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Identification and expression analysis revealed drought stress-responsive Calmodulin and Calmodulin-like genes in maize

ABSTRACT Calmodulin (CaM) and Calmodulin-like (CML) genes are the primary families of Calcium (Ca2+) sensors which are found to be involved in response to various stresses. Some genes involved in Ca2+ signal transduction have been genome-wide characterized in various species. However, the detailed identification, characterization, and expression profilings of ZmCaM and ZmCML genes in maize remain poorly understood, especially in the response to drought stress. In this study, a total of 7 ZmCaMs and 46 ZmCMLs are identified in maize and unevenly located on 10 chromosomes. ZmCaM and ZmCML proteins are divided into 9 groups. Protein structures analysis shows that the EF-hand motif number of ZmCaMs/ZmCMLs ranges from 3 to 4 and 2 to 4, respectively. A large number of cis-regulatory elements are found in the promoter regions of ZmCaM and ZmCML genes. ZmCaM and ZmCML genes display highly diversified tissue-specific expression patterns. Furthermore, ZmCaM2, ZmCML3, ZmCML6, ZmCML8, ZmCML19, ZmCML24, ZmCML27, ZmCML28, ZmCML36, ZmCML39, and ZmCML40 are induced significantly under drought stress through RNA-seq data and RT-qPCR. Taken together, these results will help to understand the critical roles of ZmCaM and ZmCML genes played in drought resistance and provide valuable candidate genes that could be used to develop drought-resistant maize.


Introduction
Plants are subjected to various abiotic and biotic stresses in the process of growth and development, such as pathogens, low or high temperature, and drought stresses (Zhu 2016). In order to adapt to stress conditions, plants have evolved multiple biochemical and physiological processes, including plant hormone regulation and cell signal transduction (Shukla et al. 2019;Berens et al. 2017). Calcium (Ca 2+ ) as an important second messenger plays a key role in the transduction and amplification of cell signals (La Verde et al. 2018). A wide range of external stimuli can induce rapid and transient changes in cytosolic Ca 2+ concentration which are recognized by several types of Ca 2+ binding proteins (Hashimoto and Kudla 2011). Typically, Ca 2+ binding proteins can be divided into four major groups, including calmodulin (CaM), calmodulin-like protein (CML), Ca 2+ dependent protein kinase (CDPK), and calcineurin-B-like protein (CBL) (Heyer et al. 2018;Aldon et al. 2018;Wang et al. 2019). The majority of Ca 2+ binding proteins have EF-hand motifs which are found to bind Ca 2+ . CaM is a highly conserved Ca 2+ binding protein that is composed of approximately 150 amino acid residues with four EF-hand motifs (Chin and Means 2000). Although CaM has no enzymatic function of its own, it can transduce the signals by interacting with other target proteins (Perochon et al. 2011). CMLs are a large subgroup of Ca 2+ binding proteins which normally possess 1-6 EF-hand motifs (Hashimoto and Kudla 2011). Previous studies have shown that CaMs and CMLs do not have any functional domains except for EF-hand motifs . At present, CaMs and CMLs have been identified in many plant species. For example, 7 CaMs and 50 CMLs have been identified in the whole genome of Arabidopsis (McCormack and Braam 2003), 5 CaMs and 32 CMLs have been identified in rice (Boonburapong and Buaboocha 2007), 6 CaMs and 144 CMLs have been identified in soybean (Zeng et al. 2017), and 6 CaMs and 45 CMLs have been identified in Solanum lycopersicum .
CaM and CML genes have been shown to be involved in plant developmental process and response to various stresses. Overexpression of AtCaM1 and AtCaM4 in Arabidopsis results in enhanced leaf senescence (Dai et al. 2018;Koo et al. 2017). AtCML24 and AtCML25 are involved in pollen germination and pollen tube growth (Yang et al. 2014;Wang et al. 2015). AtCaM2 and AtCaM7 play vital roles in pollen germination and seedling development, respectively (Landoni et al. 2010;Kushwaha et al. 2008). The knockout mutants of AtCML9 display enhanced tolerance to drought and salinity stresses (Magnan et al. 2008). AtCML20 is a negative regulator of ABA-induced stomatal movement and drought stress tolerance (Wu et al. 2017). AtCML39 positively regulates ABA-mediated signaling pathway and negatively regulates GA 3 -mediated signaling pathway (Midhat et al. 2018). OsCML4, OsCML5, OsCML8, and OsCML11 in rice are involved in osmotic and salt stresses (Chinpongpanich et al. 2012). OsCML16 positively regulates root growth and drought tolerance under the regulation of transcription factor OsERF48 (Jung et al. 2017). Transgenic tomato plant overexpressing ShCML44 shows increased tolerance to drought and low-temperature stresses ). Overexpression of GsCML27 in Arabidopsis confers enhanced bicarbonate tolerance, while it decreases salt and osmotic tolerance during seed germination and early growth (Chen et al. 2015). The expression level of TaCMl20 is regulated by drought stress, and TaCMl20 overexpression transgenic wheat lines enhance water-soluble carbohydrate accumulation (Kalaipandian et al. 2019). VaCML21 positively regulates the plant tolerance to cold stress through increasing the expression of the cold stress-responsive marker genes (Aleynova et al. 2020). In addition, CaM and CML genes are found to be involved in response to pathogen infection. The expression level of AtCML8 is induced after infection with Pseudomonas syringae . In cotton, GhCML11 positively participates in defense reaction to Verticillium dahliae (Cheng et al. 2016).
Maize (Zea mays) is an important food and economic crop on earth, but its production is seriously constrained by drought stress. Ca 2+ signals play significantly important roles in stress tolerance in maize (Li et al. 2004), and some genes (CAMTA) involved in Ca 2+ signal transduction have been genome-wide characterized (Yue et al. 2015). However, detailed characterization and expression patterns of ZmCaM and ZmCML genes family in maize are largely unknown, especially their potential roles in response to drought stress. Here, we identified 7 ZmCaM and 46 ZmCML genes in the maize B73 genome and performed comprehensive bioinformatics analysis, including molecular characterization, gene structure, phylogenetic classification, chromosome location, and cis-regulatory elements. In addition, the expression levels of ZmCaM and ZmCML genes in different tissues and in response to drought stress were analyzed based on RNA-seq database and real-time quantitative PCR (RT-qPCR). These results will help us to understand the functional characterization of ZmCaM and ZmCML genes in response to drought stress in maize.

Plant materials and stress treatments
The seeds of maize inbred line B73 were provided by Maize Breeding Innovation Team of Jilin Agricultural University and were collected in accordance with relevant national regulations. The seeds of maize were sterilized with 6% sodium hypochlorite for 10 min and rinsed with distilled water. The seeds were grown in germination box for 4-5 days with 16 h light (28°C) and 8 h dark (21°C). For drought treatment, the seedlings were soaked with Polyethylene glycol (PEG) 8000 at a water potential of −0.2 MPa (moderate stress) and −0.8 MPa (severe stress) when the length of the main root reached 2-4 cm (Opitz et al. 2014). The mock-treated plants were treated with sterile water. The roots were sampled at 0, 6, and 24 h, respectively. The whole roots were frozen in liquid nitrogen immediately and stored at −80°C until RNA was extracted. The experiment was performed using three biological replicates with three technical replicates.

Analysis of cis-regulatory elements of ZmCaM and ZmCML genes
The promoter regions up to 2000 bp in the ZmCaMs and ZmCMLs genes were used to analyze potential cisregulatory elements by the online tool PlantCARE (http:// bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Expression pattern analysis of ZmCaM and ZmCML genes
To identify the expression patterns of ZmCaM and ZmCML genes in different tissues, the transcription data of ZmCaM and ZmCML genes were derived from Walley's report (Walley et al. 2016). The RNA-seq data for maize roots with low water potentials were downloaded from the NCBI Gene Expression Omnibus under accession PRJNA226757 (Opitz et al. 2014). FPKM values were used to compare ZmCaM and ZmCML expression levels.

RT-qPCR assay
RNA was extracted by the method of TRIzol and was reverse transcribed into cDNA by TOYOBO kit. RT-qPCR was analyzed using the SYBR Green Supermix kit (Genstar) according to the instruction manual with QuantStudio 3. The housekeeping gene of maize ZmTub (GRMZM2G066191) was used as an internal control to normalize the data. The relative expression level was calculated using the 2 − ΔΔCT method. The experiment was performed using three biological replicates with three technical replicates, and was statistically analyzed using Student's t-test (*p < 0.05, **p < 0.01,). Bars indicate the standard error of the mean. The primers used in the experiment are shown in Table S4.

Gene interaction network of the ZmCaM/ZmCML proteins
The interaction network of ZmCaM/ZmCML proteins was analyzed using online STRING database (https://string-db. org/).

A total of 53 ZmCaMs and ZmCMLs are identified in mazie
To identify the ZmCaM and ZmCML genes in the maize genome, the AtCaM and AtCML genes of Arabidopsis were used as query sequences. A total of 53 ZmCaMs/ZmCMLs were identified, including 7 ZmCaMs and 46 ZmCMLs, which were named ZmCaM1-ZmCaM7 and ZmCML1-ZmCML46, respectively. The coding sequences of ZmCaMs/ZmCMLs were analyzed, including AA, pI, MW, GRAVY, and protein location. The sizes of ZmCaMs/ZmCMLs ranged from 80 (ZmCML2 and ZmCML23) to 398 (ZmCaM5) AA residues. The average molecular weight of ZmCaMs/ZmCMLs was 20.60 kDa, varying between 8.77 (ZmCML23) and 44.88 (ZmCaM5) kDa. The pI of these proteins ranged from 4.17 to 4.87 and 3.89 to 9.12, respectively. Hydrophilicity analysis showed that most ZmCaMs/ZmCMLs are hydrophilic proteins, except ZmCML10, ZmCML29, ZmCML30 and ZmCML32. To further investigate the potential functions of ZmCaMs/ZmCMLs, the subcellular localization was predicted using Cell-PLoc 2.0. We found that all 7 ZmCaM proteins were predicted to be in the cell membrane and cytoplasm. The ZmCML proteins were predicted to be in different organelles, including the nucleus, vacuole, and cell membrane (Table S1). These results indicate that identified ZmCaMs/ ZmCMLs may have diverse functions in Ca 2+ signaling transduction.

ZmCaMs and ZmCMLs are divided into nine groups and carry different numbers of the EF-hand motif
To gain insights into the potential functions of ZmCaMs/ ZmCMLs, the phylogenetic tree was built using the CaM/ CML protein sequences from Arabidopsis, rice, and maize, respectively. The phylogenetic tree showed that ZmCaMs/ ZmCMLs are divided into nine groups, namely groups I-IX, according to their similarities and relationships with Arabidopsis and rice members (Shi and Du 2020;Sun et al. 2020). Groups I-IX contained 2, 3, 12, 4, 3, 9, 7, 3, and 11 members, respectively. All seven ZmCaMs were divided into group I (Figure 1). These results demonstrate that CaM/CML proteins from Arabidopsis, rice, and maize show highly homologous.
EF-hand motif is the main domain of calcium-binding protein, which is found to be involved in binding Ca 2+ . As shown in Figure 2(A), the EF-hand motif number of ZmCaMs/ZmCMLs ranged from 3 to 4 and 2 to 4, respectively. In total, 17, 16, and 12 ZmCMLs were found to have 4, 3, and 2 EF-hand motifs, respectively. In addition, ZmCaM2 had four EF-hand motifs, the remaining ZmCaMs contained 3 EF-hand motifs. These results demonstrate that all identified ZmCaM and ZmCML proteins carry typical EFhand domains, which may have function in binding Ca 2+ . The different numbers of the EF-hand motif may lead to functional differences among ZmCaMs/ZmCMLs.
The gene structures of 53 ZmCaMs/ZmCMLs were analyzed based on the arrangement of exon/introns. The majority of the ZmCaMs/ZmCMLs lacked introns, while 1-7 introns were found in 21 ZmCaMs/ZmCMLs. ZmCML3, ZmCML17, ZmCML27, ZmCML46, ZmCaM3, ZmCaM6, and ZmCaM7 had 1 intron, while ZmCML19 and ZmCML39 contained the largest number of introns, up to 7 ( Figure 2B). These results show that the number of introns in ZmCaMs/ZmCMLs are different, which may exist in gene structure differentiation in the evolution process.
ZmCaM and ZmCML genes are unevenly distributed on 10 chromosomes To determine the distribution of ZmCaMs/ZmCMLs in maize genome, their chromosomal location was analyzed by mg2c (http://mg2c.iask.in/mg2c_v2.0/). The results demonstrated that 7 ZmCaMs and 46 ZmCMLs are distributed on 10 chromosomes, but the distribution appears to be unbalanced. Most ZmCaM and ZmCML genes were located on chromosome 2, 3, and 4, whereas a single ZmCML4 was found on chromosome 10. There are only four genes each on chromosome 1, 5, 7, 8, and 9. In addition, six genes (ZmCaM3, ZmCaM4, ZmCML34, ZmCML35, and ZmCML37) were located on chromosome 6 ( Figure 3). The uneven distribution of ZmCaMs/ZmCMLs demonstrates that genetic variations exist in the process of evolution.

ZmCaM and ZmCML genes experience purification selection
Previous studies have shown that gene duplication can lead to generating new functional genes and drive species evolution (Panchy et al. 2016). We performed synteny analysis for exploring duplications within the ZmCaMs and ZmCMLs family. As shown in Figure 4, most of ZmCaMs/ZmCMLs were found to exist segmental-duplicated gene pairs, except ZmCML6 and ZmCML19. The nonsynonymous substitution rate (Ka) and the synonymous substitution rate (Ks) represented the selection  and evolution directions of protein-coding genes (Li et al. 2009). There were three directions of gene evolution: positive selection (Ka/Ks >> 1), neutral evolution (Ka/Ks = 1), and purify selection (Ka/Ks << 1). Table S2 showed that the average Ka/Ks ratio of 172 collinear gene pairs is 0.20. These results indicate that ZmCaM and ZmCML genes experience purification selection.
More than 110 cis-regulatory elements are found in the promoter regions of ZmCaM and ZmCML genes To investigate the function of ZmCaMs/ZmCMLs, 2000 bp sequences of promoter regions for the ZmCaMs/ZmCMLs were analyzed using the PlantCARE tool. As shown in Table S3, more than 110 cis-regulatory elements were found in the promoter regions of ZmCaMs/ZmCMLs. They were classified into five categories according to different functions, including core promoter element, light response element, hormone response element, stress response element, and other regulatory element. The cis-regulatory elements were involved in hormone response including auxin (AuxRE and AuxRR-core), gibberellin (GARE-motif and TATC-box), ethylene (ERE), salicylic acid (SARE and TCA-element), and MeJA (TGACG-motif). Stress response elements are mainly composed of hypoxia-induced response element (ARE), drought response element (MBS), dehydration response element (DRE), low-temperature response element (LTR), trauma-induced response element (WUNmotif), pressure response element (STRE), defense and stress response elements (TC-rich repeats). These results indicate that ZmCaMs/ZmCMLs may respond to various stresses.

ZmCaM and ZmCML genes have different expression patterns in various tissues
To further reveal potential function of ZmCaMs/ZmCMLs in maize, the transcription levels of ZmCaMs/ZmCMLs were analyzed in different tissues via RNA-seq database (Walley et al. 2016). As shown in Figure 5, most of ZmCaMs/ ZmCMLs were expressed in endosperm, leaf, root, and other tissues, whereas some ZmCMLs (ZmCML9, ZmCML14, ZmCML33, ZmCML43 and ZmCML44) were not expressed in tissues. ZmCML2, ZmCML4, ZmCML8, ZmCML11, ZmCML20, ZmCML23, ZmCML25, ZmCML27, and ZmCML40 showed high expression levels in maize natural pollination. ZmCML10, ZmCML21, ZmCML26, ZmCML30, ZmCaM1, ZmCaM7, ZmCML44, and ZmCML35 were highly expressed in root elongation zone and cortex. ZmCML6, ZmCML32, ZmCML19, and ZmCML39 had high expression profiles in mature leaves and leaf zone. Interestingly, we also found that some ZmCaMs/ZmCMLs displayed different expression patterns in the developmental process of leaf and roots ( Figure 5). The expression profiles of ZmCaMs/ZmCMLs in different tissues demonstrate that ZmCaMs/ZmCMLs may have different biology functions in plant developmental processes.

ZmCaM and ZmCML genes are regulated under drought stress
To illuminate the possible functions of ZmCaMs/ZmCMLs in response to drought stress, their expression patterns were analyzed in maize roots under drought stress via RNA-seq database (Li et al. 2009). RNA-seq database revealed that most of ZmCaMs/ZmCMLs are regulated under drought stress, except ZmCML4 and ZmCML9. As shown in Figure 6, the expression levels of ZmCML12, ZmCML22, and ZmCML30 were up-regulated under moderate stress and severe stress at 6 h. The expression patterns of ZmCML3, ZmCML8, and ZmCML26 were upregulated under moderate stress and severe stress at 24 h. The expression patterns of ZmCaM7 and ZmCML10 were down-regulated at 6 h under moderate stress and severe stress. The expression levels of ZmCaM5 and ZmCML2 were higher under moderate stress at 24 h than under severe stress at 24 h. The expression levels of ZmCML14 and ZmCML38 were increased under moderate stress at 24 h, while decreased under severe stress at 24 h. These results suggest that ZmCaMs/ZmCMLs have distinct expression patterns under moderate and severe stresses, which may play different roles in drought tolerance.
To confirm the results of RNA-seq, the expression levels of ZmCaMs/ZmCMLs (ZmCaM2, ZmCML3, ZmCML6, ZmCML8, ZmCML19, ZmCML24, ZmCML27, ZmCML28, ZmCML36, ZmCML39, and ZmCML40) were detected in maize under drought stress using RT-qPCR. As shown in Figure 7, the expression levels of ZmCaMs/ ZmCMLs were strongly induced in maize roots under drought stress, which were consistent with RNA-seq data. The relative expression level of ZmCML39 was significantly increased under moderate drought stress at 6 h, while was decreased under severe drought stress at 6 h. The relative expression levels of ZmCML3 and ZmCML8 were increased under moderate and severe drought stress at 24 h. In addition, we also found that ZmCaM2, ZmCML3, ZmCML6, ZmCML8, ZmCML19, ZmCML24, ZmCML28, ZmCML36, ZmCML39, and ZmCML40 have the highest expression levels under severe drought stress at 24h, which indicate that they are responsive to severe drought stress. These results indicate that identified ZmCaMs/ZmCMLs may play important roles in drought resistance.

The interaction network of ZmCaM and ZmCML proteins
Interaction network analysis is helpful to study the function of genes and discover the core regulatory genes (Zhao et al. 2018). The relative expression levels of ZmCaM2, ZmCML3, ZmCML8, and ZmCML40 reached the highest under severe drought stress at 24 h, which indicate they may play important roles in response to drought stress. To further explore the function of these genes, we conducted the network interaction analysis using STRING database. As shown in Figure 8, ZmCaM2, ZmCML3, ZmCML8, and ZmCML40 proteins were associated with 10 functional proteins, respectively. The cal2 (homologous protein of ZmCaM2) was associated with plant defense signaling network protein (CBL6 and Si496006g11), which plays roles in transmitting ROS signals ( Figure 8A) (Sewelam et al. 2016). The GRMZM2G149923_P01 (homologous to ZmCML3) was associated with CBL proteins, which participates in response to drought stress ( Figure 8B) (Moumeni et al. 2011). Similarly, the GRMZM2G115628_P01 (homologous to ZmCML8) was predicted to be interacting with CBL1, which is a positive regulator of salt and drought stresses ( Figure 8C) (Cheong et al. 2003). In addition, the pco068771 (homologous to ZmCML40) was predicted to be interacting with GRMZM2G120922_P01, which plays crucial roles in maize root under drought stress ( Figure  8D) (Zaidi et al. 2016). These results indicate that ZmCaM2, ZmCML3, ZmCML8, and ZmCML40 proteins may regulate the maize drought resistance through interaction with these proteins.

Identification and characterization of ZmCaM and ZmCML genes
Plants have evolved a series of physiological and biochemical mechanisms to cope with stress during evolution. Previous studies have shown that Ca 2+ is a crucial second messenger in eliciting response to environmental stimuli. CaMs and CMLs as conserved Ca 2+ sensor proteins involved in Ca 2+ signaling transduction in plants (Cheval et al. 2013). However, the comprehensive identification and expression of ZmCaM/ZmCML genes in maize are largely unclear, especially in the response to drought stress. (Mohanta et al. 2017) have studied the evolution of CaM and CML genes in various species, including in maize (Mohanta et al. 2017). In Mohanta's report, only 8 ZmCaMs and 21 ZmCMLs are identified in maize, however, the characterization and potential roles of these genes are unclear. In this study, more detailed ZmCaM/CML genes (7 ZmCaMs and 46 ZmCMLs) were identified in maize through BLASTP in B73-Reference-Graphene-4.0 genome database (http:// alpha.maize-gdb.org/) (Table S1). Furthermore, a comprehensive analysis of ZmCaM/CML genes was accomplished including subcellular localization phylogenetic tree, conserved motifs, exon-intron structure, chromosome location, synteny analysis, cis-regulatory elements, expression patterns in various tissues and under drought stress and interaction network. These results will provide novel and valuable information that will help understand the function and regulatory mechanism of ZmCaM/CML genes in maize, especially in drought resistance.
Previous studies showed that CaM and CML proteins contain the conserved EF-hand motif which is a key Ca 2+ binding site and the analysis of gene structure and conserved motifs can help us to understand the function of gene family (Nelson et al. (2002)). Here, three to four typical EF-hand motifs were identified in ZmCaMs, and two to four typical EF-hand motifs were identified in ZmCMLs (Figure 2A). Gene structure analysis showed that most ZmCMLs lack introns, while ZmCaMs contain 1, 2, or 6 introns (Figure 2B). These findings demonstrate that the conservation and divergence of motif and intron numbers may lead to similar or different biological functions among ZmCaM/CML family members. In principle, evolving more genes are the correct direction for the plant to adapt to different environments (Li et al. 2019). More ZmCML genes in the maize genome indicate that ZmCML genes may evolve before ZmCaM genes (Figure 1).
The cis-elements in the promoters of ZmCaM and ZmCML genes CaM and CML genes play critical roles in the plant developmental process and response to various stresses. These functions are associated with the cis-regulatory elements which are present in the promoter regions. Various hormone response elements and stress response elements have been identified in the promoter regions of CaM and CML genes in plants (He et al. 2020). The LTRs were enriched in the promoter regions of MtCML16 and MtCML33, and the transcript expression levels of MtCML16 and MtCML33 were induced under cold treatment at 6 h (Sun et al. 2020). The transcriptional expression levels of OsCML4, OsCML5, and OsCML11 were significantly increased under dehydration (Chinpongpanich et al. (2012)). In this study, the AuxRE, GARE-motif, ERE, TCA-element, MBS, DRE, LTR, and TC-rich repeats were found in the promoter regions of ZmCaMs/ZmCMLs (Table S3). We speculate that they may involve in response to abiotic stresses such as low temperature, drought, and salt stresses.  (Guo et al. 2018). In tomato, SlCML15, SlCML46, SlCML24, SlCML20, SlCML19, and SlCML14 were expressed in various tissues, including young leaves and flowers ). In addition, VviCML9a, VviCML9b, and VviCML79 were specifically expressed in senescent leaves, roots and fruit setting rate, respectively (Vandelle et al. 2018). In this study, we found that most ZmCaMs/ZmCMLs were expressed in plant tissues, except ZmCML9, ZmCML14, ZmCML33, ZmCML43, and ZmCML44 ( Figure 5). For instance, ZmCML3, ZmCML29, and ZmCML45 were highly expressed in endosperm, indicating that these genes may be involved in endosperm synthesis. ZmCML16, ZmCML12, ZmCML41, ZmCML37, ZmCML17, ZmCML13, ZmCML42, ZmCML36, ZmCML30, ZmCML3, ZmCML1, ZmCML46, and ZmCML24 showed high expression in embryos, indicating that these genes may play a crucial role in the development of embryos. These results demonstrate that ZmCaMs/ ZmCMLs may play important and different roles in the process of plant growth and development. ZmCML40. The relative expression is calculated using the 2 -ΔΔCT method. STDEV is used to calculate the standard deviation of sample-based estimates (*p < 0.05, **p < 0.01, Student's t-test). The experiment was performed using three biological repeats.

Expression profiles of ZmCaM and ZmCML genes under drought stress
Previous studies demonstrated that CaM and CML genes as key regulators are involved in drought stress. AtCML20 played a negative role in drought tolerance, and overexpression of AtCML20 led to hypersensitive to drought stress (Wu et al. 2017). AtCML37 and AtCML42 responded to drought stress, and they interacted with each other under drought stress (Scholz et al. 2015). MsCML46 was localized in the cytoplasm and its expression was significantly increased under drought treatment (Du et al. 2021). Under waterdeficit condition, the expression levels of VaCML57, VaCML66, and VaCML88 were significantly decreased (Dubrovina et al. 2019). Rice Calmodulin-like gene OsMSR2 enhanced drought resistance in Arabidopsis thaliana (Xu et al. 2011). To further understand whether ZmCaMs and ZmCMLs respond to drought stress, their expression patterns were analyzed through RNA-seq and RT-qPCR. The relative expression levels of ZmCaM2, ZmCML3, ZmCML6, ZmCML8, ZmCML19, ZmCML24, ZmCML27, ZmCML28, ZmCML36, ZmCML39, and ZmCML40 were induced significantly under drought stress (Figures 6 and 7). Furthermore, drought stess-responsive elements are found in the promoter regions of 11 ZmCaMs and ZmCMLs genes. These results indicate that they may have critical functions in drought stress.

Conclusion
CaMs and CMLs protein are primary Ca 2+ binding protein family, which are involved in response to various stresses.
In this study, we identified 7 ZmCaM and 46 ZmCML genes in maize, and they were unbalanced distributed on 10 chromosomes. Phylogenetic analysis showed that ZmCaMs/ZmCMLs are divided into nine subgroups. Gene structure analysis revealed that the EF-hand motif number of ZmCaMs/ZmCMLs ranged from 3 to 4 and 2 to 4, respectively. ZmCaMs/ZmCMLs showed different expression patterns among various tissues. A total of 11 ZmCaMs/ ZmCMLs were significantly induced under drought stress based on RNA-seq data and qRT-PCR, which indicate that they may play crucial roles in drought resistance. Overall, these results may provide an in-depth understanding of the roles of ZmCaM/ZmCML genes played in drought resistance.

Author contributions
L. Y. J. and W. G. Y conceived and designed the experiments. Z. W., L. H. W., and J. X. L. performed the experiments and drafted the manuscript. W. Y., J. B. C., X. J. R., W. W., and Y. B. W. analyzed the data.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This work was supported by Youth Growth Technology Project of Science and Technology Department of Jilin Province, China (20210508020RQ), Innovation and Entrepreneurship Training Program for College Students in Jilin Province (S202110193120, S202110193125).