Introduction
============

Organism evolution has led to the formation of complex nutrient absorption and transport systems, including ion channels, ion pumps, and carriers. It has been previously shown that these systems respond to endogenous and abiotic stimuli ([@ref-47]). The cyclic nucleotide-gated channel (CNGC) is a Ca^2+^-permeable cation transport channel, and it has been suggested that it is one of the fundamental mechanisms in organism systems ([@ref-61]; [@ref-43]). Secondary messengers, such as cyclic nucleotide monophosphates (3′,5′-cAMP and 3′,5′-cGMP) and Ca^2+^/calmodulin (CaM), can regulate CNGCs by acting as molecular switches. The CNGCs are activated by directly binding cyclic nucleotides and are inhibited when CaM binds to the CaM binding domain ([@ref-47]; [@ref-6]; [@ref-13]; [@ref-28]).

In plants, CNGCs are composed of six transmembrane (TM) domains and one pore region between the fifth and sixth TM domains. The cyclic nucleotide-binding domain (CNBD) is a highly conserved region and has a phosphate-binding cassette (PBC) and a hinge region ([@ref-47]). Unfortunately, although the existence of these domains is necessary for CNGC function, they cannot be used to identify a CNGC protein because other ion transporters such as potassium AKT/KAT channels (Shaker type) also contain both a CNBD and a TM domain ([@ref-51]; [@ref-9]). However, previous studies have proposed that a plant CNGC-specific motif, \[LIMV0\]-X(2)-\[GSANCR\]-X-\[FVIYASCL\]-X-G-X(0,1)-X(0,1)-\[EDAQGH\]-L-\[LIVFA\]-X-\[WRCMLS0\]-X-\[LMSIQAFT0\]-X(7,37)-\[SAC\]-X(9)-\[VTIALMS\]-X(0,1)-\[EQDN\]-\[AGSVT\]-\[FYL\]-X-\[LIVF\], in the PBC and hinge region within the CNBD of CNGC proteins only exists in plant CNGCs and does not occur in other ion transporters ([@ref-62]; [@ref-47]).

The first plant CNGC was identified in *Hordeum vulgare* and named *HvCBT1* ([@ref-48]). The use of bioinformatics tools has led to the identification of the *CNGC* gene family members in *Arabidopsis (20)*, rice (16) and other plants (18 in tomato and 26 in *Brassica oleracea*) ([@ref-7]; [@ref-43]; [@ref-57]; [@ref-63]; [@ref-46]; [@ref-8]; [@ref-62]; [@ref-21]; [@ref-27]).

Previous studies have shown that CNGCs are key components of plant development. Most *CNGC* s have been characterized by genetic methods. They have been shown to play vital roles related to plant physiological and molecular functions, such as multiple physiological processes involved in signal pathways, plant development, and responses to environmental stresses. For example, *Arabidopsis CNGC7* and *CNGC8* are essential for male reproductive fertility ([@ref-54]); *CNGC16* and *CNGC18* participate in pollen development ([@ref-55]; [@ref-18]; [@ref-20]); *AtCNGC2* is involved in jasmonic acid induced apoplastic Ca^2+^ influx in epidermal cells ([@ref-37]; [@ref-56]); and *Arabidopsis CNGC6*, *CNGC19*, and *CNGC20* are involved in abiotic stress response ([@ref-30]; [@ref-19]). *Arabidopsis* CNGC structures have six TM domains and a pore domain. They also possess a cyclic nucleotide-binding domain and CaM-binding domain in the C-terminus. These various domains have diverse functions ([@ref-53]; [@ref-10]; [@ref-25]; [@ref-29]). For example, *AtCNGC2* plays a key role in stress signaling pathways, including changes to the cytosolic free Ca^2+^ in *Arabidopsis*. In contrast, *CNGC4* is permeable to K^+^ and Na^+^, and is activated by both cGMP and cAMP ([@ref-4]; [@ref-1]).

In recent years, efforts had been made to study the *CNGC* gene family in plants. However, there have been few studies on the maize *CNGC* gene family, even though maize is an important food crop and a source of industrial materials worldwide. This study used maize genome-wide sequence information, research information on *Arabidopsis* and rice CNGC families, and comprehensive bioinformatics analysis techniques to conduct a genome-wide identification of CNGCs in maize. To the best of our knowledge, this is the first systematic study of *CNGC* genes in maize and provides the basis for future research on the *ZmCNGC* gene family.

Materials and Methods
=====================

Identification of *CNGC* genes in the maize genome
--------------------------------------------------

A total of 20 *Arabidopsis* and 16 rice *CNGC* protein sequences were retrieved from the *Arabidopsis* Information Resource (TAIR10) database (<http://www.arabidopsis.org/>) and the Rice Genome Annotation Project (RGAP) database (<http://rice.plantbiology.msu.edu/>), respectively. This information was then used to identify the *CNGC* genes in maize. Two methods were utilized to search the maize protein sequences. One used a Hidden Markov Model (HMM) to search against maize protein sequences and the other used the local BLASTP method with a threshold *e*-value \<1e−5. After the searches were conducted, a manual correction was performed to remove any redundancy and proteins without PBC and hinge regions within the CNBD of the CNGC proteins. To further confirm whether the ZmCNGC proteins contained the CNBD domain, the putative ZmCNGC protein sequences were submitted to the SMART ([@ref-35]) and NCBI-CDD databases ([@ref-39]). The proteins without CNBD domains or with amino acid (aa) numbers below 200 were removed and the ZmCNGCs confirmed. Another 11 gramineae plant CNGCs were identified by applying the same method as that described above.

The PI (theoretical isoelectric point), MW (molecular weight), and GRAVY (grand average of hydropathy) of the ZmCNGCs were predicted by ExPASy ([@ref-2]). CELLO v.2.5 software was used to predict the subcellular location of the ZmCNGCs ([@ref-59]). Information about the chromosome distribution of ZmCNGCs and genetic sequences, such as DNA sequences, CDS, cDNA, and up-stream 1500 base pair (bp) DNA sequences obtained from a BLASTN search of the Ensembl Plant database ([@ref-5]).

Multiple alignments, phylogenetic analysis and gene duplication analysis
------------------------------------------------------------------------

Multiple sequence alignments were performed using the T-COFFEE method ([@ref-14]) and visualized by ESPript using the default program setting ([@ref-45]). A maximum likelihood (ML) phylogenetic tree was constructed using the MEGA 7 software program with 1,000 bootstrap replications and the Jones-Taylor-Thornton model ([@ref-31]). To further validate the accuracy of the ML tree, an un-rooted phylogenetic tree was **c**onstructed with 1,000 bootstrap replications using the MEGA 7 software program and was based on full-length protein sequence alignments. The ML analysis of 12 gramineae plants was performed using the IQTree program with the LG  + G4 and state frequencies were determined from the amino acid matrix and other default parameters ([@ref-32]). The tree was visualized by Evolview online ([@ref-22]). Segmental duplication between maize genes and the synteny block between maize and *Sorghum*, and rice and *Brachypodium* were obtained from the Plant Genome Duplication database ([@ref-33]). The substitution rates (Ka/Ks) for duplication events were calculated by the DnaSP v5 software program ([@ref-36]), and the divergence times (Mya) were calculated using the formula: Mya = Ks ∕2*λ* × 10^−6^, where *λ* = 6.5 × 10^−9^ ([@ref-38]).

Gene structure and conserved motif analyses
-------------------------------------------

The gene structures (exon-intron) of the *ZmCNGC* genes were determined by the Gene Structure Display Server ([@ref-24]) using the CDS and DNA sequences from the *ZmCNGC* genes. The conserved motifs of the ZmCNGC proteins were subjected to the MEME Suite web server ([@ref-3]) with the maximum number of motifs set at 10 and an optimum width range for the motifs of 6--200 aa.

Cis-acting regulatory elements and the prediction of protein--protein interaction in ZmCNGCs
--------------------------------------------------------------------------------------------

The up-stream 1,500 bp DNA sequences in the *ZmCNGC* genes were used to locate cis-acting regulatory elements by the 'Signal Scan Search' programs in the NEW PLACE database ([@ref-23]). The interactions between ZmCNGCs and other maize proteins were predicted using the STRING 10 online program ([@ref-52]) and visualized by the Cytoscape v3.4.0 software program ([@ref-50]).

*ZmCNGC* gene expression profiles and network interaction analysis
------------------------------------------------------------------

To understand the expression of *ZmCNGC* genes in different tissues, two high-throughput datasets for of maize were obtained from the Expression Atlas datasets (<https://www.ebi.ac.uk/gxa/home/>) under accession numbers E-MTAB-3826 and E-MTAB-439. These data were used to analyze the expression of *ZmCNGC* genes in six different tissues (ear, embryo, endosperm, pollen, root, and tassel) and at different developmental stages (embryo, endosperm, and seed). The FPKM values were used to calculate the *ZmCNGC* genes expressions and these were visualized by OmicShare tools, which is a free online platform for data analysis (<http://www.omicshare.com/tools>).

Results
=======

Identification of the *CNGC* genes in maize
-------------------------------------------

A total of 20 *Arabidopsis* and 16 rice CNGC protein sequences which had been BLAST aligned against maize protein sequences, were used to obtain an overview of the *CNGC* genes in maize. After BLAST alignment, 18 putative *ZmCNGC* genes were identified in the maize genome. The 18 putative *ZmCNGC* genes were confirmed by using SMART and NCBI CDD to determine whether they contained the CNGC-specific domains (CNBD and TM). After removing redundant genes, 12 *ZmCNGC* genes were finally identified, which was lower than the number of *CNGC* genes in rice and *Arabidopsis* ([@ref-44]; [@ref-60]). The predicted *ZmCNGC* genes were designated as ZmCNGC1 to ZmCNGC12 based on their family classification ([Table 1](#table-1){ref-type="table"}). To further confirm the existence of the ZmCNGCs, we identified all the expressed sequence tags (ESTs) that had aligned to the *ZmCNGC* genes by using the BLASTN program provided by the NCBI. The results demonstrated that only ZmCNGC3 had showed no EST hits, whereas the other ZmCNGCs had more than 13 representative matches to ESTs. Five of them were located on chromosome 5, whereas the others were unevenly located on chromosomes 1, 2, 4, 6, 7, and 8. The characteristic features of these 12 *ZmCNGC* genes are listed in [Table 1](#table-1){ref-type="table"}. The ZmCNGC protein lengths ranged from 326 to 745 aa with an average of 612 aa. The molecular weights of these proteins ranged from 38.63 kDa (ZmCNGC2) to 85.52 kDa (ZmCNGC8) and the calculated pI values ranged from 8.92 (ZmCNGC4) to 9.75 (ZmCNGC12). Subcellular localization prediction analysis showed that all the ZmCNGCs were localized in the plasma membrane except for ZmCNGC3 which was localized in the nuclear fraction. These results are similar to *Arabidopsis* ([@ref-34]).

10.7717/peerj.5816/table-1

###### Characteristic features of *ZmCNGC* genes in maize.

![](peerj-06-5816-g006)

  Group   Gene ID    Gene name       Chr[^a^](#table-1fn1){ref-type="fn"}   Start[^b^](#table-1fn2){ref-type="fn"}   end[^c^](#table-1fn3){ref-type="fn"}   Length (aa)[^d^](#table-1fn4){ref-type="fn"}   MW (Da)[^e^](#table-1fn5){ref-type="fn"}   pI[^f^](#table-1fn6){ref-type="fn"}   GRAVY[^g^](#table-1fn7){ref-type="fn"}   Localization[^h^](#table-1fn8){ref-type="fn"}   EST[^i^](#table-1fn){ref-type="fn"}
  ------- ---------- --------------- -------------------------------------- ---------------------------------------- -------------------------------------- ---------------------------------------------- ------------------------------------------ ------------------------------------- ---------------------------------------- ----------------------------------------------- -------------------------------------
  I       ZmCNGC1    GRMZM2G148118   4                                      230194909                                230205383                              701                                            79914.66                                   9.18                                  −0.046                                   PlasmaMembrane                                  17
          ZmCNGC2    GRMZM2G129375   6                                      109824359                                109825921                              626                                            38405.22                                   9.17                                  −0.484                                   PlasmaMembrane                                  23
          ZmCNGC3    GRMZM2G066269   4                                      230382717                                230384596                              329                                            38632.18                                   9.59                                  −0.52                                    Nuclear                                         0
  II      ZmCNGC4    GRMZM2G023037   2                                      5966501                                  5978989                                723                                            82856.52                                   8.92                                  −0.114                                   PlasmaMembrane                                  29
          ZmCNGC5    GRMZM2G077828   5                                      17453069                                 17455739                               699                                            80110.81                                   9.42                                  −0.091                                   PlasmaMembrane                                  15
  III     ZmCNGC6    GRMZM2G005791   5                                      218834435                                218840917                              700                                            80057.39                                   8.97                                  −0.078                                   PlasmaMembrane                                  31
          ZmCNGC7    GRMZM2G068904   5                                      191609046                                191611784                              689                                            80038.95                                   9.8                                   −0.117                                   PlasmaMembrane                                  15
          ZmCNGC8    GRMZM2G135651   7                                      150652512                                150657060                              739                                            85523.26                                   9.33                                  −0.148                                   PlasmaMembrane                                  61
  IVa     ZmCNGC9    GRMZM2G141642   5                                      217119224                                217125465                              463                                            53303.64                                   9.48                                  −0.059                                   PlasmaMembrane                                  13
  IVb     ZmCNGC10   GRMZM5G858887   5                                      6938133                                  6943695                                745                                            83672.62                                   9.46                                  0.081                                    PlasmaMembrane                                  84
          ZmCNGC11   GRMZM2G074317   1                                      283401507                                283408034                              730                                            81440.82                                   9.43                                  0.067                                    PlasmaMembrane                                  74
          ZmCNGC12   GRMZM2G090528   8                                      177244867                                177247281                              505                                            57042.47                                   9.75                                  0.014                                    PlasmaMembrane                                  48

**Notes.**

chromosome location.

star of gene location.

end of gene location.

the length of ZmCNGC proteins.

the molecular weight of ZmCNGC proteins.

the Grand average of hydropathicity of ZmCNGC proteins.

the prediction the ZmCNGCs subcellular location.

expressed sequence tags blast results.

Multiple alignments of maize CNGCs and potassium AKT/KAT channel genes
----------------------------------------------------------------------

Many ion transporters other than CNGCs also have a CNBD in the C-terminus and a hexa-TM in the N-terminus. For example, potassium AKT/KAT channels (Shaker type) also contain a CNBD and a TM domain. All AKT/ KAT-type channels consist of six TM regions with one P region ([@ref-51]). Therefore, 11 maize AKT/KAT proteins were obtained from the NCBI resource and aligned with the 12 ZmCNGC protein sequences ([File S1](#supp-1){ref-type="supplementary-material"}). The results showed that the proteins were highly conserved and that all of them contained six TM domains (S1-S6) and a pore region. The PBC and hinge domain were also highly conserved. A ML phylogenetic tree indicated that maize CNGC and AKT/KAT-type channel genes were clustered into two separate sections ([File S1](#supp-1){ref-type="supplementary-material"}).

The CNBD is a gene structural feature element in plant CNGCs that contains the PBC and hinge region ([@ref-15]). [Figure 1](#fig-1){ref-type="fig"} illustrates that within the maize PBCs, a conserved phenylalanine (F), a stabilizing glycine (G) and an acidic residue (D or E), and two aliphatic leucines (L) were 100% conserved inside the PBCs. Additionally, aromatic phenylalanine (F) and leucine (L) were 100% conserved within the hinge region. These two conserved regions occurred between the CNBD and CaMBD regions. Based on corresponding alignments with other plants, a stringent motif (L-X(2)-G-\[ED\]-ELL-\[TSG\]-W-\[ACY\]-L-X(10,20)-\[SA\]-X-T-X(7)-\[EQ\]-\[AG\]-F-X-L) which was recognized in the 12 maize CNGCs that included the PBC and hinge domain, was found to be consistent with other plant species ([@ref-47]; [@ref-43]). The maize, rice, and *Arabidopsis* CNGCs were also aligned. The results showed that no positions were specific to the maize CNGC consensus, which suggested that the PBCs and hinge domain were highly conserved among plants ([File S2](#supp-2){ref-type="supplementary-material"}).

![The maize CNGC-specific motif spans the putative PBC and the hinge within the CNBD of the 12 ZmCNGCs.\
The diagram at the top represents three regions of plant CNGCs: the six transmembrane domains (TM), a CNBD containing a PBC and the hinge, and CaMBD. The maize CNGC-specific amino acid motif is shown below the cartoon. The asterisk indicate 100% identity among the 12 ZmCNGCs. Below is the alignment of CNBD domains of 12 ZmCNGCs. Residues in blue and yellow highlighted indicate the PBC and hinge domain, respectively.](peerj-06-5816-g001){#fig-1}

Phylogenetic and duplication analyses of ZmCNGCs
------------------------------------------------

Large phylogenetic trees with minimal homologous characters increase the likelihood of confounding relationships between different species. Therefore, we constructed a ML phylogenetic tree based on the alignment of CNGC proteins from 12 gramineae plants. The tree included 24 CNGC proteins in *Aegilops tauschii*, 16 in *Brachypodium distachyon*, 20 in *Hordeum vulgare*, 23 in *Leersia perrieri*, 21 in *Nicotiana attenuata*, 16 in *Oryza sativa,* 28 in *Setaria italic*, 13 in *Sorghum bicolor*, 79 in *Triticum aestivum*, 21 in *Triticum urartu*, and 12 maize CNGCs identified by this study ([Table S1](#supp-5){ref-type="supplementary-material"}). The phylogenetic tree ([File S4](#supp-4){ref-type="supplementary-material"}), was used to cluster these plant CNGC proteins into six groups, which were named Groups I, II, III, IV, IVa, and IVb, all of which had significant bootstrap values. The results showed that the CNGC proteins from *B. distachyon*, *O. sativa*, *S. bicolor*, and maize did not cluster in Group IV, and the *N. attenuata* CNGCs did not cluster in Group I. Additionally, the number in each group was unevenly distributed. Group IV was the largest with 86 genes, followed by 69 in Group III, 44 in Group II, 33 in Group IVb, 25 in Group I, and 16 in Group IVa. These data demonstrate that gramineae plant CNGC gene expansion appeared to have occurred unequally during evolution.

To better understand the evolutionary relationship among CNGC proteins, a ML phylogenetic tree was created based on the full-length protein alignments of 12 *ZmCNGC* s, 19 *AtCNGCs* and 16 *OsCNGCs* ([@ref-43]; [@ref-40]). The phylogenetic tree shows that the 47 CNGC proteins could be classified into five groups with significant bootstrap values ([Fig. 2](#fig-2){ref-type="fig"}). These were named Groups I, II, III, IVa, and IVb. This was consistent with what has been previously reported for flowering plant CNGCs ([@ref-47]). Group I contained three maize *CNGC* genes (*ZmCNGC1*, *ZmCNGC2*, and *ZmCNGC3*), five *Arabidopsis*, and two rice *CNGC* genes; Group II contained two maize (*ZmCNGC4* and *ZmCNGC5*), five *Arabidopsis*, and three rice *CNGC* genes. Group III contained three maize *CNGC* genes (*ZmCNGC6*, *ZmCNGC7*, and *ZmCNGC8*); and Group IV contained five rice and four *Arabidopsis CNGC* genes. The maize genes in Group IV were separated into two groups, Group IVa contained *ZmCNGC9* and Group IVb contained three (*ZmCNGC10*, *ZmCNGC11*, and *ZmCNGC12*).

![Phylogenetic relationships among the ZmCNGCs, OsCNGCs and AtCNGCs.\
The multiple alignment was performed by ClustalX program. MEGA 7.0 was used to create maximum likelihood (ML) under the Jones-Taylor-Thornton (JTT) model. The tree was constructed with 1,000 bootstrap replications. Each group identified is indicated on the right.](peerj-06-5816-g002){#fig-2}

The phylogenetic tree also showed that the maize *CNGC* genes could also be grouped into five groups ([Fig. 3A](#fig-3){ref-type="fig"}). Consistent with other plant *CNGC* genes, Group IVa contained only one or two gene members and formed the smallest group ([@ref-47]). The results suggested that during the evolution of CNGCs, Group IV *CNGC* genes may have emerged first whereas Groups I and II *CNGC* genes appeared later. Furthermore, the tree topology produced by the neighbor joining analyses was the same as the ML tree in [Fig. 2](#fig-2){ref-type="fig"}, and it contained all the groups ([File S3](#supp-3){ref-type="supplementary-material"}).

![Phylogenetic relationships (A), motif compositions (B), gene structure (C), and (D) motif logo of ZmCNGCs.\
The maximum likelihood (ML) tree under the Jones-Taylor-Thornton (JTT) model was constructed with 1,000 bootstrap replications using MEGA7 based on the full-length protein sequence. The exon-intron structures of these genes were graphically displayed by the Gene Structure Display Server using the CDS and genome sequence of ZmCNGC genes. The protein sequences of ZmCNGC genes were used to predict the conserved motifs by using the MEME Suite web server.](peerj-06-5816-g003){#fig-3}

Additionally, only one segmental duplication gene pair, *ZmCNGC10*-*ZmCNGC11*, was formed in the maize genome ([Table 2](#table-2){ref-type="table"}). The evolutionary process between maize CNGCs and other gramineae plants was further explored by investigating the genome synteny among *Sorghum*, rice, and *Brachypodium*. The results showed that there were two, two and one *ZmCNGC* genes that showed syntenic bias towards particular *Sorghum*, rice, and *Brachypodium* chromosomes, respectively ([Table 2](#table-2){ref-type="table"}). In addition, Ka/Ks was used to evaluate their specific positions under positive selection pressure after duplication ([@ref-41]). Ka/Ks values that =1, \<1 or \>1 indicate neutral, purifying, and positive selection, respectively ([@ref-38]). The Ka/Ks value of each gene pair was calculated and the Ka/Ks value for all gene pairs was less than 1, which suggested that these genes had evolved under strong purifying selection. Furthermore, the results indicated that the divergence of maize CNGCs from other gramineae plants did not occur evenly.

10.7717/peerj.5816/table-2

###### The Ka/Ks ratios and estimated divergence time for orthologous CNGC proteins between maize and other Gramineae plants.

![](peerj-06-5816-g007)

  Gene ID    Gene ID            Ka       Ks       Ka/Ks      Mya
  ---------- ------------------ -------- -------- ---------- ----------
  ZmCNGC11   ZmCNGC10           0.0258   0.1829   0.141061   14.06923
  ZmCNGC5    Sobic.001G155100   0.5298   0.8917   0.594146   68.59231
  ZmCNGC12   Sobic.009G188800   0.095    0.4878   0.194752   37.52308
  ZmCNGC12   Sobic.003G317700   0.0306   0.0993   0.308157   7.638462
  ZmCNGC5    LOC_Os03g44440     0.7415   1.2253   0.605158   94.25385
  ZmCNGC12   LOC_Os05g42250     0.1187   0.4278   0.277466   32.90769
  ZmCNGC12   LOC_Os01g57370     0.073    0.3017   0.241962   23.20769
  ZmCNGC5    Bradi1g13740       0.7912   1.1721   0.675028   90.16154

*ZmCNGC* gene structures and the conserved motif analyses
---------------------------------------------------------

Gene structure analysis could improve our understanding of gene function and evolution. The number of introns ranged from 0 to 7 ([Fig. 3B](#fig-3){ref-type="fig"}), which was different from rice and *Arabidopsis* CNGCs. In rice, *the OsCNGC* s introns numbers ranged from 1 to 11, whereas *Arabidopsis* CNGCs ranged from 4 to 10 ([@ref-43]). The Group IVa and IVb ZmCNGCs had distinct gene structures compared to those of the other groups, with more introns at different phases and lengths, which is consistent with most flowering plant species Group IV *CNGC* genes ([@ref-47]).

The motif-based recognition of proteins can improve understanding of their the evolutionary history ([@ref-49]). Ten putative motifs were characterized and named as motif1 to motif10 in the ZmCNGCs. The relative positions of the motifs in the five groups were found to have various patterns ([Figs. 3C](#fig-3){ref-type="fig"} and [3D](#fig-3){ref-type="fig"}), for example, all the ZmCNGCs contained motif1, motif2, motif3, and motif4, which meant they presented typical ZmCNGC domains. Motif3 was a combination of the calmodulin binding (CaMB) domain and the IQ domain (QWRTWAA\[CV\]FIQ\[AL\]AW\[RH\]RY), and motif4 was the cyclic nucleotide-binding (CNB) domain, which was located in the C-terminal. Furthermore, motif10, 9, 5, 8 (or 6), 2, and 1 were the motif logos of the transmembrane domains. They represented the S1, S2, S3, S4, S5, and S6 regions of the transmembrane domain in the N-terminal. Motif7 represented the ion transport protein (ITP) domain. In addition, 10 of the ZmCNGCs, but not *ZmCNGC2* and *ZmCNGC3*, possessed motif 5 and 6 that are associated with ion transport ([@ref-43]). Although other motifs have not been reported in plants or animals, they undertake important functions within the organism.

Prediction of cis-acting regulatory elements and protein-protein interactions that involve ZmCNGC proteins
----------------------------------------------------------------------------------------------------------

Cis-acting regulatory elements are important molecular switches that are associated with the transcriptional regulation of genes when environmental stresses are encountered ([@ref-42]). To better understand the possible biological processes undertaken by the ZmCNGCs involved, 1.5 kb sequences upstream of the *ZmCNGC* gene genomic sequences were used to identify cis-regulatory elements. A total of 137 different putative cis-elements were found to be associated with the identified *ZmCNGC* genes and only 12, including CACTFTPPCA1, EBOXBNNAPA, DOFCOREZM, MYCCONSENSUSAT, CAATBOX1, GTGANTG10, WRKY71OS, GT1CONSENSUS, ROOTMOTIFTAPOX1, POLLEN1LELAT52, MYBCORE, and OSE2ROOTNODULE, were in the promoter region of the *ZmCNGC* genes ([Table S2](#supp-5){ref-type="supplementary-material"}), which was highly consistent with rice CNGCs. Additionally, five cis-elements were gene-specific. ACGTCBOX, TATABOX3, CTRMCAMV35S, and HDZIP2ATATHB2 were unique to *ZmCNGC6*, *ZmCNGC7*, *ZmCNGC8*, and *ZmCNGC11*, respectively. Additionally, some cis-elements were involved in responses to different abiotic/biotic stimuli, including hormones (e.g., abscisic acid, auxin, ethylene), stress (e.g., drought, temperatures, disease) and development (e.g., mesophyll specific, tissue specific) responses, which indicated that these *ZmCNGC* genes might be involved in regulating diverse stress responses.

The ZmCNGC protein-protein interactions were predicted in order to gain a better understanding of ZmCNGC protein roles in the plant. A total of 120 protein pairs were predicted to interact between the 12 ZmCNGC proteins and the other 11 maize proteins, and ZmCNGC8 was found to interact with *ZmCNGC10* by prediction analysis ([Fig. 4](#fig-4){ref-type="fig"} and [Table S3](#supp-5){ref-type="supplementary-material"}). We also used genes in *Arabidopsis* that were homologous to the *ZmCNGC* genes to predict the protein-protein interactions. The results showed that three were validated by the experimental data ([Table S3](#supp-5){ref-type="supplementary-material"}).

![The protein--protein interaction network of ZmCNGC genes in maize.\
The protein--protein interaction network were constructed based on STRING database.](peerj-06-5816-g004){#fig-4}

Expression profiles of *ZmCNGC* genes in different tissues
----------------------------------------------------------

The physiological function of *ZmCNGC* genes was investigated using transcriptome sequencing which evaluated the tissue-specific expression levels of *ZmCNGC* genes in different tissues (see Materials and Methods section). The expression levels among the *ZmCNGC* genes are shown in [Fig. 5A](#fig-5){ref-type="fig"} and [Table S4](#supp-5){ref-type="supplementary-material"} suggested that their expression levels were tissue-specific. For example, *ZmCNGC5* was specifically expressed in pollen compared to other tissues, which implied that it played a particular role in pollen physiological development, whereas *ZmCNGC2*, *ZmCNGC4*, *ZmCNGC6*, and *ZmCNGC8* had higher expression levels in the roots than in the other tissues, which suggested that they had important roles in root growth and development. All Group IVb *ZmCNGC* genes, including *ZmCNGC10*, *ZmCNGC11*, and *ZmCNGC12*, were relatively highly expressed in the embryo, which implied that these genes played crucial roles in the growth and development of the maize embryo.

![Expression profiles of ZmCNGCs in six different tissues and different development stages in embryo, endosperm and seed after pollination, respectively.\
(A) Expression profile in six different tissues, including ear, embryo, endosperm, pollen, root and tassel. Expression profile in (B) embryo, (C) endosperm and (D) seed of some days after pollination.](peerj-06-5816-g005){#fig-5}

We also evaluated the expression of several *ZmCNGC* genes in the embryo, endosperm, and seed for several days after pollination. *ZmCNGC3*, *ZmCNGC5* and *ZmCNGC7* were not detected or had no expression in any of the tissues. However, *ZmCNGC5* was expressed in pollen ([Figs. 5A](#fig-5){ref-type="fig"}, [5B](#fig-5){ref-type="fig"}, [5C](#fig-5){ref-type="fig"}, and [5D](#fig-5){ref-type="fig"}). Cis-acting regulatory elements analysis showed that only *ZmCNGC5* and *ZmCNGC7* did not contain CANBNNAPA, which is the element required for embryo and endosperm-specific transcription ([@ref-16]). This might be the reason why they did not show any expression in these tissues. The embryo specific-expression gene *ZmCNGC10* gradually increased over time in the embryo ([Fig. 5B](#fig-5){ref-type="fig"}). *ZmCNGC8* was highly expressed in the embryo, endosperm, and seeds after pollination, and the Group IVb gene showed a similar expression pattern.

Discussion
==========

Features and evolution of plant CNGC family genes
-------------------------------------------------

Plant CNGC family genes are characterized by the presence of a CNBD in the C-terminal and a hexa-TM in the N-terminal ([@ref-47]). A total of 12 *ZmCNGC* genes were identified after a BLAST search against the maize genome protein sequences. Among them, ZmCNGC3 showed no ESTs and was not expressed in all the tissues, which suggested that it was a non-expressed pseudogene. In *Arabidopsis*, AtCNGC16 (AT3G48010) had no EST alignments and might also be a pseudogene ([@ref-40]). Many other ion transporters also possess these domains. For example, potassium AKT/KAT channels (Shaker type) contain both a CNBD domain and a TM domain. All AKT/ KAT-type channels consist of six transmembrane (TM) regions with one P region ([@ref-51]). The ML phylogenetic tree showed that the maize CNGC and the AKT/KAT-type channel genes were clustered into two separate sections ([File S1](#supp-1){ref-type="supplementary-material"}). Previous studies have shown that the CNGC-specific motif with a PBC and a hinge domain (L-X(2)-G-\[ED\]-ELL-\[TSG\]-W-\[ACY\]-L-X(10,20)-\[SA\]-X-T-X(7)-\[EQ\]-\[AG\]-F-X-L) only exists in maize plant CNGCs and does not occur in the ion transporters found in other plants, such as rice, *Arabidopsis*, and tomato ([@ref-47]; [@ref-43]; [@ref-62]). In this study, 12 ZmCNGCs were identified and the PBCs and hinge domains were highly conserved after alignment, which further confirmed the previous hypothesis ([@ref-47]). The conserved motif analysis showed that the motif3 (QWRTWAA\[CV\]FIQ\[AL\]AW\[RH\]RY) pattern was the IQ domain among these 12 ZmCNGCs. The IQ domain is conserved among plant CNGCs and enhances the changeable Ca^2+^-dependent channel control mechanisms in plants ([@ref-17]). The results from this study showed that all CNGC proteins contained the IQ motif, which suggested that they bind CaM in a Ca^2+^-dependent manner. Notably, motif4 is the sequence logo of the CNBD domain, which is conserved in most plants and animals ([@ref-26]).

The results showed that the CNGC family in 12 gramineae plants at various evolutionary nodes is adequate for analyzing the phylogeny and evolution of the CNGC gene family in gramineae plants. In [Table S1](#supp-5){ref-type="supplementary-material"} shows that 4 out of 12 plants under analysis contained \<20 CNGC genes. These were *B. distachyon* (16), *O. sativa* (16), *S. bicolor* (13) and maize (12). The ML tree for the 273 CNGCs clearly indicated that gramineae CNGCs clustered into six groups (I, II, III, IV, IVa, and IVb) with significant bootstrap values; Groups IV, IVa and IVb had particularly high bootstrap support values, but group IV CNGCs did not cluster with *B. distachyon*, *O. sativa*, *S. bicolor*, and maize CNGCs. This suggested that there are missing CNGCs that need to be identified or there was duplication during evolution. [@ref-12] showed that gene duplication can lead to the generation of significant numbers of novel genes. Therefore, these results imply that duplication events play a principal role in gene evolution. Phylogenetic, gene structure, and conserved motifs analyses were used to classify the 12 *ZmCNGC* s into five groups with significant bootstrap values. Among these five groups, Group IV *ZmCNGC* s emerged first and had more introns than the genes in the other groups.

*CNGC* genes play an important role in plant development
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The CNGCs are involved in the regulation of plant growth and development ([@ref-11]). This study focused on the role that maize CNGCs play in different tissues by investigating its expression in plant embryo, endosperm and seed expression. The results showed that some *ZmCNGC* genes have tissue-specific expression. For example, *ZmCNGC2*, *ZmCNGC4*, *ZmCNGC6* and *ZmCNGC8* were highly expressed in the roots; *ZmCNGC5* was specifically expressed in pollen; and all Group IVb genes were expressed in the embryo. Group IVb genes are the evolutionary ancestors of CNGC genes and were mainly expressed in the embryo and seeds after pollination, which suggested that Group IVb genes play a significant role in embryo development. In addition, all Group IVb genes were linked to gene duplication and they had a similar expression pattern in the different tissues, which indicated that their function was to enhance adaptability during gene evolution.

Most previous studies have shown that *CNGC* genes are related to pollen development and responses to environmental stimuli. For example, *Arabidopsis CNGC16* is critical for pollen fertility under heat and drought stress ([@ref-55]), and *CNGC18* has been shown to play a role in pollen tube tip growth ([@ref-18]). In rice, *OsCNGC13* promotes the seed-setting rate by facilitating pollen tube growth in stylar tissues ([@ref-58]). *ZmCNGC1* and *ZmCNGC5*, two homologous genes of *CNGC16* and *CNGC18*, are mainly expressed in pollen, which indicates that they are predominantly involved in pollen development. Previous studies have shown that *AtCNGC3* is mainly expressed in the embryo, leaves, and roots, and *ZmCNGC4* expression level is similar to that of *AtCNGC3* which was highly expressed during plant development ([@ref-28]). These results imply that these genes play crucial roles during the growth and development of maize.

Conclusion
==========

A total of 12 CNGC genes were identified in maize via bioinformatics. The results were based on the presence of a plant CNGC-specific motif that spanned the PBCs and hinge domain in the CNBD of CNGC proteins. Phylogenetic analyses showed that Group IV ZmCNGCs emerged first and had more introns than the other ZmCNGC groups, whereas Groups I and II evolved later. Phylogenetic analysis of 12 gramineae plants revealed that some CNGCs are probably missing or have been duplicated during evolution. Significantly, the *ZmCNGC* genes had diversity in gene structures, protein lengths and sizes. We modified a maize stringent motif (L-X(2)-G-\[ED\]-ELL-\[TSG\]-W-\[ACY\]-L-X(10,20)-\[SA\]-X-T-X(7)-\[EQ\]-\[AG\]-F-X-L) that contained a PBC and a hinge domain to better characterize classification. Expression profiles of the *ZmCNGC* genes were tissue-specific and were related to pollen development. This study provides important information about plant CNGCs during gene evolution.

Supplemental Information
========================

10.7717/peerj.5816/supp-1

###### A ML phylogenetic tree, conserved regions and multiple sequences alignments between maize AKT/KAT channels genes and ZmCNGCs

The AKT and CNGC indicated maize AKT/KAT channel genes and CNGC genes. S1--S6 indicated six transmembrane (TM) regions with one P region of AKT/KAT or CNGC proteins.

###### 

Click here for additional data file.

10.7717/peerj.5816/supp-2

###### Multiple sequences alignments among maize, Arabidopsis and rice CNGCs by using T-COFFEE method

Residues in highlighted indicate most conserved among these CNGCs.

###### 

Click here for additional data file.

10.7717/peerj.5816/supp-3

###### The un-root neighbor joining (NJ) tree based on the maize, rice and Arabidopsis CNGCs protein sequences by using MEGA7

The multiple alignment was performed by ClustalX program. MEGA 7.0 was used to create the neighbor joining (NJ) under the poisson model. The tree was constructed with 1,000 bootstrap replications.

###### 

Click here for additional data file.

10.7717/peerj.5816/supp-4

###### The Maximum likelihood (ML) tree based on the maize and other 11 gramineous plants CNGCs protein sequences by using IQTREE

The multiple alignment was performed by ClustalX program. IQTREE was used to create the Maximum likelihood (ML) with JTT+LG4 model. The tree was constructed with 1,000 bootstrap replications.

###### 

Click here for additional data file.

10.7717/peerj.5816/supp-5

###### The supplemental information in this study

Table S1. The GNGC information of 12 gramineae plants.

Table S2. Numbers of known stress-related elements in the promoter regions of ZmCNGCs.

Table S3. The protein--protein interaction information of maize CNGC protein with other maize proteins.

Table S4. The FPKM data of ZmCNGC genes in different tissues.

###### 

Click here for additional data file.

10.7717/peerj.5816/supp-6

###### The logo of ten motifs in study

The conserved motifs of ZmCNGC proteins were using the MEME Suite web server (<http://meme-suite.org/index.html>) with the maximum number of motif sets at 10 and optimum width of motifs from 6 to 200 amino acids.

###### 

Click here for additional data file.

10.7717/peerj.5816/supp-7

###### The gene sequences used in this research

Including DNA, CDS, Cdna, protein sequences of ZmCNGCS, and protein sequences of maize AKT/KAT channel genes and other plant CNGC gens.

###### 

Click here for additional data file.

We extend our gratitude to Dr. Lihe Yu for providing valuable suggestions.

Additional Information and Declarations
=======================================

The authors declare there are no competing interests.

[Lidong Hao](#author-1){ref-type="contrib"} conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.

[Xiuli Qiao](#author-2){ref-type="contrib"} conceived and designed the experiments, authored or reviewed drafts of the paper, approved the final draft.

The following information was supplied regarding data availability:

The information of chromosome distribution of ZmCNGCs and the sequences including DNA sequences, CDS, cDNA, up-stream 1,500 bps of ZmCNGC genes were obtained from BLASTN (the Ensembl Plant database, <http://plants.ensembl.org/index.html>): accession numbers GRMZM2G148118, GRMZM2G129375, GRMZM2G066269, GRMZM2G023037, GRMZM2G077828, GRMZM2G005791, GRMZM2G068904, GRMZM2G135651, GRMZM2G141642, GRMZM5G858887, GRMZM2G074317, and GRMZM2G090528.

13 maize AKT/KAT genes were downloaded from Ensembl Plant database with gene ID: C234152.1_FGT002,GRMZM2G020859,GRMZM2G081666,GRMZM2G178356, GRMZM2G310569,GRMZM2G156255,GRMZM2G093313, GRMZM2G022915, GRMZM2G171279,GRMZM2G028258,GRMZM2G342375,GRMZM2G428859, GRMZM5G838773,GRMZM2G095861.

The 20 Arabidopsis and 16 rice CNGC protein sequences were obtained from TAIR10 (<http://www.arabidopsis.org>) and RGAP (<http://rice.plantbiology.msu.edu/>) respectively. Arabidopsis asscession numbers: AT5G53130, AT3G48010,AT2G23980, AT2G24610, AT5G54250, AT2G28260, AT5G15410, AT1G15990, AT4G30560, AT5G57940, AT3G17700, AT1G01340, AT1G19780, AT2G46450, AT4G30360, AT5G14870, AT4G01010, AT3G17690, AT2G46430, AT2G46440, LOC_Os06g33570, LOC_Os06g33610, LOC_Os03g44440, LOC_Os12g28260, LOC_Os04g55080, LOC_Os02g41710, LOC_Os12g06570, LOC_Os09g38580, LOC_Os02g54760, LOC_Os06g08850, LOC_Os02g53340, LOC_Os06g10580, LOC_Os03g55100, LOC_Os01g57370, LOC_Os05g42250.
