Genome-Wide Identification and Characterization of CDPK Family Reveal Their Involvements in Growth and Development and Abiotic Stress in Sweet Potato and Its Two Diploid Relatives

Calcium-dependent protein kinase (CDPKs) is one of the calcium-sensing proteins in plants. They are likely to play important roles in growth and development and abiotic stress responses. However, these functions have not been explored in sweet potato. In this study, we identified 39 CDPKs in cultivated hexaploid sweet potato (Ipomoea batatas, 2n = 6x = 90), 35 CDPKs in diploid relative Ipomoea trifida (2n = 2x = 30), and 35 CDPKs in Ipomoea triloba (2n = 2x = 30) via genome structure analysis and phylogenetic characterization, respectively. The protein physiological property, chromosome localization, phylogenetic relationship, gene structure, promoter cis-acting regulatory elements, and protein interaction network were systematically investigated to explore the possible roles of homologous CDPKs in the growth and development and abiotic stress responses of sweet potato. The expression profiles of the identified CDPKs in different tissues and treatments revealed tissue specificity and various expression patterns in sweet potato and its two diploid relatives, supporting the difference in the evolutionary trajectories of hexaploid sweet potato. These results are a critical first step in understanding the functions of sweet potato CDPK genes and provide more candidate genes for improving yield and abiotic stress tolerance in cultivated sweet potato.


Introduction
Ca 2+ is an important second messenger in plants, and signaling pathways mediated by Ca 2+ have been shown to play an important role in plant growth and development in response to abiotic and biotic stresses [1,2]. When cells sense external changes, the Ca 2+ concentration in the cytoplasm changes, resulting in a series of physiological and biochemical reactions to plant tolerance improvement. There are three families of calcium-sensing proteins in plants, including calmodulin (CaM) and calmodulin-like protein (CaML), calciumdependent protein kinase (CDPK), and calcineurin B-like proteins (CBLs)/CBL-interacting proteins (CIPK) [3][4][5][6][7]. Among these sensors, CDPKs are Ser/Thr protein kinases, serving as special sensors as they can directly convert upstream Ca 2+ signals into downstream protein phosphorylation events [8]. Genome-wide analysis led to the identification of CDPK genes in various plant species. There are 34 genes in Arabidopsis thaliana [3], 31 in rice (Oryza total of 39, 35, and 35 CDPKs were identified in I. batatas, I. trifida, and I. triloba, respectively (named after "Ib", "Itf ", "Itb"). The physicochemical properties of CDPKs were analyzed using the sequences from I. batatas (Table 1 All the CDPKs were separately mapped on 15 chromosomes of I. batatas, I. trifida, and I. triloba (Figure 1). In the I. batatas genome, 39 IbCDPKs genes were distributed across every chromosome except LG4. In the I. trifida and I. triloba genome, 35 IbCDPKs genes were distributed across every chromosome except Chr13. In I. batatas, four IbCDPKs were detected on LG1, three on LG2, three on LG3, three on LG5, four on LG6, three on LG7, one on LG8, three on LG9, two on LG10, one on LG11, two on LG12, one on LG13, six on LG14, and three on LG15 ( Figure 1A). The numbers of Itf/ItbCDPKs located on the chromosomes were the same in two diploid relatives. One CDPK was detected on Chr01, Chr02, and Chr11; two on Chr04, Chr06, Chr07, Chr08, and Chr14; three on Chr03, Chr10, and Chr12; four on Chr05 and Chr15; and five on Chr09 ( Figure 1B,C). The results indicated that the distribution of CDPKs was different on chromosomes in sweet potato and its two diploid relatives, whereas it was similar in two diploid relatives.   Table S1.

Phylogenetic Relationship of CDPKs in Sweet Potato and Its Two Diploid Relatives
To study the evolutionary relationship of CDPKs in I. batatas, I. trifada, I. triloba, and Arabidopsis, we constructed a phylogenetic tree for 143 CDPKs (i.e., 39 in I. batatas, 35 in I trifida, 35 in I. triloba, and 34 in Arabidopsis) ( Figure 2). All CDPKs were unevenly distrib uted and divided into five subgroups (group I to V) according to the evolutionary dis tance. The specific distribution of CDPKs was as follows (total: I. batatas, I. trifida, I. triloba Arabidopsis): group I (54: 16,14,14,10); group II (42:11, 9, 9, 13); group III (32:8, 8, 8, 8) group IV (12:3, 3, 3, 3), and group V (3:1, 1, 1, 0) ( Figure 2; Table S1). We named IbCDPKs ItfCDPKs, and ItbCDPKs based on their homology with homologs in Arabidopsis, and AtCDPK15/19/21/22/23/27/31 from Arabidopsis have no homologous protein in I. batatas, I trifada, I. triloba, and only Ib/Itf/ItbCDPK35 have no homologous proteins in Arabidopsis One additional IbCDPK sequence (IbCDPK25.4) was identified in I. batatas that had no homology protein in I. trifida and I. triloba. Two sequences (IbCDPK34.1 and IbCDPK34.2 in I. batatas were homologous with ItfCDPK34 and ItbCDPK34. We speculated that large differences in number and type of CDPKs divided in five subgroups between Arabidopsi and sweet potato and its two diploid relatives were due to species specificity. Moreover the discrepancy showed in sweet potato and its two diploid relatives might be due to chromosomal hybridization during evolution. Figure 2. Phylogenetic analysis of the CDPK family in I. batatas, I. trifida, I. triloba, and Arabidopsis. A total of 143 CDPKs were divided into five subgroups (group I to V) according to the evolutionary distance. The pink pentagrams, blue cycles, yellow triangles, and green squares represent IbCDPKs in I. batatas, ItfCDPKs in I. trifida, ItbCDPKs in I. triloba, and AtCPKs in Arabidopsis, respectively.

Conserved Motif and Exon-Intron Structure Analysis of CDPKs in Sweet Potato and Its Two Diploid Relatives
To illustrate the structural characteristics of the 109 CDPKs from I. batatas, I. trifida, and I. triloba, we performed motif and domain analysis using the MEME website ( Figure 3). A total of 10 motifs were identified ( Figure 3A and Figure S2). Overall, the protein structure of this family was relatively conserved, and most of the CDPKs contained six protein kinase domains and four EF-hands (three EF-hand_1 and one EF-hand_2) except ItbCDPK13. Most CDPKs contained even numbers of EF-hand except IbCDPK11. in group III, and IbCDPK28 in group IV lacked at least one protein kinase domain. In group I, ItfCDPK5.1, ItbCDPK20.1, and ItbCDPK25.1 contained two EF-hand_1 and one EF-hand_2. The C-terminus of IbCDPK12.3 and the N-terminus of ItbCDPK20.2 contained one more EF-hand. Ib/ItbCDPK11.1 lacked one and two protein kinase domains, respectively. Ib/Itf/ItbCDPK25.2 lacked one, two, and two protein kinase domains, respectively. In group II, the composition of the EF-hand of ItbCDPK3 was distinct, with two EF-hand_1 and two EF-hand_2. IbCDPK24 in group III lacked two protein kinase domains and one EF-hand_1. IbCDPK32 lacked one protein kinase domain and two EF-hands. Itf/ItbCDPK34 lacked two and three protein kinase domains, respectively. In group V, Ib/ItfCDPK35 lacked two protein kinase domains and two EF-hands, and ItbCDPK35 lacked three protein kinase domains and two EF-hands.
The exon-intron structures of IbCDPKs varied from those of Itf/ItbCDPKs, with the coding DNA sequence (CDS) composition ranging from five to twenty-three exons ( Figure 3B). Ib/Itf/ItbCDPKs contained seven to ten, six to eight, and six to thirteen exons in group I; eight to twelve, seven to nine, and six to eight exons in group II; six to twelve, five to eight, and five to eight exons in group III; thirteen to twenty-three, twelve, and twelve exons in group IV; eight, eight, and eight exons in group V, respectively ( Figure 3B).

Cis-Element Analysis in the Promoter of IbCDPKs in Sweet Potato
Cis-acting elements are nucleotide sequences that are found upstream or downstream of genes and can regulate their transcription levels. They work through combining with some specific transcription factors when plants respond to various development processes and stresses. To reveal how CDPKs function in growth and development and abiotic stress adaption in sweet potato, 2000 bp upstream sequences of 39 IbCDPKs in I. batatas were extracted and the cis-element analysis was performed using PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 30 January 2022). According to the prediction function, all cis-elements were divided into core elements and binding sites, development, light-responsive, hormonal-responsive, and abiotic/biotic stress-responsive elements ( Figure 4). The degree of red colors represented the number of cis-elements upstream of the IbCDPKs. The majority of 39 CDPKs possessed a large number of core promoter elements and binding sites, such as AT-TATA-box, TATA-box, and CAAT-box ( Figure 5). TATA-box and CAAT-box are the binding sites of RNA polymerase and are involved in the transcription initiation and frequency of genes [39]. Some development elements (i.e., The majority of 39 CDPKs possessed a large number of core promoter elements and binding sites, such as AT-TATA-box, TATA-box, and CAAT-box ( Figure 5). TATA-box and CAAT-box are the binding sites of RNA polymerase and are involved in the transcription initiation and frequency of genes [39]. Some development elements (i.e., CCAAT-box, CATbox, circadian, O2-site, RY-element, and A-box) were found in IbCDPKs ( Figure 4). Lightresponsive elements (i.e., AAGAA-motif, Box 4, G-box, CATA-motif, GT1-motif, Sp1, TCCCmotif, and TCT-motif) were found in most of IbCDPKs ( Figure 4). In addition, hormonalresponsive elements (i.e., ABA-responsive element ABRE, GARE-motif, MeJA-responsive elements CGTCA-motif and TGACG-motif, GA-responsive elements P-box and TATC-box, SA-responsive element TCA, auxin-responsive element TGA-element) were found. The majority of IbCDPKs except IbCDPK1, IbCDPK12.3, and IbCDPK13, processed at least two hormone-responsive elements ( Figure 4). These results indicated that IbCDPKs might be involved in the crosstalk between different hormone signaling pathways. Furthermore, anaerobic induction-responsive element ARE, low temperature-responsive element LTR, drought-responsive elements MBS, MYB and MYC, stress-responsive element STRE, injury and defensive-responsive elements WRE3, and WUN-motif were found in most IbCDPKs ( Figure 4). All IbCDPKs processed at least three drought-responsive elements. These results suggested that IbCDPKs might be involved in the crosstalk between hormone signaling pathways to regulate the growth and development and stress adaption in sweet potato, particularly in drought stress. IbCDPKs ( Figure 4). Light-responsive elements (i.e., AAGAA-motif, Box 4, G-box, CATAmotif, GT1-motif, Sp1, TCCC-motif, and TCT-motif) were found in most of IbCDPKs (Figure 4). In addition, hormonal-responsive elements (i.e., ABA-responsive element ABRE, GARE-motif, MeJA-responsive elements CGTCA-motif and TGACG-motif, GA-responsive elements P-box and TATC-box, SA-responsive element TCA, auxin-responsive element TGA-element) were found. The majority of IbCDPKs except IbCDPK1, IbCDPK12.3, and IbCDPK13, processed at least two hormone-responsive elements ( Figure 4). These results indicated that IbCDPKs might be involved in the crosstalk between different hormone signaling pathways. Furthermore, anaerobic induction-responsive element ARE, low temperature-responsive element LTR, drought-responsive elements MBS, MYB and MYC, stress-responsive element STRE, injury and defensive-responsive elements WRE3, and WUN-motif were found in most IbCDPKs ( Figure 4). All IbCDPKs processed at least three drought-responsive elements. These results suggested that IbCDPKs might be involved in the crosstalk between hormone signaling pathways to regulate the growth and development and stress adaption in sweet potato, particularly in drought stress.

Protein Interaction Network of IbCDPKs
To explore the potential regulatory network of IbCDPKs, we constructed an IbCDPKs interaction network based on Arabidopsis orthologous proteins ( Figure 5). We speculated IbCDPK11 might interact with IbCDPK24. They also interacted with JA biosynthesis-related protein (i.e., ACX1 and ACX5), ABA-responsive element-binding factor 1 (ABF1), potassium channel protein (KAT2), and stomatal movement protein (ELUS3). They could interact with L-ascorbate peroxidase 3 (APX3) and catalase (F5M15.5/ROG1) to scavenge hydrogen peroxide in plants. IbCDPK11.1, IbCDPK11.2, IbCDPK12.1, IbCDPK12.2, and IbCDPK12.3 might interact with DI19 (DEHYDRATION- INDUCED 19) in response to drought stress. IbCDPK2 might generate a complex with IbCDPK20.1/20.2 through SALH3. SALH3, encoding S-type anion channel protein, was an essential negative regulator of inward potassium channels in guard cells. IbCDPK2, IbCDPK3, IbCDPK13, and IbCDPK20.1/20.2 may be essential for efficient stomatal movement in guard cells. IbCDPK3 might also interact with ORP2A to be involved in the transport of sterols. IbCDPK32 could interact with CNGC18 to regulate pollen growth in sweet potato. IbCDPK3 and IbCDPK8 might play important roles in disease resistance through their Network nodes represent proteins, green nodes represent AtCPKs and other colored nodes represent interacting proteins. Lines represent protein-protein interaction which was experimentally determined.

Protein Interaction Network of IbCDPKs
To explore the potential regulatory network of IbCDPKs, we constructed an IbCDPKs interaction network based on Arabidopsis orthologous proteins ( Figure 5). We speculated IbCDPK11 might interact with IbCDPK24. They also interacted with JA biosynthesis-related protein (i.e., ACX1 and ACX5), ABA-responsive element-binding factor 1 (ABF1), potassium channel protein (KAT2), and stomatal movement protein (ELUS3). They could interact with L-ascorbate peroxidase 3 (APX3) and catalase (F5M15.5/ROG1) to scavenge hydrogen peroxide in plants. IbCDPK11.1, IbCDPK11.2, IbCDPK12.1, IbCDPK12.2, and IbCDPK12.3 might interact with DI19 (DEHYDRATION- INDUCED 19) in response to drought stress. IbCDPK2 might generate a complex with IbCDPK20.1/20.2 through SALH3. SALH3, encoding S-type anion channel protein, was an essential negative regulator of inward potassium channels in guard cells. IbCDPK2, IbCDPK3, IbCDPK13, and IbCDPK20.1/20.2 may be essential for efficient stomatal movement in guard cells. IbCDPK3 might also interact with ORP2A to be involved in the transport of sterols. IbCDPK32 could interact with CNGC18 to regulate pollen growth in sweet potato. IbCDPK3 and IbCDPK8 might play important roles in disease resistance through their involvement in stomatal movement and JA biosynthesis. These results indicated that IbCDPKs might play an important role in plant growth and development, such as stomatal movement, icon transport, and participate in hormone signaling pathways (i.e., JA and ABA) in response to abiotic and biotic stresses. Therefore, interacting proteins of IbCDPKs are still worth exploring.
In I. triloba, the expression pattern of ItbCDPKs was similar to that of ItfCDPKs in I. trifida except for individual genes. ItbCDPK1 showed low expression in the flower but not the leaf. ItbCDPK2 was highly expressed in the stem but not flower. ItbCDPK12.2 and ItbCDPK25.2 were highly expressed in the leaf ( Figure 7B). These results suggested that Variation in gene expression between homolog CDPKs was also observed. RNA-seq data of six tissues (i.e., flower, flower bud, leaf, root 1, root 2, and stem) were used to   1, -20.2, -25.1, -25.2, -25.3, -17.1, -17.2, -33.2, -34, -7, -24, and -16) were highly expressed in flowerbud. Furthermore, ItfCDPK12.3, -8, and -30 showed low expression in all tissues, and ItfCDPK12.3 showed the lowest expression in the flower, while ItfCDPK30 had the lowest expression in the flower bud. ItfCDPK5.1 and ItfCDPK3 were highly expressed in the stem and flower, while ItfCDPK13 was highly expressed in the stem and flowerbud ( Figure 7A).

Expression Analysis under Abiotic Stresses
To evaluate the possible function of IbCDPKs, the level of transcript accumulation was determined using quantitative real-time PCR in leaf tissues at 0, 1, 3, 6, 12, and 48 h under NaCl, PEG, H 2 O 2 , and cold treatments ( Figure 10 Figure 10D). In general, twenty IbCDPKs were induced by all four abiotic stress treatments in sweet potato, while only IbCDPK34.1 was down-regulated in three abiotic stress treatments (NaCl, PEG, and cold). These results indicated IbCDPKs might be key players in abiotic stress resistance.
In addition, we also analyzed the expression patterns of CDPKs using the RNA-seq data of I. trifida and I. triloba under cold, heat, drought, and salt treatments [38]. In I. trifida, under cold treatment, compared with the control, in group I, ItfCDPK1, -2, -5. , and -28 showed lower expression levels. ItfCDPK2 was induced by cold, heat, salt stresses. ItfCDPK5.2 was induced by cold, salt, and drought stresses. ItfCDPK12.1 and ItfCDPK28 were induced by cold and heat stresses. ItfCDPK29.2 was induced by salt and drought stresses. ItfCDPK30 was induced by cold, heat, salt, and drought ( Figure 11A). In I. triloba, ItbCDPK2 showed an opposite expression pattern in cold, heat, salt, and drought stresses compared with ItfCDPK2. ItbCDPK12.2 was induced by cold stress. ItbCDPK20.1 was repressed by cold stress. ItbCDPK20.2 was repressed in salt stress. ItbCDPK3 and ItbCDPK9 were induced by cold stress. ItbCDPK17.1 and ItbCDPK17.2 were repressed by cold stress. ItbCDPK7 was repressed by salt stress. ItbCDPK16 was induced by cold stress and repressed by heat stress. ItbCDPK18 was induced by drought stress ( Figure 11B). These results indicated that CDPKs showed commonalities and differences in response to abiotic stresses in I. trifida and I. triloba. was not responsive to any hormone. ItbCDPK10 was up-regulated by ABA but not BAP and down-regulated by IAA. ItbCDPK30 was induced by ABA but not BAP. ItbCDPK32 was induced but ItfCDPK32 was repressed by ABA ( Figure 9B). Furthermore, IbCDPKs and their homologous CDPKs in I. trifida and I. triloba showed different expression patterns in response to ABA, GA, IAA, and MeJA. These results indicated that CDPKs might function in developmental processes through various hormone signaling pathways between sweet potato and its two diploid relatives. In addition, we also analyzed the expression patterns of CDPKs using the RNA-seq data of I. trifida and I. triloba under cold, heat, drought, and salt treatments [38]. In I. trifida, under cold treatment, compared with the control, in group I, ItfCDPK1, -2, -5.2, -11.1, -12.1, -20.1, -20.2, -25.1, -25.2 were induced and ItfCDPK5.1, -12.2, -12.3, -25.3 were repressed. In group II, ItfCDPK17.1 and ItfCDPK33.1 were induced and ItfCDPK3, -9, and -17.2 were repressed. ItfCDPK10, ItfCDPK14, and ItfCDPK32 were induced while ItfCDPK8 and ItfCDPK13 were repressed. ItfCDPK28 was induced. The results suggested these ItfCDPKs might be involved in the response to cold stress. ItfCDPK2, -12.1, -12.3, -25.2, -Figure 10. Gene expression patterns of IbCDPKs in response to abiotic stresses, i.e., (A) NaCl, (B) PEG, (C) H 2 O 2 , and (D) cold, of I. batatas. The values were determined by RT-qPCR from three biological replicates consisting of pools of three plants, and the results were analyzed using the comparative C T method. The expression of 0 h in each treatment was considered "1". The fold change is shown in the boxes. Different lowercase letters indicate significant differences (p < 0.05; Student's t-test).

Discussion
CDPKs are essential in the regulation of plant growth and development, as well as in response to biotic and abiotic stresses. In the previous study, CDPKs were identified in Arabidopsis, rice, wheat, maize, polar, pear, and grape. However, the functional roles of the CDPKs family are still poorly understood in sweet potato. The modern cultivated sweet potato (I. batatas) is an autohexaploid (2n = 6x = 90) varying from I. trifida NCNSP0306 (2n = 2x = 30) and I. triloba NCNSP0323 (2n = 2x = 30) ( Figure S1) and is an

Discussion
CDPKs are essential in the regulation of plant growth and development, as well as in response to biotic and abiotic stresses. In the previous study, CDPKs were identified in Arabidopsis, rice, wheat, maize, polar, pear, and grape. However, the functional roles of the CDPKs family are still poorly understood in sweet potato. The modern cultivated sweet potato (I. batatas) is an autohexaploid (2n = 6x = 90) varying from I. trifida NCNSP0306 (2n = 2x = 30) and I. triloba NCNSP0323 (2n = 2x = 30) ( Figure S1) and is an important crop because of its tuberous roots [38]. In this work, we characterized the CDPKs family in sweet potato and its two diploid relatives using whole-genome sequence data. To investigate physicochemical properties of CDPKs, the protein physiological properties, chromosome localization, phylogenetic relationships, conserved motifs, and protein interaction networks were predicted. We also analyzed the expression patterns of CDPKs in different tissues and relatives cross-talking of multiple stress signaling pathways. Genome-wide identification of CDPKs in sweet potato and its two diploid relatives will facilitate further genetic studies of growth, development, and stress resistance.

Evolution of the CDPK Gene Family in Sweet Potato and Its Two Diploid Relatives
A total of 109 CDPKs (i.e., 39 in I. batatas, 35 in I. trifida, and 35 in I. triloba) were identified from the cultivated hexaploid sweet potato and its two diploid relatives. According to the evolutionary distance to AtCPKs, these CDPKs were classified into five subgroups (group I to V), with one more group than CDPKs in other species (group I to IV) [3,[9][10][11][12]. Ib/Itf/ItbCDPK35 in group V have no homologous protein in Arabidopsis, meaning group V in sweet potato is unique. We also observed that the number of CDPKs varied between sweet potato and its two diploid relatives. Thirty-nine IbCDPKs were distributed across the genome in I. batatas ( Figure 1A). While the number of CDPKs identified in I. trifida was the same as that in I. triloba but was less than that in I. batatas., supporting the distinct origin during the evolution of hexaploid sweet potato.
In this study, a total of 10 motifs were identified in the 109 CDPKs from I. batatas, I. trifida, and I. triloba ( Figure 3A and Figure S2), including six protein kinase domains and four EF-hands. Moreover, these motifs were highly conserved in I. batatas, I. trifida, and I. triloba. The majority of CDPKs contained a protein kinase domain and EF-hand except for ItbCDPK13, which contained a protein kinase domain only. The EF-hand in the C-terminus is a Ca 2+ binding site [59], which means IbCDPK13 might be Ca 2+ insensitive. There was no significant difference in each group except for Ib/Itf/ItbCDPK35 in group V. The number of protein kinase domains and EF-hands varied in sweet potato and its two diploid relatives, especially in group I (i.e., ItfCDPK5.1, Ib/ItbCDPK11.1, IbCDPK11.3, IbCDPK12.1, IbCDPK12.3, ItbCDPK20.1, Ib/ItbCDPK20.2, ItbCDPK25.1, Ib/Itf/ItbCDPK25.2, and IbCDPK25.4) ( Figure 3A).
Besides, the exon-intron structures of some homologous CDPKs were different between sweet potato and its two diploid relatives and varied from I. trifida and I. triloba also. Some CDPKs contained the same number of exons and introns in I. trifida and I. triloba but less than that in I. batata, while some CDPKs contained different exons-introns in I. batatas, I. trifida, and I. triloba. These results suggested that CDPKs produced more changes during the evolution from diploid to hexaploid, while the structures of other groups were relatively conserved. Moreover, CDPKs in group I might play more critical roles in plant growth and development and response to environmental stress.

Different Functions of CDPKs on Growth and Development between Sweet Potato and Its Two Diploid Relatives
CDPKs could be detected on roots, stems, leaves, fruits, and seeds of plants. The expression levels of OsCDPK2 increased with the seed development period. The expression patterns of OsCDPK2 were opposite in green leaves exposed to light and darkness. OsCDPK2 might function in seed development and in response to light in leaves [60]. PiCDPK1 and PiCDPK2 were specifically expressed at the pollen stage in Petunia hybrida. Overexpression of PiCDPK1 disturbed the growth polarity of pollen tubes, while overexpression of PiCDPK2 inhibits the elongation ability of pollen tubes but had no effect on the growth polarity of pollen tubes [52]. PnCDPK1 was accumulated mainly in petals and sepals, which means that PnCDPK1 may be an important component in the signal transduction pathways for flower morphogenesis [30]. Here, the expression levels of CDPKs in different tissues of I. batatas, I. trifida, and I. triloba were shown (Figures 6 and 7). In I.batatas, no similar expression trends were observed between subgroups in five tissues. The majority of IbCDPKs were highly expressed in leaf, petiole, and pigmented root. Many IbCDPKs showed tissue-specific expression, and some IbCDPKs showed higher expression levels in the same tissue at the same time except for in tuberous root. Interestingly, only IbCDPK28 showed the highest expression in tuberous root ( Figure 6). IbCDPK28 might be a key regulator in the development of tuberous root. In I. trifida, most ItfCDPKs in group I and group II might play key roles in flower and flowerbud growth and development. ItfCDPKs in group III might play roles in stem and flower and ItfCDPKs in group IV and V might be involved in root growth and development ( Figure 7A). In I. triloba, the expression pattern of ItbCDPKs was similar to that of Itf CDPKs in I. trifida except for ItbCDPK1, -2, -12.2, and -25.2 ( Figure 7B). Some homologous CDPKs showed similar or opposite tissue-specific expression (Figures 6 and 7). Ib/Itf/ItbCDPK5.1 were highly expressed in stem and Ib/Itf/ItbCDPK18 were highly expressed in leaf. IbCDPK3 was lowly expressed, whereas ItfCDPK3 was highly expressed in leaf. Diverse gene expression patterns among homologous CDPKs across different tissues suggest that CDPK proteins might be specialized for different biological responses.

Different Functions of CDPKs on Hormone Crosstalk between Sweet Potato and Its Two Diploid Relatives
Protein interaction prediction was performed to further reveal the potential function of IbCDPKs ( Figure 5). IbCDPKs might interact with each other, such as IbCDPK11 and IbCDPK24, although it has not been reported that CDPKs could form homodimers. They might interact with JA biosynthesis-related proteins (i.e., ACX1 and ACX5) or ABAresponsive element-binding factor 1 (ABF1). IbCDPK11.1, -11.2, -12.1, -12.2, and -12.3 might interact with DI19 in response to drought stress. In rice, OsDi19-4 regulated the expression of OsASPG1 and OsNAC18, two ABA-responsive genes, by directly binding to their promoters. The regulation was further enhanced by the increased phosphorylation of OsDi19-4 after the treatment of ABA [53]. Furthermore, All IbCDPKs were induced by at least two hormones ( Figure 8). The majority of IbCDPKs possessed at least two hormone-responsive elements, such as ABA-responsive elements (ABRE, GARE-motif), MeJA-responsive elements (CGTCA-motif and TGACG-motif), GA-responsive elements (P-box and TATC-box), SA-responsive element (TCA), auxin-responsive elements (TGAelement) ( Figure 4). Moreover, most of them peaked within 12 h (Figure 8), indicating that IbCDPKs could sense fluctuations quickly in hormone levels. SLAC1 was regulated by two AtCDPK protein kinases (CPK21 and CPK23), with distinct Ca 2+ affinities in response to drought stress through ABA signaling pathways [54]. In Arabidopsis, disruption of the CPK6 gene impaired MeJA-induced stomatal closure. MeJA-induced transient cytosolic free calcium concentration increments were reduced in the cpk6-1 mutant. MeJA failed to activate slow-type anion channels in the cpk6-1 guard cells. AtCPK6 functions as a positive regulator of MeJA signaling in Arabidopsis guard cells [26]. Overexpression of ZmCPK4 in the transgenic Arabidopsis enhanced ABA sensitivity in seed germination, seedling growth, and stomatal movement. The transgenic plants also enhanced drought stress tolerance [55]. Thus, IbCDPKs might also participate in hormone signaling pathways in response to environmental stress.
However, some of their homologous CDPKs in I. trifida and I. triloba showed different expression patterns in response to ABA, GA, IAA, and MeJA. Under IAA treatment, Itf CDPKs and ItbCDPKs were insensitive. For example, Ib/Itf/ItbCDPK1, -5.1, -25.3, -16, and -35 under ABA treatment, -12.3, -9, and -10 under IAA treatment, and -18 under GA treatment showed opposite expression trends (Figure 8; Figure 11). In addition, expression patterns of ItfCDPKs were not exactly the same as those of ItbCDPKs (i.e., Itf/ItbCDPK30, -32 under ABA treatment, -33.1 under IAA treatment, -2 under GA treatment, and -30 under BAP treatment) ( Figure 11). These results indicated that CDPKs participated in multiple hormones crosstalk, and homologous CDPK genes were involved in different hormonal pathways in sweet potato and its two diploid relatives.

Different Functions of CDPKs on Multiple Abiotic Stress Response between Sweet Potato and Its Two Diploid Relatives
There have been many reports that CDPKs were related to abiotic stress resistance [32][33][34]. In Chenopodium glaucum, CgCDPK interacted with CgbHLH001 in the signal transduction pathway in response to salt and drought stress [56]. Overexpression of SiCDPK24 in Arabidopsis enhanced drought resistance and improved the survival rate under drought stress [61]. In this study, we analyzed the level of transcript accumulation using qRT-PCR at different time points post treatments ( Figure 10). Under NaCl, H 2 O 2 , and cold stresses, the expression of IbCDPKs peaked at 1 h ( Figure 10A), 12/48 h ( Figure 10C), and 3 h ( Figure 10D), meaning that the regulation of IbCDPKs was mainly activated on the prophase of NaCl and cold treatments, and the anaphase of H 2 O 2 treatment, respectively. In general, thirty-eight IbCDPKs were up-regulated by at least two abiotic stresses, consistent with CDPK genes in other species.  Figure 9). In protein interaction prediction, IbCDPKs might interact with a potassium channel protein (KAT2), stomatal movement protein (ELUS3) ( Figure 5). These results indicated that IbCDPKs might be key players in response to abiotic stresses by regulating stomatal movement and icon transport.
In addition, the expression patterns of homologous CDPKs in diploid I. trifida and I. triloba using RNA-seq were distinct ( Figure 11). The numbers of Itf/ItbCDPKs induced by salt and cold stresses were less than those of IbCDPKs, which may be due to the fact that only one time point was detected.
Differences in expression patterns of CDPKs in sweet potato and its two diploid relatives might provide potential candidate genes for further functional characterization and for improving abiotic stress tolerance of sweet potato and other species.

Protein Properties Prediction of CDPKs
The MW, pI, and the number of EF-hands of CDPKs were calculated by ExPASy (https://www.expasy.org/ accessed on 23 December 2021). The N-myristoylation and Palmitoylation sites of CDPKs were predicted by GPS-Lipid 1.0 with a high threshold (http://lipid.biocuckoo.org/ accessed on 24 December 2021) [63].

Phylogenetic Analysis of CDPKs
The phylogenetic analysis of CDPKs from I. batatas, I. trifida, I. triloba, and Arabidopsis was performed using ClustalW in MEGA X [64] with default parameters, the maximum likelihood method, and the Poisson correction model. Bootstrapping was performed with 1000 replicates. Then, the phylogenetic tree was constructed by iTOL (http://itol.embl.de/ accessed on 13 January 2022).

Domain Identification and Conserved Motifs Analysis of CDPKs
The conserved motifs of CDPKs were analyzed by MEME (https://meme-suite.org/ meme/ accessed on 29 January 2022), the maximum number of motifs parameter was set to 10.

Protein Interaction Network of CDPKs
The protein interaction network of CDPKs was predicted by STRING (https://cn. string-db.org/, accessed on 25 January 2022) based on Arabidopsis homologous proteins. The network map was built by Adobe Illustrator CC2019 software (Adobe Systems Incorporated, San Jose, CA, USA).

qRT-PCR Analysis of CDPKs
The salt-tolerant sweet potato (I. batatas) line 'ND98' was used for qRT-PCR analysis in this study [45]. In vitro-grown ND98 plants were cultured on Murashige and Skoog (MS) medium at 27 ± 1 • C under a photoperiod consisting of 13 h of cool-white fluorescent light at 54 µmol m -2 s -1 and 11 h of darkness. Sweet potato plants were cultivated in a field at the campus of China Agricultural University, Beijing, China.
For expression analysis in various tissues, total RNA was extracted from the pigmented root, tuberous root, stems, leaves, and petioles tissues of 3-month-old field-grown ND98 plants using the TRIzol method (Invitrogen). For expression analysis of hormone and abiotic treatment, the leaves were sampled at 0, 1, 3, 6, 12, and 48 h after being treated with 200 mM NaCl, 20% polyethylene glycol (PEG) 6000, 10 mM H 2 O 2 , 4 • C, 100 µM ABA, 100 µM GA, 100 µM MeJA, and 100 µM IAA, respectively. Three independent biological replicates were taken, each with three plants. qRT-PCR was conducted using the SYBR detection protocol (TaKaRa, Kyoto, Japan) on a 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The reaction mixture was composed of first-strand cDNA, primer mix, and SYBR Green M Mix (TaKaRa; code RR420A) to a final volume of 20 µL. A sweet potato actin gene (GenBank AY905538) was used as an internal control. The relative gene expression levels were quantified with the comparative C T method [66]. The specific primers of qRT-PCR analysis are listed in Table S2. The heat maps of gene expression profiles were constructed using TBtools software (v.1.098696) [62].

Transcriptome Analysis
The RNA-seq data of ItfCDPKs and ItbCDPKs in I. trifida and I. triloba were downloaded from the Sweetpotato Genomics Resource (http://sweetpotato.plantbiology.msu.edu/ accessed on 20 January 2022). The expression levels of CDPKs were calculated as fragments per kilobase of exon per million fragments mapped (FPKM). The heat maps were constructed by TBtools software (v.1.098696) [62].

Conclusions
Here, we identified 39, 35, and 35 CDPKs in cultivated hexaploid sweet potato and its two diploid relatives, I. trifida and I. triloba, respectively. There were differences in chromosome localization, phylogenetic relationship, and gene structure of these 109 CDPKs. The expression profiles of the identified CDPKs indicated that CDPKs showed tissue specificity and various expression patterns in sweet potato and its two diploid relatives. These results indicated that homologous CDPKs might be involved in distinct hormone crosstalk and abiotic stress responses to regulate plant growth and development. Moreover, the identification of interacting proteins of each CDPK might be their phosphorylation targets to help identify the mechanism. This work provided valuable insights into the structure and function of CDPK genes and provided more potential candidate genes for improving field and abiotic stress tolerance in sweet potato and its two diploid relatives.