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Article

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

by
Shuangcheng Ding
1,2,3,†,
Fengyu He
1,2,3,†,
Wenlin Tang
1,2,3,
Hewei Du
4 and
Hongwei Wang
1,2,3,*
1
Hubei Key Laboratory of Waterlogging Disaster and Agricultural Use of Wetland, Agricultural College, Yangtze University, Jingzhou 434000, China
2
Engineering Research Center of Ecology and Agricultural Use of Wetland, Ministry of Education, Agricultural College, Yangtze University, Jingzhou 434000, China
3
Hubei Collaborative Innovation Center for Grain Industry, Agricultural College, Yangtze University, Jingzhou 434000, China
4
Department of Biotechnology, College of Life Science, Yangtze University, Jingzhou 434025, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Genes 2019, 10(8), 610; https://doi.org/10.3390/genes10080610
Submission received: 9 July 2019 / Revised: 1 August 2019 / Accepted: 7 August 2019 / Published: 12 August 2019
(This article belongs to the Special Issue Abiotic Stress in Plants: Current Challenges and Perspectives)
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Abstract

:
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.

1. 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 (SOD), catalase (CAT), ascorbic acid peroxidase (APX), and glutaredoxin (GRX) [4]. GRX is a small redox enzyme composed of approximately 100 amino acid residues. In the action to keep its substrates from being oxidized, GRX transfers electrons to its substrates and becomes oxidized; it returns to the reduced state by acquiring electrons from glutathione. GRXs are not only ubiquitous in plants but an essential component of the plant antioxidation system to keep proteins in their properly reduced state [5,6]. GRXs have been studied for their involvement in response to oxidative stress [6,7,8]. GRXs play a critical role in scavenging reactive oxygen species (ROS) to prevent damage [4] when plants suffer from drought stress accompanied by a massive accumulation of ROS [9]. GRXs were suggested to form one of the most important protein modification systems in plants [5]. Meanwhile, GRXs were found to participate in the oxidation-reduction homeostasis and ROS signal transduction in plant cells [10]. Besides, the protein substrates of GRXs are involved in all aspects of plant growth, including primary metabolism, iron/sulfur cluster formation, development, environmental adaptation, and stress response [6]. GRXs are; therefore, a class of global regulators.
Several studies have reported genome-wide identification of the GRX gene family [11,12,13]. Based on the sequences at their redox activity centers, GRXs are classified into six categories, namely CSY(C/S)-, CGF-, CC-type, and other groups with unknown functions [11,12,13]. CC-type GRXs are a plant-specific subgroup and are also known as the Roxy family in Arabidopsis thaliana [14,15]. In sequence composition, CC-type GRXs have their unique conservative active site motif. For example, they contain unique CC(M/L)(C/S) conservative active site motifs in Arabidopsis [16]; whereas in rice, the conservative active site motifs extends to C(C/G/F/Y/P)(M/L)(C/S/I/A) [5,11,15,17]. The first CC-type GRX was identified as a regulator of fetal development [14]. However, CC-type GRXs also participate in jasmonic acid (JA)/ethylene (ET)-mediated abiotic stress response through its interaction with TGA transcription factors (TFs) [11,18]. Additionally, CC-type GRXS13 is crucial in limiting ROS production induced by basic and photooxidative stress [19]. Therefore, CC-type GRXs play a vital role in the crosstalk between ROS and ET. CC-type GRXs also participate in organ development and abiotic stress responses in other plants [11,20,21,22,23].
Drought stress greatly affects the productivity of crops, including maize, which is a primary staple food and provides industrial raw materials [24,25,26]. The completion of the maize genome sequence provides the opportunity to annotate and analyze the whole genome of maize [27]. In addition, previous evidence suggests that CC-type GRXs are candidate genes that regulate maize growth and response to environmental stresses [11,20,21,22,23]. With the goal to understand the role of GRXs in maize, here we comprehensively analyzed the phylogeny, gene structure, and chromosome location of the CC-type GRX family. We also analyzed the molecular evolution of the gene family, promoter analysis, gene expression patterns in various tissues, and response to drought stress. These results will provide a useful reference for future research on the function of CC-type GRX genes in maize.

2. Materials and Methods

2.1. Identification of CC-Type GRX Protein-Coding Genes in the Maize Genome

CC-, CG-, and CP-type GRXs of rice and Arabidopsis were retrieved from the report by Garg et al. (2010) [13]. The protein sequences of CC-, CG-, and CP-type GRXs were obtained from the Rice Genome Annotation Project database 6 for rice and from the TAIR database 9 for Arabidopsis. These sequences were used as queries to search the maize B73 genome (genome assembly: AGPv3) in Phytozome V12 by TBLASTN. All the retrieved maize sequences were further curated using the NCBI Conserved Domain Database (CDD) [28] to determine whether there was a PF00462 protein domain. Those genes encoding proteins containing a CC-type redox site motif were considered ZmGRXCC genes.

2.2. Gene Structure and Phylogenetic Relationships Analysis

Protein sequences of maize ZmGRXs were downloaded from Phytozome V12. (Berkeley, California, USA) To display the exon/intron structure, the coding sequence of each ZmGRXCC gene was aligned 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).

2.3. Identification of Cis-Regulatory Elements in Promoters of ZmGRXCCs

To identify potential cis-regulatory elements in the promoter sequences of ZmGRXCC genes, the 1500 bp sequences of each ZmGRXCC gene upstream of the ATG start codon were selected from the maize genome as the promoter, and the promoter sequence was screened using PlantCARE [32]. The elements searched included Skn-1_motif (-GGGCGG-), CCGTCC-box (-CCGTCC-box-), CAT-box (-GCCACT-), and RY-element (-CATGCATG-) for development and metabolism; TGACG-motif (-TGACG-) and CGTCA-motif (-CGTCA-) for jasmonic acid responsiveness; GARE-motif (-AAACAGA- or -TCTGTTG-) for gibberellin responsiveness; TGA-element (-AACGAC-) for auxin responsiveness; ABRE (ABA-responsive element, -CACGTG- or -TACGTG-) and CE3 (coupling element 3, -CACGCG-) for ABA responsiveness; ERE (ethylene-responsive element, -ATTTCAAA-) for ethylene responsiveness; GC-motif (-CCCCCG-), LTR (-CCGAAA-) and box S (-AGCCACC-) for stress responsiveness; and MBS (MYB-binding site, -CAACTG- or -TAACTG-), DRE (dehydration-responsive element, -GCCGAC- or -ACCGAC-), T/G Box (-CACGTT-), EE (evening element, -AATATC-), MYCR (MYC-binding site, -CACATG-), and NACR (binding site of drought-inducible NAC TFs, -CACGCA-) for drought/dehydration responsiveness [33,34,35,36,37].

2.4. Expression Profile Analysis

2.4.1. 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.

2.4.2. 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−ΔCT 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.

2.5. 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].

3. Results

3.1. 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.

3.2. 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].

3.3. 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).

3.4. 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]. Furthermore, a large number of elements involved in various stress responses are also found in the ZmGRXCC promoters, such as stress-responsive elements (LTR and box S), MYB-binding site (MBS), dehydration-responsive element (DRE and T/G Box), evening element (EE), MYC-binding site (MYCR), and binding site of drought-inducible NAC TFs (NACR). These results indicate that the ZmGRXCC genes may participate in plant abiotic stress responses.

3.5. 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.

3.6. 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).
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, −log10P = 2.86) (Figure 6B).

3.7. 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.

4. 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.

5. 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.

Supplementary Materials

Supplementary Materials can be found online at https://www.mdpi.com/2073-4425/10/8/610/s1. Table S1. Sequence alignment used to generate figure 1, Table S2. PCR Primer Sequences Used for This Research.

Author Contributions

S.D. and F.H. designed the experiments, performed the experiments, analyzed the data, and prepared figures and tables. H.D. advised on cis-element analysis, expression profile analysis, and qPCR analysis. W.T. prepared the figures and tables. H.W. wrote the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation (31701062 and 31701439), Hubei Province Natural Science Foundation (grant to Hongwei Wang) and the Opening Fund of Engineering Research Center of Ecology and Agricultural Use of Wetland, Ministry of Education (grant to Hongwei Wang, ID: KF201912). 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.

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Figure 1. Phylogenetic tree of glutaredoxins (GRXs) from maize, rice, and Arabidopsis. Members of GRXs were classified by their redox activity site. The names used for GRX genes in rice and Arabidopsis followed the report of Garg et al. [13].
Figure 1. Phylogenetic tree of glutaredoxins (GRXs) from maize, rice, and Arabidopsis. Members of GRXs were classified by their redox activity site. The names used for GRX genes in rice and Arabidopsis followed the report of Garg et al. [13].
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Figure 2. Predicted ZmGRXCC protein phylogeny, expression profiles, and gene structures. (A) Neighbor-joining (NJ) phylogeny of maize ZmGRXCC proteins. (B) Expression profile of maize ZmGRXCC genes. Normalized gene expression values [41] are shown in different colors that represent the relative levels of expression indicated on the scale bar. (C) Position of exons, introns, and untranslated region (UTR) in the ZmGRXCC genes. Introns were indicated by black boxes, UTR by gray box.
Figure 2. Predicted ZmGRXCC protein phylogeny, expression profiles, and gene structures. (A) Neighbor-joining (NJ) phylogeny of maize ZmGRXCC proteins. (B) Expression profile of maize ZmGRXCC genes. Normalized gene expression values [41] are shown in different colors that represent the relative levels of expression indicated on the scale bar. (C) Position of exons, introns, and untranslated region (UTR) in the ZmGRXCC genes. Introns were indicated by black boxes, UTR by gray box.
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Figure 3. Alignment of ZmGRXCC protein sequences. Black boxes indicate identified conserved positions. The letters above the sequence indicate motif names.
Figure 3. Alignment of ZmGRXCC protein sequences. Black boxes indicate identified conserved positions. The letters above the sequence indicate motif names.
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Figure 4. 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.
Figure 4. 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.
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Figure 5. Transcriptomic analysis of ZmGRXCC genes. The log2-based fold change (FC) values were supplied to build the heatmap. The color scale ranged from blue, representing low expression, and passes through white and finally to red, representing high expression. The gray color shows missing data.
Figure 5. Transcriptomic analysis of ZmGRXCC genes. The log2-based fold change (FC) values were supplied to build the heatmap. The color scale ranged from blue, representing low expression, and passes through white and finally to red, representing high expression. The gray color shows missing data.
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Figure 6. Association analysis of genetic variations in ZmGRXCC14 and ZmGRXCC16 with maize drought tolerance. (A), Quantile-quantile plots of estimated −log10(P) from ZmGRXCC gene family-based association analysis using three methods. The gray line is the expected line under the null distribution. The white square represents the observed p values using GLM; the gray square represents the GLM model with the first two principal components (PC2); the black diamond represents the observed p values using the MLM model incorporating both PC2 and a Kinship matrix. Schematic diagrams of ZmGRXCC14 (B) and ZmGRXCC16 (C), including protein coding regions (thick black line), are presented in the x-axis. The p value is shown on a −log10 scale.
Figure 6. Association analysis of genetic variations in ZmGRXCC14 and ZmGRXCC16 with maize drought tolerance. (A), Quantile-quantile plots of estimated −log10(P) from ZmGRXCC gene family-based association analysis using three methods. The gray line is the expected line under the null distribution. The white square represents the observed p values using GLM; the gray square represents the GLM model with the first two principal components (PC2); the black diamond represents the observed p values using the MLM model incorporating both PC2 and a Kinship matrix. Schematic diagrams of ZmGRXCC14 (B) and ZmGRXCC16 (C), including protein coding regions (thick black line), are presented in the x-axis. The p value is shown on a −log10 scale.
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Figure 7. Expression patterns of ZmGRXCC14 and 17 in seedlings (A), V12 (B), V14 (C), V16 (D), and R1 (E) under drought. ZmUbi-2 was used as an internal control for data analysis. The mean and SD were calculated from data of three biological replicates. t-test, * p ≤ 0.05, ** p ≤ 0.01.
Figure 7. Expression patterns of ZmGRXCC14 and 17 in seedlings (A), V12 (B), V14 (C), V16 (D), and R1 (E) under drought. ZmUbi-2 was used as an internal control for data analysis. The mean and SD were calculated from data of three biological replicates. t-test, * p ≤ 0.05, ** p ≤ 0.01.
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Table
Table 1. Detailed information for the 21 ZmGRXCC genes identified in the Zea mays genome.
Table 1. Detailed information for the 21 ZmGRXCC genes identified in the Zea mays genome.
IdentifierGene SymbolChromosomeClassRedox SiteALWL-Motif
GRMZM2G413315ZmGRXCC11CCCCMCALWV
GRMZM2G469994ZmGRXCC21CCCCMCALWV
GRMZM2G480903ZmGRXCC32CCCCMCALWL
GRMZM5G892308ZmGRXCC42CCCCMCALWL
GRMZM2G303044ZmGRXCC53CCCCMAALCA
GRMZM2G110286ZmGRXCC63CCCCMCALWV
GRMZM2G052796ZmGRXCC73CCCCMCALWV
GRMZM2G403680ZmGRXCC83CCCCMCALWL
GRMZM2G023237ZmGRXCC93CCCCLSALWL
GRMZM2G371063ZmGRXCC104CCCSMGALWL
GRMZM5G860607ZmGRXCC114CCCSMGALWL
GRMZM2G470756ZmGRXCC125CCCCMCALWL
GRMZM2G318213ZmGRXCC136CCCCMCALCG
GRMZM2G318180ZmGRXCC146CCCCLSALWL
GRMZM2G442791ZmGRXCC157CCCCMCALWL
GRMZM2G441906ZmGRXCC168CCCCMAALCA
GRMZM2G311898ZmGRXCC178CCCCLSALWL
GRMZM2G178886ZmGRXCC188CCCCLSALWL
GRMZM2G337706ZmGRXCC1910CCCPMCAIWL
GRMZM2G457898ZmGRXCC2010CCCCMCALWV
GRMZM2G303536ZmGRXCC2110CCCPMCDIWL
Table
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.
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 IDGene NamePolymorphicGLMPC2MLM
p ≤ 0.01p ≤ 0.01p ≤ 0.01p ≤ 0.005
GRMZM2G413315ZmGRXCC11----
GRMZM2G469994ZmGRXCC2-----
GRMZM2G480903ZmGRXCC311---
GRMZM5G892308ZmGRXCC4-----
GRMZM2G303044ZmGRXCC543----
GRMZM2G110286ZmGRXCC61----
GRMZM2G052796ZmGRXCC73----
GRMZM2G403680ZmGRXCC8-----
GRMZM2G023237ZmGRXCC923----
GRMZM2G371063ZmGRXCC10-----
GRMZM5G860607ZmGRXCC11-----
GRMZM2G470756ZmGRXCC121----
GRMZM2G318213ZmGRXCC1311----
GRMZM2G318180ZmGRXCC142010111
GRMZM2G442791ZmGRXCC15-----
GRMZM2G441906ZmGRXCC16204-1-
GRMZM2G311898ZmGRXCC171----
GRMZM2G178886ZmGRXCC183410---
GRMZM2G337706ZmGRXCC19-----
GRMZM2G457898ZmGRXCC20-----
GRMZM2G303536ZmGRXCC21-----

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Ding, S.; He, F.; Tang, W.; Du, H.; Wang, H. Identification of Maize CC-Type Glutaredoxins That Are Associated with Response to Drought Stress. Genes 2019, 10, 610. https://doi.org/10.3390/genes10080610

AMA Style

Ding S, He F, Tang W, Du H, Wang H. Identification of Maize CC-Type Glutaredoxins That Are Associated with Response to Drought Stress. Genes. 2019; 10(8):610. https://doi.org/10.3390/genes10080610

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Ding, Shuangcheng, Fengyu He, Wenlin Tang, Hewei Du, and Hongwei Wang. 2019. "Identification of Maize CC-Type Glutaredoxins That Are Associated with Response to Drought Stress" Genes 10, no. 8: 610. https://doi.org/10.3390/genes10080610

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