Identification of Maize CC-Type Glutaredoxins That Are Associated with Response to Drought Stress

Global maize cultivation is often adversely affected by drought stress. The CC-type glutaredoxin (GRX) genes form a plant-specific subfamily that regulate plant growth and respond to environmental stresses. However, how maize CC-type GRX (ZmGRXCC) genes respond to drought stress remains unclear. We performed a TBLASTN search to identify ZmGRXCCs in the maize genome and verified the identified sequences using the NCBI conservative domain database (CDD). We further established a phylogenetic tree using Mega7 and surveyed known cis-elements in the promoters of ZmGRXCCs using the PlantCARE database. We found twenty-one ZmGRXCCs in the maize genome by a genome-wide investigation and compared their phylogenetic relationships with rice, maize, and Arabidopsis. The analysis of their redox active sites showed that most of the 21 ZmGRXCCs share similar structures with their homologs. We assessed their expression at young seedlings and adult leaves under drought stress and their expression profiles in 15 tissues, and found that they were differentially expressed, indicating that different ZmGRXCC genes have different functions. Notably, ZmGRXCC14 is up-regulated at seedling, V12, V14, V16, and R1 stages. Importantly, significant associations between genetic variation in ZmGRXCC14 and drought tolerance are found at the seedling stage. These results will help to advance the study of the function of ZmGRXCCs genes under drought stress and understand the mechanism of drought resistance in maize.


Introduction
Abiotic stresses, especially drought, seriously affect plant growth and reproduction. Drought stress has far-reaching impact and strong destructive power, mainly due to the increased levels of reactive oxygen species in plants, such as hydrogen peroxide and singlet oxygen [1]. These oxidative substances are highly active and toxic because they can lead to lipid peroxidation and damage of proteins, carbohydrates, and nucleic acids, thereby destroying biofilm, affecting cell structure and function, and ultimately resulting in oxidative stress [2,3]. Therefore, studying the mechanism of antioxidation employed by plants is critical to the improvement of drought tolerance in plants.
The main ways plants employ to alleviate oxidative stress include both enzymatic and non-enzymatic systems [4]. The enzymatic system in plant cells includes superoxide dismutase to its corresponding genome sequence, and then the schematic was generated using GSDS 2.0 [29]. To build a phylogenetic tree, rice, Arabidopsis, and maize GRX proteins were aligned by ClustalX2 [30] and manually adjusted. The phylogenetic tree was built using this alignment output based on a maximum likelihood method in MEGA7 [31]. The parameters used were as follows: pairwise deletion and bootstrap (1000 replicates).

Plant Treatments
Seedling and adult leaves of maize inbred line B73 were subjected to drought. Seedling growth conditions and drought stress treatments of B73 were conducted according to the protocol of Wang et al. [38]. Briefly, hydroponic-cultured seedlings at the three-leaf stage were placed on a plate and subjected to dehydration (40%-60% relative humidity and 28 • C). To study the effect of drought stress on adult leaves of B73, water was intercepted after the eight-leaf stage (V8), and plants with sufficient water (40% of soil moisture) were used as control. Three replicates of the middle section of the flag leaf were collected from both drought-stressed and well-watered plants (control) at the twelve-leaf stage (V12), the fourteen-leaf stage (V14), the sixteen-leaf stage (V16), and the silking stage (R1). Leaf samples of seedlings or flag leaves of at least three replicates were frozen in liquid nitrogen and stored at −80 • C until RNA isolation.

RNA Isolation and Real-Time PCR Analysis
Total RNA was isolated from leaf samples from seedlings and adult leaves using the TriZol Reagent (Invitrogen, Carlsbad, California, USA) according to the product manuals. The relative expression of ZmGRXCCs was measured using quantitative real time-PCR (RT-qPCR) in 96-well plates using the ABI7500 Real-Time PCR Systems (Applied Biosystems, Foster, California, USA). The PCR reaction system consists of 1 µg cDNA, 200 nM primers, and 5 µL SYBR Premix Ex Taq II (Takara, Dalian, China), and the reaction volume is 10 µL. The PCR reaction was conducted with the following conditions: 10 min at 94 • C, 40 cycles of 15 s at 94 • C, and 30 s at 60 • C. The expression level of the ZmUbi-2 gene (UniProtKB/TrEMBL; ACC: Q42415) was used as the internal control. For quantification, the 2 −∆CT formula was used, and variations (standard errors) were calculated from three technical replicates for each of the three biological replicates. Primers for qRT-PCR are listed in Table S2.

Association Analysis
Association analysis for ZmGRXCCs was conducted by using a previously reported mapping population [38]. The mapping population, containing 367 maize inbred lines and corresponding survival rate under drought, contains 556,000 single nucleotide polymorphism (SNP) markers, with the minor allele frequency (MAF) ≥ 0.05. The identified ZmGRXCCs harbored 159 SNPs in the coding region and both the 5 -, and 3 -untranslated region (UTR). Three statistical models including the general linear model (GLM) model, adjusting the first two principal components (PC2), and the mixed linear model (MLM) model (incorporating PC2 and a kinship matrix) were selected to identify the SNPs significantly associated with drought tolerance by using the TASSEL4.0 program [39,40].

Forty-Five Maize GRXs, Including Twenty-One CC-Type GRX (ZmGRXCC) Genes, Were Identified
We systematically surveyed the maize genome to identify putative maize GRX genes. To further assess the phylogenetic relationship between GRX, we constructed a phylogenetic tree of maize, rice, and Arabidopsis thaliana GRXs ( Figure 1, Table S1). As shown in Figure 1, a total of 106 GRX genes were divided into three types: CC, CP, and CG ( Figure 1). We found that the CC-type subgroup had the most members among all the subgroups in maize. All Arabidopsis and rice GRX genes fell in the same class or clade as previously reported, in agreement with previous work [11,12]. Interestingly, the Arabidopsis GRXs were isolated from the two monocotyledons and clustered into a single branch in Figure 1. Among them, 21 contain the CC-type redox site and are thus ZmGRXCCs ( Figure 1, Table 1). The physical location of each ZmGRXCC in the maize genome was determined based on the physical coordination provided by Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html). Besides, the gene symbols are named according to their chromosomal locations ( Figure 2, Table 1). The results showed that these 21 ZmGRXCC genes are unevenly distributed without clustering in the ten maize chromosomes except chromosome 9. Chromosome 3 possesses five ZmGRXCC genes, accounting for the largest number of ZmGRXCCs on a single chromosome. Chromosomes 8 and 10 each contain three genes, while chromosomes 5 and 7 each contain one ZmGRXCC. Table 1. Detailed information for the 21 ZmGRXCC genes identified in the Zea mays genome.

Identifier
Gene Symbol Chromosome

Phylogenetic Relationship Analysis Showed High Conservation in ZmGRXCC Genes
We further studied the phylogenetic relationship, expression profile, and gene structure of the identified ZmGRXCCs ( Figure 2). Tissue-specific expression profiles [39] based on the transcriptomic data of maize B73 were also used to reveal the roles of ZmGRXCC genes. An expression heatmap based on the normalized gene expression value was constructed for the 21 ZmGRXCC in different tissues, as well as developmental stages ( Figure 2B). As shown in Figure 2B, the expression pattern of ZmGRXCC genes varied greatly. ZmGRXCC5, ZmGRXCC9, ZmGRXCC13, ZmGRXCC14, ZmGRXCC16, ZmGRXCC17, and ZmGRXCC18 were expressed higher than other ZmGRXCCs. Interestingly, there were similarities between protein clustering and expression patterns. Proteins clustered together have similar expression patterns. Additionally, there is only one exon in each ZmGRXCC gene ( Figure 2C), consistent with previous reports [12].

ZmGRXCC Proteins Were Highly Conserved Containing an Active Site and a C-Terminal ALWL Sequence
Using the protein sequence of GRXs from rice and Arabidopsis, we found 21 ZmGRXCCs that contain a conserved redox activity site in the maize B73 genome (genomic version: AGPV3.0). To analyze the conserved motifs of identified ZmGRXCCs, we performed multiple sequence alignments of ZmGRXCCs and AtROXY1 (Figure 3). It was found that 11 of the 21 ZmGRXCCs shared an ALWL motif at the C terminus and was further extended to A(D)L(I)W(C)L(A/G/V) ( Figure 3, Table 1). ZmGRXCCs also shared a distinctive CC(S/P)M(C/S) redox site ( Figure 3, Table 1). The fact that the redox motif of ZmGRXCCs shares high sequence similarity with AtROXY1 demonstrates that CC-type GRXs undergo a relatively conserved evolutionary history ( Figure 3, Table 1).

Promoter Analysis of the ZmGRXCC Gene Family
We identified a total of twenty-one major regulatory elements in the promoters of ZmGRXCC genes; these elements are associated with phytohormones, growth, development, and abiotic stress ( Figure 4). As shown in Figure 4, the genes belonging to the same subfamily have a diverse profile of cis-elements enriched in the promoters, suggesting a possible role for promoter sequences in functional diversification of the ZmGRXCC genes in the same subfamily. The presence of several major hormone-responsive elements, including the jasmonic acid-responsive element (TGACG-motif and CGTCA-motif), gibberellin-responsive element (GARE-motif), auxin-responsive element (TGA-element), ABA-responsive elements (ABRE and CE3), and ethylene-responsive elements (ERE) are present in the ZmGRXCC promoters, indicating that these ZmGRXCC genes could be involved in various phytohormone signaling pathways. Development-and metabolism-regulatory elements, such as Skn-1_motif, CCGTCC-box, CAT-box, and RY-element, are also present in the ZmGRXCC promoters. These results are consistent with previous reports showing that CC-type GRXs may participate in the control and regulation of organ development [20].  Cis-regulatory elements identified in the ZmGRXCC promoters. The 1.5 kb regions upstream of the translation start codons of the ZmGRXCC genes were used to search for putative cis-regulatory elements using the plant cis-acting regulatory DNA elements. The chart was drawn based on the presence and number of cis-regulatory elements that are responsive to specific elicitors or conditions. The color scale ranged from gray, representing minimum number of insertion, and finally to black, representing maximum number of insertion. The white color shows missing data.

Expression Profile of ZmGRXCCs under Stress Treatments
Studies have shown that the expression levels of CC-type GRXs are regulated by stress [12,13]. Based on the published RNA-seq transcriptome data (PRJNA244661 for drought and salt; PRJNA335771 for cold and heat), the log2 fold change relative to control were used to examine the gene expression profiles of the ZmGRXCCs in maize ( Figure 5). In general, genes within the same subgroup (Figure 2A) showed similar expression patterns. ZmGRXCC5, 9,13,14,16,17, and 18 were up-regulated above two-fold under drought, salinity, cold and heat, while others were down-regulated or undetected, indicated functional divergence of ZmGRXCCs in maize. Notably, ZmGRXCC14 and 17 were up-regulated above two-fold by all the four treatments.

Natural Variations in ZmGRXCC14 Are Associated with Drought Tolerance in Maize
In order to further investigate whether the natural variations in any of the ZmGRXCCs are associated with the different drought tolerance levels of maize varieties, we conducted an association analysis for these genes. To assess potential associations between survival rates and ZmGRXCCs, we utilized previously reported methods and data [38,42]. Among the 21 identified ZmGRXCC genes, 12 were found to be polymorphic (Table 2), while the polymorphic information of the other nine genes was currently absent (minor allele frequency, MAF ≥ 0.05). Table 2. Association analysis of natural variation in ZmGRXCC genes with respect to drought tolerance at the seedling stage in the maize diversity panel. GLM, PC2, and MLM stand for general linear model, general linear model with the first two principal components, and mixed linear model, respectively.

Locus ID
Gene Name Polymorphic GLM PC2 MLM Subsequently, three statistical models [42][43][44] were applied to identify significant genotypic and phenotypic associations ( Figure 6A). The candidate gene association analysis detected significant associations between the genetic variations of ZmGRXCC14 and 16 and drought tolerance under different models with a p-value ≤ 0.01 (Table 2; Figure 6B,C). However, under the standard mixed linear model (MLM), one significantly associated SNP contributing to the phenotype of drought tolerance was located at the CDS region of ZmGRXCC14, which suggests that this candidate gene is significantly associated with drought tolerance (p-value ≤ 0.005, −log 10 P = 2.86) ( Figure 6B).

Expression Profile of ZmGRXCC14 and 17 Are Both Induced by Drought Stress
ZmGRXCC14 and 17 were consistently up-regulated by drought, salt, heat, and cold; we further analyzed their expression under drought treatment by RT-qPCR. Maize seedlings were treated with drought stress for 5 or 24 h and their RNAs analyzed to monitor their expression profiles. As shown in Figure 7A, ZmGRXCC14 and 17 responded to drought stress in young maize seedling. Their expression levels were further assessed in adult leaves at four growth stages: V12, V14, V16, and R1. At the V8 stage, drought stress was applied by intercepting water, with the corresponding control being irrigated adequately (40% soil moisture). As shown in Figure 7, ZmGRXCC14 and 17 were up-regulated by drought stress. These results showed that ZmGRXCC14 and 17 may be involved in response to drought.

Discussion
Many reports indicate that CC-type GRXs are a class of small proteins that play an important role in plant development and abiotic stress responses [5,6]. However, there was no detailed analysis of the CC-type GRX gene family in maize, especially on their gene expression profile and genetic variation under drought stress. Therefore, we were prompted to conduct this study in an attempt to address this issue. Here, we identified a total of 21 maize CC-type GRX genes. Subsequently, we further systematically determined their phylogenetic relationship with rice, and Arabidopsis GRXs, pattern of drought-responsiveness, and association analysis of their natural variations with drought tolerance. Collectively, our data demonstrate that a few ZmGRXCC family proteins are likely to be involved in plant drought tolerance; in particular, ZmGRXCC14 may function as an important gene for drought tolerance. The ZmGRXCC14 gene will be of top priority for functional validation through overexpression and/or gene knockout in transgenic plants. Our results will not only facilitate the genetic improvement of drought resistance in maize but also increase our understanding of the biological functions of the CC-type GRX gene family.
At present, GRX family members described in various species are classified into five types, among which the CC-type are plant-specific [11][12][13]. In previous studies, there were 17 CC-type GRX genes in rice and 21 CC-type GRX genes in Arabidopsis [13]. Compared to rice or Arabidopsis, there is about one-fold duplication of CC-type GRX genes in maize. The unchanged ratio of gene numbers in species may suggest that the ZmGRXCC gene family has undergone a relatively conserved evolutionary history. On the other hand, as shown in our phylogenetic analysis (Figure 1), monocot GRX proteins cluster independently from Arabidopsis GRXs, suggesting a potential functional diversification between dicot and monocot GRXs.
In the present study, a total of 21 major regulatory elements are identified in the ZmGRXCC upstream regions, among which we find several types of motifs that may be responsive to drought stress and/or ABA hormone signaling (Figure 4). It is noteworthy that most of the ZmGRXCC gene promoters contain the ABRE motif. ABRE is a well-studied cis-element involved in ABA-induced gene expression, and it has been found to require a coupling element (CE) to achieve ABA induction [45,46]. Interestingly, we find that 12 ZmGRXCC promoters contain both ABRE and its CE3, suggesting that these genes may function in the ABA-mediated drought signaling pathway [12]. More importantly, 17 of the 21 ZmGRXCC promoters contain the JA-responsive element, implying that these genes may also be involved in the jasmonic acid signaling pathway [11,18]. Taken together, the mechanism by which ZmGRXCC genes respond to drought stress is likely through the ABA-and/or JA-mediated pathway.
To our knowledge, although the relationship between CC-type GRX proteins and plant stress was studied [21,23,47], the dynamic drought-responsive expression patterns of ZmGRXCC genes had not been reported. Analysis of the expression patterns of ZmGRXCC genes can give clues to their possible functions, and pave the way for future research. In general, different members of ZmGRXCCs respond differently to drought, salinity, cold, and heat, suggesting that they not only carry different functions between subgroups but also among members of the same subgroup ( Figure 5). Seven of the twenty-one ZmGRXCCs, including ZmGRXCC14 and 17, are up-regulated by at least one of the drought, salinity, cold, and heat treatments, while others are not ( Figure 3). Notably, ZmGRXCC14 and 17 are further validated to be up-regulated at seedlings and adult leaves by qPCR analysis (Figure 7). Importantly, the rice orthologs of ZmGRXCC14 and ZmGRXCC17 are also induced by plant hormones and abiotic stress [13], indicating that these genes may function as key mediators of drought tolerance in maize.
Identification of the key genetic components underlying drought is critical and will serve as the foundation for crop genetic improvement. Among the 21 ZmGRXCCs analyzed in this paper, the genetic variations of ZmGRXCC14 and ZmGRXCC16 are significantly associated with drought tolerance (p-value ≤ 0.01, MLM) ( Table 2). While ZmGRXCC14 is the most significantly (p-value ≤ 0.005, MLM) associated with drought tolerance among the ZmGRXCC gene family, the natural variation in the ZmGRXCC14 CDS may contribute to maize drought tolerance ( Figure 6). In comparison with ZmGRXCC16, ZmGRXCC14 is detected at a higher level under various stress ( Figure 5). More notably, ZmGRXCC14 is significantly induced by drought stress at both young seedlings and adult leaves (Figure 7). These results indicate that ZmGRXCC14 is involved in plant drought response. In summary, we suggest that ZmGRXCC14 may be an important positive regulator of drought tolerance through analyses of gene expression and natural variations.

Conclusions
In this report, we identified 21 maize CC-type GRX genes in the maize genome. Phylogenetic analyses revealed that the ZmGRXCC gene family had a conserved evolutionary history. Protein domain analysis indicates that most of the 21 ZmGRXCCs share similar structures to their homologs. Promoter analysis show that ZmGRXCCs have a diverse profile of cis-elements associated with phytohormones, growth, development, and abiotic stress. Analysis of their differential expression profiles upon stress at various developmental stages, including seedlings and adult leaves, as well as the expression profiles in 15 tissues, suggest a functional divergence of ZmGRXCC genes. Importantly, ZmGRXCC14 is up-regulated in both seedlings and adult leaves, and the natural variations in ZmGRXCC14 are significantly associated with drought-stress tolerance, implying ZmGRXCC14 is an important candidate gene for maize drought tolerance improvement. The findings presented here will enhance understanding of the role of ZmGRXCCs under drought.   The funders had no role in the study design, data collection, and analysis, the decision to publish, or the preparation of the manuscript.

Conflicts of Interest:
The authors declare no conflicts of interest.