK7 and AiCIPK5) were uncovered (Figure 1). The gene length of the peanut CIPKs ranged from 1,146 to 3,177 bps, of which the shortest was AiCIPK4 with 1,146 bps and the longest length was AhCIPK22 with 3,177 bps. The amino acid length of CIPKs varied from 381 to 570. The isoelectric point ranged from 5.8 (AdCIPK11) to 9.79 (AiCIPK8), and the molecular weight ranged from 45,067.06 to 64,189.82 Da. All CIPKs do not contain a TMD.

TABLE 1

Information on CIPK genes identified in peanuts.

Gene name	Gene locus	CDS length (bp)	AAa	MWb (kDa)	pI c	TMD d	Chr
AdCIPK1	Aradu.9W61Z	1,386	461	51.86	8.38	0	Aradu.A01
AdCIPK2	Aradu.TL55R	1,582	453	51.10	8.48	0	Aradu.A01
AdCIPK3	Aradu.K8K3S	2002	463	52.10	8.64	0	Aradu.A01
AdCIPK4	Aradu.SX4F9	1,670	452	50.61	8.35	0	Aradu.A01
AdCIPK5	Aradu.MRA83	1,645	434	49.26	8.44	0	Aradu.A02
AdCIPK6	Aradu.T9ESX	1,221	406	46.38	8.80	0	Aradu.A02
AdCIPK7	Aradu.HS592	2,199	457	51.51	9.22	0	Aradu.A03
AdCIPK8	Aradu.8B2M9	2,311	466	51.93	8.84	0	Aradu.A03
AdCIPK9	Aradu.73JAV	1982	441	50.46	6.42	0	Aradu.A07
AdCIPK10	Aradu.7W2Z9	2,110	456	50.40	8.30	0	Aradu.A07
AdCIPK11	Aradu.V638G	1,378	457	51.72	5.85	0	Aradu.A08
AdCIPK12	Aradu.Z7XZ9	1,681	461	52.41	8.75	0	Aradu.A09
AdCIPK13	Aradu.Q5XDE	1,653	550	61.66	8.17	0	Aradu.A10
AiCIPK1	Araip.L2Z00	1,582	455	51.24	8.06	0	Araip.B01
AiCIPK2	Araip.X0WZQ	1,386	461	51.93	8.38	0	Araip.B01
AiCIPK3	Araip.I6C5W	1,591	427	47.72	8.37	0	Araip.B01
AiCIPK4	Araip.J6DER	2,212	456	51.33	8.09	0	Araip.B01
AiCIPK5	Araip.MS6UX	1,146	381	43.51	8.81	0	Araip.B01
AiCIPK6	Araip.A3V01	1,239	412	46.65	8.52	0	Araip.B02
AiCIPK7	Araip.CMC8E	1,317	413	47.25	6.54	0	Araip.B02
AiCIPK8	Araip.B16CX	1,663	508	57.37	9.79	0	Araip.B03
AiCIPK9	Araip.M3K7N	2,359	465	51.84	8.84	0	Araip.B03
AiCIPK10	Araip.7IS5A	1991	441	50.46	6.42	0	Araip.B07
AiCIPK11	Araip.WP1GX	2021	456	50.40	8.02	0	Araip.B07
AiCIPK12	Araip.Z7THM	2,259	452	51.10	6.72	0	Araip.B07
AiCIPK13	Araip.KS6V8	1,677	461	52.56	8.49	0	Araip.B09
AiCIPK14	Araip.W1LHP	1,653	550	61.63	8.17	0	Araip.B10
AiCIPK15	Araip.882H9	1,387	436	48.93	9.00	0	Araip.B10
AhCIPK1	Arahy.TE3LXI	1,386	461	51.86	8.38	0	Arahy.01
AhCIPK2	Arahy.N6YX8I	2,735	453	51.10	8.48	0	Arahy.01
AhCIPK3	Arahy.069RBA	2014	446	50.44	8.79	0	Arahy.01
AhCIPK4	Arahy.HIWA31	3,017	570	64.19	9.22	0	Arahy.01
AhCIPK5	Arahy.MA0DIS	2,252	448	50.59	9.21	0	Arahy.02
AhCIPK6	Arahy.T0XBGT	1,516	398	45.07	9.31	0	Arahy.02
AhCIPK7	Arahy.9ML3HZ	2,762	497	55.49	8.90	0	Arahy.03
AhCIPK8	Arahy.KQQ5DM	1,218	405	46.26	6.26	0	Arahy.07
AhCIPK9	Arahy.50QZS1	2,813	456	50.40	8.30	0	Arahy.07
AhCIPK10	Arahy.YEIN47	2,295	549	61.97	7.56	0	Arahy.08
AhCIPK11	Arahy.R30FAJ	2,874	461	52.41	8.75	0	Arahy.09
AhCIPK12	Arahy.L0AIUK	1,653	550	61.66	8.17	0	Arahy.10
AhCIPK13	Arahy.RMG3R2	1,365	454	51.14	8.18	0	Arahy.11
AhCIPK14	Arahy.M8C26Q	1,386	461	51.90	8.38	0	Arahy.11
AhCIPK15	Arahy.4 × 641H	2,976	501	56.15	8.66	0	Arahy.11
AhCIPK16	Arahy.QIVE9X	2015	446	50.44	8.79	0	Arahy.11
AhCIPK17	Arahy.I9I2G1	1948	417	47.05	8.97	0	Arahy.12
AhCIPK18	Arahy.LMT476	1,318	427	48.48	9.13	0	Arahy.12
AhCIPK19	Arahy.84D15R	1,365	454	51.18	8.79	0	Arahy.13
AhCIPK20	Arahy.JQ1SFF	2,694	465	51.84	8.84	0	Arahy.13
AhCIPK21	Arahy.EY5MJ2	1,218	405	46.26	6.26	0	Arahy.17
AhCIPK22	Arahy.42B8G7	2,818	456	50.40	8.02	0	Arahy.17
AhCIPK23	Arahy.9NB62H	3,177	492	55.53	6.95	0	Arahy.17
AhCIPK24	Arahy.XP1WSF	2,874	461	52.56	8.49	0	Arahy.19
AhCIPK25	Arahy.VY9V4D	1,653	550	61.63	8.17	0	Arahy.20
AhCIPK26	Arahy.D01KFK	1,361	436	48.93	9.00	0	Arahy.20

Length of the amino acid sequence.

Molecular weight of the amino acid sequence.

Isoelectric point of the AhCIPKs.

Number of transmembrane domains, as predicted by the TMHMM Server v2.0.

CDS, coding sequence.

FIGURE 1

Chromosomal locations of peanut calcineurin B-like protein–interacting protein kinase (CIPK) genes. Chromosomal positions of the peanut CIPK genes were mapped based on GFF data downloaded from PeanutBase. The chromosome number is indicated above each chromosome. Genes in red mean wild species specific.

Phylogenetic and gene structure analysis of CIPKs in peanut

To determine the evolutionary relationship of CIPKs among A. hypogaea, A. duranensis, and A. ipaensis, the phylogenetic tree of the 54 CIPKs was constructed. The results indicated that the CIPKs can be classified into two clades (I and Ⅱ) (Figure 2A). Clades I and II consisted of 31 CIPKs (15 AhCIPKs, 8 AdCIPKs, and 8 AiCIPKs) and 23 CIPKs (11 AhCIPKs, 5 AdCIPKs, and 7 AiCIPKs), respectively. It is interesting that the results of the gene structure based on the genome annotations also showed that the CIPK genes can be divided into two groups, corresponding to the two phylogenetic families (the intron-rich group corresponded to phylogenetic clade I, and the intron-less group corresponded to phylogenetic clade Ⅱ). The intron numbers of the intron-less group were less than 3 (0 to 2), while those of the intron-rich group were more than 10 (Figure 2B). Further, the phylogenetic relationship and classification of peanut CIPKs were supported by motif analysis. A total of 20 motifs were identified (Figure 2C); in general, peanut CIPKs had 11–17 motifs. Motif 1, Motif 2, and Motif 4 were the most common, present in all CIPK proteins. Otherwise, the vast majority of CIPKs included Motifs 3, 5, 6, 7, and 8, which covered more than 50 CIPK members. Motifs 13 and 19 were clade-specific elements in clade II, and Motif 17 only existed in AhCIPK15 of clade I but 13 CIPKs in clade II.

FIGURE 2

Comparison of the gene structure and motif of 54 CIPK genes in peanut. (A) unrooted phylogenetic tree with over 50% bootstrap value above the branch. Clades I and II are displayed in pink and blue colors, respectively. The names of the species are abbreviated to two letters, named as Arachis duranensis (Ad), Arachis ipaensis (Ai), and Arachis hypogaea L. Tifrunner (Ah). (B) exon/introns and untranslated regions (UTRs) of CIPKs. Blue boxes denote UTR (untranslated region); yellow boxes denote coding sequence (CDS); and black lines denote introns. The length of protein can be estimated using the scale at the bottom. (C) motif architectures of all CIPK genes. Each motif is illustrated with a specific color, and the distribution of identified motifs corresponds to their positions.

Biological evolution analysis of CIPKs in peanut and other plant species

To further understand the relationship of CIPK members among different species, the phylogenetic tree of CIPK proteins of Arabidopsis, rice, grape, soybean [AtCIPK (26), OsCIPK (31), VvCIPK(16), and GmCIPK (52)], and peanut was constructed using maximum parsimony (Figure 3). The results showed that CIPK proteins of these species can be divided into two subfamilies (Ⅰ and Ⅱ). The analysis of the phylogenetic tree revealed that all the peanut CIPKs were clustered together (Figure 3). The relationships between the two wild species A. duranensis and A. ipaensis and cultivated species A. hypogaea were closer than that between the other four species. In addition, many CIPK members of peanut and soybean clustered together, suggesting that the two legume species were evolutionarily closer than others. TThe second evolutionary closest of peanut was Arabidopsis. There were more family members in peanut and soybean than in Arabidopsis, rice, and grape plants, suggesting a specific linear amplification of the gene family in legume plant. Whether these additional members of the genes have additional functions as well or whether they are produced only because functional redundancy requires further experimental verification.

FIGURE 3

Phylogenetic tree of CIPKs in peanut and other plant species. The plants in the square frame indicate that the CIPK members outside of peanuts have the closest evolutionary relationship with soybean CIPKs.

Gene duplication and synteny analyses of peanut CIPKs

Chromosomal location analyses revealed that the 26 AhCIPKs distributed unevenly on 13 chromosomes (chromosomes 01, 02, 03, 07, 08, 09, 10, 11, 12, 13, 17, 19, and 20). The 13 AdCIPKs presented on chromosomes A01, A02, A03, A07, A08, A09, and A10, and the 15 AiCIPKs distributed on chromosomes B01, B04, B06, B09, and B10 (Figure 4). A total of 16 chromosomal fragment repeat gene pairs were identified without tandem repeats (Figure 4, Supplementary Table S1). Further, we calculated the Ks (synonymous) and Ka (nonsynonymous) values of the duplicated gene pairs and found that the Ka/Ks ratio for duplicated AhCIPK gene pairs ranged from 0.00 to 0.57 with an average of 0.17 (Supplementary Table S2). The ω values of all duplicated gene pairs were less than 1, showing that purifying selection occurred on these duplicated gene pairs. Synteny analysis with Arabidopsis, rice, grape, and soybean revealed one conserved CIPK gene (AhCIPK14) in these species (Figure 5, Supplementary Table S1). BLASTP methods were used to identify peanut CIPK gene orthologs between peanut and Arabidopsis. In total, we found 54 orthologous gene pairs between peanut and Arabidopsis (Table 2). The orthologs in Arabidopsis included AtCIPK12/AtWL4 and AtCIPK5 participating in pollen germination and tube growth (Wang et al., 2008; Steinhorst et al., 2015), AtCIPK24/AtSOS2 required for salt tolerance in A. thaliana (Halfter et al., 2000; Ishitani et al., 2000; Liu et al., 2000; Guo et al., 2001), and AtCIPK1 and AtCIPK3 relating to the ABA signal transduction (D'Angelo et al., 2006; Sanyal et al., 2017; Pandey et al., 2008; Kim et al., 2003). Therefore, we speculated that these AhCIPK homologous genes might play multiple roles not only in peanut growth and development but also in plant hormone and stress resistance.

FIGURE 4

Chromosomal distribution and gene duplications of the AdCIPK, AiCIPK, and AhCIPK genes. The scales on the circle are in megabases. Each colored bar represents a chromosome as indicated. Gene IDs are labeled on the basis of their positions on the chromosomes. Red arrows indicate wild species–specific CIPK genes.

FIGURE 5

Comparative physical mapping showed orthologous relationships of AhCIPK genes with (A) Arabidopsis, (B) rice, (C) grape, and (D) soybean. Red arrows indicate common collinear CIPK orthologs.

TABLE 2

The function of AhCIPKs genes homologous to Arabidopsis.

Peanut	Arabidopsis	Function	References
AdCIPK4/AiCIPK3/AhCIPK4/AhCIPK15	AtCIPK1 (At3G17510)	Controls abscisic acid-dependent and independent stress responses	D'Angelo et al. (2006)
AdCIPK5/AdCIPK6/AiCIPK6/AiCIPK7/AhCIPK5/AhCIPK6/AhCIPK17/AhCIPK18	AtCIPK9 (At1G01140)	A calcium sensor-interacting protein kinase required for low-potassium tolerance	Pandey et al. (2007)
			Singh et al. (2018)
			Lara et al. (2020)
			Kanwar et al. (2022)
AdCIPK7/AdCIPK8/AiCIPK8/AiCIPK9/AhCIPK7/AhCIPK19/AhCIPK20	AtCIPK23 (At1G30270)	Serves as a positive regulator of the potassium transporter AKT1 by directly phosphorylating AKT1	Sánchez-Barrena et al. (2020)
			Ragel et al. (2015)
			Tian et al. (2016)
			Wang et al. (2016)
			Xu et al. (2006)
			Cheong et al. (2007)
AhCIPK8/AhCIPK21/AdCIPK9/AiCIPK10	AtCIPK3 (At2G26980)	Regulates Abscisic Acid and Cold Signal Transduction	Sanyal et al. (2017)
			Pandey et al. (2008)
			Kim et al. (2003)
AdCIPK2/AdCIPK10/AiCIPK1/AiCIPK11/AhCIPK2/AhCIPK9/AhCIPK13/AhCIPK22/	AtCIPK11 (At2G30360)	A positive regulator in cadmium stress response	Zhou et al. (2015)
			Gu et al. (2021)
			Liu and Guo, (2011)
AdCIPK13/AiCIPK14/AhCIPK12/AhCIPK25	AtCIPK12/AtWL4 (At4G18700)	Required for Polarized Pollen Tube Growth	Steinhorst et al. (2015)
AdCIPK11/AiCIPK12/AhCIPK10/AhCIPK23	AtCIPK8 (At4G24400)	Regulates the low-affinity phase of the primary nitrate response	Hu et al. (2009)
			Gong et al. (2002)
AiCIPK5	AtCIPK6 (At4G30960)	Required for development and salt tolerance	Sardar et al. (2017)
			Chen et al. (2013)
			Tsou et al. (2012)
			Held et al. (2011)
			Tripathi et al. (2009a)
			Tripathi et al. (2009b)
AdCIPK12/AiCIPK13/AhCIPK11/AhCIPK24	AtCIPK5 (At5G10930)	Regulates potassium homeostasis under low oxygen	Schlücking et al. (2013)
			Tagliani et al. (2020)
AdCIPK3/AiCIPK4/AhCIPK3/AhCIPK16	AtCIPK24/AtSOS2 (At5G35410)	SOS2 gene encodes a protein kinase that is required for salt tolerance	Liu et al. (2000)
			Halfter et al. (2000)
			Ishitani et al. (2000)
			Guo et al. (2001)
AdCIPK1/AiCIPK2/AiCIPK15/AhCIPK1/AhCIPK14/AhCIPK26	AtCIPK5 (At5G58380)	Gene expression to accompany pollen germination and tube growth	Wang et al. (2008)

Cis-acting elements prediction of CIPK genes in peanut

Cis-acting elements in a promoter as the binding target of transcription factors are essential in the regulation of gene expression. In order to understand the regulation mechanisms of peanut CIPK genes, 2-kb upstream sequences of the peanut CIPK genes were analyzed via the PlantCARE database. In total, 54 cis-regulatory elements were detected. Four main categories were defined as the light responsiveness element, phytohormone responsiveness, abiotic stress responsiveness, and plant growth groups (Figures 6A–C, Supplementary Table S3). In the promoter region of the AdCIPKs, the largest subdivision was the light responsiveness group, containing 53.2% of the predicted cis-elements; phytohormone responsiveness elements ranked second (24.2%) (Figure 6A); abiotic stress response elements were 15.9%; and elements involved in plant growth accounted for 6.7% (Figure 6A). AdCIPK2 had the greatest number of elements with 38 in total, which contained six abscisic acid responsiveness elements (ABREs) (Supplementary Table S3). For AiCIPKs, the percentage of light, phytohormone, abiotic stress, and plant growth responsiveness cis-elements was 57.7, 21.5, 16.6, and 4.2% (Figure 6B). AiCIPK14 had the greatest number of elements at 40 in total, which also contained six ABREs. In AhCIPKs, the proportions were 56.0, 20.9, 16.0, and 7.1% (Figure 6C). In the light response category, Box 4 (light-responsive element) and GT1-motif (part of a module for light response) were the most dominant. Meanwhile, cis-acting elements responding to auxin, abscisic acid, gibberellin, flavonoids, methyl jasmonate, and salicylic acid were detected in the phytohormone responsiveness group.

FIGURE 6

cis-acting elements in peanut CIPK genes. (A) assessment of AdCIPK different subclass and category proportions in sunburst chart. (B) assessment of AiCIPK different subclass and category proportions in sunburst chart. (C) assessment AhCIPK different subclass and category proportions in sunburst chart. The lengths of the petals are proportional to the number of elements in each subclass qualitatively. Blue, purple, green, and red represent light responsiveness, phytohormone responsiveness, abiotic stress responsiveness, and plant growth regulation, respectively.

Tissue expression profiles of AhCIPKs

To further study the expression pattern of peanut CIPKs in different tissues and explore its function in peanut growth and development, the tissue expression profiles of CIPK genes were analyzed by using the transcriptome data of 22 peanut tissues (Figure 7). The results showed that the 26 AhCIPK genes had distinct tissue-specific expression patterns across the 22 tissues (leaves, stem, roots, flower, pod, and seed). AhCIPK9 and AhCIPK25 showed a higher expression level in leaf. AhCIPK7 and AhCIPK20 were mostly expressed in reproductive shoot and pattee 1 stalk; interestingly, nearly half of AhCIPK genes were highly expressed in reproductive organs, among which AhCIPK2, AhCIPK3, AhCIPK10, AhCIPK14, AhCIPK15, AhCIPK18, and AhCIPK22 had strong expression in perianth; AhCIPK3, AhCIPK6, AhCIPK8, AhCIPK16, AhCIPK17, and AhCIPK21 were highly expressed in stamens; and AhCIPK12 and AhCIPK26 were obviously expressed in roots. AhCIPK11, AhCIPK23, and AhCIPK24 were highly expressed in nodules. AhCIPK4, AhCIPK12, AhCIPK19, and AhCIPK24 had strong expression during the relatively later pericarp developmental stage. In addition, AhCIPK1 and AhCIPK19 were enriched in the earlier seed developmental stage, while AhCIPK4 and AhCIPK13 were expressed highly in the later seed developmental stage (Figure 7).

FIGURE 7

Expression profiles of AhCIPK genes. The heatmap of the AhCIPK gene expression levels was hierarchically clustered using TBtools with the data normalization method of Z-score standardization. The color scale bar from green to red represents low and high expressions, respectively. Abbreviations of the 22 tissues used in the expression profiles of AhCIPK genes were from Ren et al. (2022).

Expression pattern of the AhCIPKs under submergence and Ralstonia solanacearum infection

Plants suffer from a wide variety of environmental stressors under natural conditions. To determine the abiotic and biotic stress responses, we detected the expression of AhCIPK genes responding to submergence and R. solanacearum infection. The results showed that except the two unexpressed members AhCIPK6 and 20, almost all other AhCIPKs respond to submergence stress (Figure 8A, Supplementary Table S4). AhCIPK3, 5, 7, 8, 9, 17, 19, 20, 21, and 22 were upregulated rapidly after 6 h of the submergence treatment, while AhCIPK10, 11, 23, and 23 reached their highest expression at 24 h (Figure 8). By contrast, the expression levels of AhCIPK1, 2, 4, 14, 15, 16, and 26 were inhibited under the whole submergence treatment process. We found it interesting that AhCIPK12, 13, and 25 were first repressed under the earlier submergence treatment stages and were induced at the later stages. In addition, seven AhCIPKs (AhCIPK1, 5, 7, 11, 14, 19, and 20) were activated after 6 h R. solanacearum infection (Figure 8B), while six AhCIPK members (AhCIPK2, 9, 11, 12, 22, and 25) were obviously upregulated and three (AhCIPK3, 16, and 26) were depressed after 48 h of R. solanacearum infection (Figure 8B, Supplementary Table S4); In total, AhCIPK genes might have functioned differentially in both abiotic and biotic environmental stress regulation.

FIGURE 8

Relative expression of AhCIPKs under submergence and Ralstonia solanacearum infection. (A) expression characteristics of AhCIPKs in response to submergence at five time points (00, 06, 24, 72, and 120 h). (B) expression of AhCIPKs in response to R. solanacearum infection at two time points (6 and 48 h). The heatmap of the AhCIPK gene expression levels was hierarchically clustered using TBtools with the data normalization method of Z-score standardization. Legend at the right of the heatmap: red means upregulation and blue shows downregulated expression.

Candidate gene association of peanut CIPKs polymorphisms with 104 traits

To further uncover the roles of CIPK genes in peanut development and stress response, we performed candidate gene association analysis using 22 single-nucleotide polymorphisms in CIPKs from transcriptome data of 146 peanut lines and 104 phenotypes related to peanut development and stress response collected in five environments. The results indicated that one polymorphic site [A09_903480(G/K/T)] was significantly associated with pod length (PL), seed length (SL), hundred seed weight (HSW), and shoot root ratio (SR) traits (Figure 9A, Supplementary Tables S5–S7). Site B09_903480 mainly formed three haplotypes [B09_903480(G/K/T)] (Figure 9B) in the population and was located in the predicted exon region of AiCIPK10 (Figure 9C). Results showed that PL, SL, HSW, and SR in haplotype G were significantly higher than those in haplotype T (Figure 9D).

FIGURE 9

Association results and the phenotypes of the polymorphic sites of the peanut CIPKs associated with hundred seed weight (HSW)/pod length (PL)/ seed length (SL)/shoot root ratio (SR) variation. (A) association results between HSW/PL/SL/SR and the polymorphisms in peanut CIPKs. (B) sequences of the site significantly associated with HSW/PL/SL/SR variation. (C) gene structures of AdCIPK12(Aradu.Z7XZ9). (D) phenotypic comparison of haplotypes of the associated site with HSW/PL/SL/SR in five environments of the population.

Discussion

Peanut CIPKs did not expand with genome duplication

CIPK genes are widely distributed widely in the biological world; however, their number varies greatly among different species. One, two, and seven CIPK genes were found in green algae, Chlorella, and Physcomitrella patens, respectively (Cheong et al., 2007; Weinl and Kudla, 2009). According to previous studies, 25, 30, 27, 43, and 79 CIPK genes were identified in A. thaliana (125–155 Mb), rice (389 Mb), poplar (416 Mb), corn (2,400 Mb), and wheat (14,500 Mb) (Kolukisaoglu et al., 2004; Xiang et al., 2007; Chen et al., 2011; Sun et al., 2015; Zhu et al., 2021). Among Solanaceae plants, there were 21 and 22 CIPK members in tomato (Liu et al., 2017; Wang and Liu, 2018). Woody plants had 27 members (Zhang et al., 2008). This study uncovered 54 CIPK genes in three peanut genomes, among which 26 were from cultivated peanut (2,540 Mb). Our results support the hypothesis that the number of CIPK members in monocotyledonous plants is more than that in dicotyledonous plants. However, the number of peanut CIPK members did not expand with peanut genome size expansion.

The phylogenetic analysis revealed that CIPK members in Arabidopsis, rice, soybean, grape, and peanut could be coincidentally clustered into two distinct groups with different numbers of introns. This cluster pattern is the same as that in the previously reported CIPK-phylogenetic trees of Arabidopsis and rice (Kolukisaoglu et al., 2004). Our results further supported the hypothesis that the ancestor of CIPKs evolutionarily formed in the plant genome prior to the separation of the lineages of monocotyledons and dicotyledons (Kolukisaoglu et al., 2004; Yu et al., 2007).

Peanut CIPK genes functioned in stress response

The prediction of cis-acting elements can provide important clues for the study of gene expression regulation (Zhu et al., 2021). Cis-acting elements of biotic and abiotic stresses presented ubiquitously in the promoter region of peanut CIPKs, indicating these impact factors may interact to act on the CIPK regulatory mechanism (Figure 6). Compared to those in the AhCIPKs, the defense, stress, low-temperature response element TC-rich repeats and LTR were distributed concentratedly in several AdCIPKs or AiCIPKs in the two diploid progenitors; for example, four and three LTRs were identified in the promoter region of AdCIPK4 and AiCIPK11, and four TC-rich repeats were found in the promoter region of AdCIPK4, indicating that the regulatory mechanism might be different between the cultivated peanut and the two diploid progenitors. Further the stress-induced expression data also elucidated the potential functions of peanut CIPKs in stress.

Among the orthologs between peanut and Arabidopsis (Table 2), the functions of the corresponding ortholog genes in Arabidopsis have been determined, and they functioned in influencing plant stress response (Table 2). AdCIPK3, AiCIPK4, AhCIPK3, and AhCIPK16 were identified as the orthologous genes of the famous salt tolerance gene AtCIPK24/AtSOS2 in A. thaliana (Halfter et al., 2000; Ishitani et al., 2000; Liu et al., 2000; Guo et al., 2001), and AhCIPK8, AhCIPK21, AdCIPK9, and AiCIPK10 were found as the orthologs of AtCIPK3 relating to the cold signal transduction (Kim et al., 2003; Pandey et al., 2008; Sanyal et al., 2017). Many other orthologs were also uncovered between peanut and the Arabidopsis abscisic acid-dependent and Arabidopsis abscisic acid-independent stress response gene AtCIPK1 (D'Angelo et al., 2006), low-potassium tolerance gene AtCIPK9 (Pandey et al., 2007; Singh et al., 2018; Lara et al., 2020; Kanwar et al., 2022), and cadmium stress response gene AtCIPK11 (Liu and Guo, 2011; Zhou et al., 2015; Gu et al., 2021). Therefore, these peanut CIPK orthologous genes may also play multiple roles in peanut stress response, especially in salt response.

Peanut CIPK genes play important roles in growth and development

Many important genes were selectively expressed in specific tissues during various physiological and developmental processes (Wan et al., 2014). Our results showed that the 26 AhCIPK genes had distinct tissue-specific expression patterns, and several AhCIPKs showed higher expression level in the leaf, reproductive shoot, root, nodule, pod, and seed. We found it interesting that nearly half of AhCIPK genes are highly expressed in reproductive organs (Figure 7), indicating AhCIPKs play important roles in multiple tissue growth and development, especially the reproductive organs.

The single-nucleotide polymorphic sites in AdCIPK12 (corresponding to AhCIPK11) were significantly associated with PL, SL, HSW, and SR variation. The polymorphic site in AdCIPK12 [A09_ 903,480 (G/K/T)], located in the predicted first exon region of the gene, A09_903480(G/K/T), led no transition (synonymous mutation) in the peanut population. Recent studies proved that synonymous mutations also have dramatic effects on protein output (Gillen et al., 2021). These results indicated that the B09_903480 (G/K/T) sequence polymorphisms might be the actual functional sites. Further, AhCIPK11 was mainly expressed in the nodule, peg, pericarp, and seed, especially in the middle pericarp and seed development stages, which provided additional evidence for its function in peanut pod development (Figure 6). Further investigation was needed to confirm the roles of AdCIPK12 (AhCIPK11) in the pod and seed development of peanut.

Conclusions

We definitely identified 54 CIPK members in cultivated and wild peanut for the first time and determined their chromosomal locations, gene structures, evolution, and expression patterns under biotic and abiotic conditions. We also focused on one gene, AiCIPK10/AhCIPK21, which was involved in pod and seed development. Our results provide valuable information for understanding the functions of the peanut CIPK gene family in regulating yield, quality, and stress responses in peanut.