Genome-wide identification, characterization and evolutionary analysis of the APETALA 2/ethylene responsive element-binding protein (AP2/EREBP), cytochrome c oxidase (COX) and lipid transfer protein (LTP) genes in Zea mays under drought stress

doi:10.21203/rs.3.rs-3973623/v1

AP2 (APETALA2)/EREBP (ethylene-responsive element-binding protein), cytochrome c oxidase (COX) and nonspecific lipid transfer proteins (LTP) playimportant roles in the response to drought stress. This is the first study to identify the COX gene in Zea mays L. via genome-wide analysis. The qRT‒PCR results indicated that AP2/EREBP, COX and LTPwere downregulated, with fold changes of 0.84, 0.53 and 0.31, respectively, after 12 hours of drought stress. Genome-wide analysis identified 78 AP2/EREBP, 6 COX and 10 LTP genes in Z. mays L.. Domain analysis confirmed the presence of the AP2 domain, Cyt_c_Oxidase_Vb domain and nsLTP1 in the AP2/EREBP, COX and LTP proteins, respectively. Motif and gene structure analyses indicated that genes with closer phylogenetic relationships exhibited more similar genetic structures. The gene structure results revealed that 21 of the 78 AP2/EREBP genes hadintrons. All the COX genes had introns, while 8 LTP genes had introns. Most of the AP2/EREBP and LTP proteins were located in the nucleus and the extracellular space, respectively, whereas COX proteins were predicted to be located in different organelles. Sixteen putative nuclear localization signals (NLSs) were predicted for 21 AP2/EREBP proteins. A total of 187 microRNAs were predicted to target the AP2/EREBP genes, 48 microRNAs were predicted to target COX proteins, and 30 microRNAs werepredicted to target LTP genes. The AP2/EREBP, COX and LTP genes contained defense and stress response elements, abscisic acid-responsive elements, methyl jasmonate (MeJA)-responsive elements, salylic acid and the MYB binding site (MBS) element, which are involved in the drought response. AP2/EREBP genes were found on the 10 chromosomes of Z. mays L.. COX genes were found on chromosomes 1, 3, 4, 5, 7 and 8. LTP genes were found on chromosomes 1, 3, 6, 8, 9 and 10. In the present study, the Ka/Ks ratios of the AP2/EREBPparalogous pairs indicated that the AP2/EREBP genes were influenced primarily by purifying selection, which indicated that the AP2/EREBP genes received strong environmental pressure during evolution. The Ka/Ks ratios of the COX-3/COX-4paralogous pairs indicate that the COX-3/COX-4 genes were influenced primarily by Darwinian selection (driving change). For the LTP genes, the Ka/Ks ratios of the LTP-1/LTP-10, LTP-5/LTP-3 and LTP-4/LTP-8 paralogous pairs indicate that these genes were influenced primarily by purifying selection, while the Ka/Ks ratios of the LTP-2/LTP-6 paralogous pairs indicate that thesegenes were influenced primarily by Darwinian selection. The duplication time of the AP2/EREBPparalogous gene pairs in Z. mays L.ranged fromapproximately 9.364 to 100.935 Mya. The duplication time of the COX-3/COX-4paralogous gene pair wasapproximately 5.217 Mya. The duplication time of the LTP paralogous gene pairs ranged from approximately 19.064 to 96.477 Mya. In the resulting phylogenetic tree, the AP2/EREBP proteins were classified into three distinct clades. The AP2/EREBP protein family (AP2) includes five different domain types according to the Phytozome-13 website: the AP2/ERF domain, the EREBP-like factor (EREBP), the ethylene responsive factor (ERF), the dehydration responsive element binding protein (DREB) and the SHN SHINE. COX and LTP proteins were classified into three distinct clades. Synteny analysis of the AP2/EREBP, COX and LTP genes revealed collinearity orthologous relationships in O. sativa, H. vulgare and A. thaliana. Gene Ontology enrichment analysis confirmed the functional role of the AP2/EREBP, COX and LTP proteins in stress responses. The results of the present study will improve the understanding of the functions of the AP2/EREBP, COX and LTP genes in response to drought stress.

Bioinformatics

Evolutionary Genetics

AP2/EREBP

COX

LTP

Genome-wide identification

evolutionary analysis

drought stress

Drought is defined as dryness (lack of water, moisture deficit, shortage of precipitation) for a period of time that affects organisms (plants, animals, humans) in the affected area [1]; [2]; [3]; [4]^. Drought is the most destructive type of hydrological hazard [5]. The development of drought-tolerant seed varieties can help farmers produce crops under drought stress. In maize (Z. mays L.), drought stress is one of the major environmental stress effects on yield reduction, and drought can affect maize at any stage of development. Breeding programmes are used to improve the drought tolerance of maize hybrids. Molecular biology techniques were used to improve breeding efficiency by identifying genes related to drought stress [6]. Climate changes such as water scarcity, extreme temperature and soil salinity have negative effects on plant growth and yield production [7]. Crop plants are exposed to several types of environmental stress, which affects their growth and development throughout their life cycle. Drought activates gene expression pathways in plants to protect cells against water deficit [8]. APETALA2 (AP2)/ ethylene-responsive element-binding protein (EREBP) is a transcription factor that has essential regulatory functions for protecting plants during stress. AP2/EREBP is one of the largest transcription factor families in plants [9]; [10]; [11]. AP2s/EREPs are involved in regulating gene expression during abiotic stress [12] and plant growth and development [13]; [11]. The AP2/EREBP protein family is classified into: the APETALA2 (AP2), ethylene-responsive factor (ERF), dehydration-responsive element binding protein (DREB), and related to ABI3/VP1 (RAV). In cotton (Gossypium raimondii), the genes are distributed on all chromosomes. In Gossypium hirsutum, the ERF and DREB genes play important roles in stress responses [11]. GhERF12 protein play crucial roles in organ development and differentiation in G. hirsutum [14]. Cytochrome c oxidase (COX) catalyzes the transfer of electrons from reduced cytochrome c (CYTc) to the final acceptor O₂ to H₂O for ATP production [15]; [16]. Restriction mapping and DNA sequencing were used to study the functional relationships of the COX mitochondrial genes. The results did not reveal a transcript of cox1; rather, a reduced level of a cox2 transcript and two different cox3 transcripts were detected. Results indicate that genomic rearrangements of the both 5' and 3' flanking regions of the cox1 gene leads to impaired of cox1 transcription [17]. The nonspecific lipid transfer protein (LTPs) are involved in different biological processes and play key roles in plant [18]. LTP2 play critical role for abiotic [19] and biotic stresses tolerance in plants [20]. LTPs are a large protein family present in all plants and are expressed in many different tissues. LTPs play important roles in signaling via their structure, which contains a N-terminal signal peptide that delivers proteins to the plasma membrane [21]. In maize, LTPs are differentially regulated by drought and salt treatments [22]. In rice, the expression of LTP is strongly induced under drought and salinity stresses [23]. In wheat, TaLTP1.2 and TaLTP1.13 are upregulated during drought [24]. In Lotus japonica, The LjLTP genes are expressed in aerial tissues under drought stress [25]. In the Moss Physcomitrium (Physcomitrella) patens, the expression of 8 LTPgs was investigated during several abiotic stresses. Three LTPg genes are significantly upregulated, which leads to the downregulation of the PpLTPg genes [26].

Identification of the AP2/EREBP, COX and LTP genes in Z. mays L.

The genomes of Z. mays L., Oryza sativa, Hordeum vulgare and Arabidopsis thaliana were downloaded from the Phytozome database [27]. The AP2/EREBP (accession number: NP_001183842.1), COX (accession number: NP_001288395.1) and LTP (accession number: ABA33850.1) proteins were used as query proteins from the NCBI database (https://www.ncbi.nlm.nih.gov/)[28] (Sheet 1 Online Resource SI 1) to screen AP2/EREBP, COX and LTP protein members in the genomes of Z. mays L. from the Phytozome database (https://phytozome.jgi.doe.gov) [27] with an E-value ≤1e^-30 and ≥ 50% identity for AP2/EREBP proteins (https://phytozome-next.jgi.doe.gov/blast-results/694644), whereas the Phytozome database parameters for COX proteins (https://phytozome-next.jgi.doe.gov/blast-results/694640) and LTP proteins (https://phytozome-next.jgi.doe.gov/blast-results/694645) had an E-value ≤1e^-30.

Characterization of the AP2/EREBP, COX and LTP proteins in Z. mays L.

Circoletto (http://tools.bat.infspire.org/circoletto/)[29]visualized the sequence identity of the AP2/EREBP, COX and LTP proteins. The physical and chemical properties of the AP2/EREBP, COX and LTP proteins, including the molecular weight, isoelectric point, total number of negatively charged residues, total number of atoms, instability and grand average hydropathicity (GRAVY), were computed using the ExPASy ProtParam Tool [30].

Phylogenetic, chromosomal distribution, evolutionary analysis and synteny analysis of the AP2/EREBP, COX and LTP genes

Multiple sequence alignments of the AP2/EREBP, COX and LTP proteins from Z. mays L. were performed via the MUSCLE method. Molecular evolutionary genetic analysis (MEGA-11) [31] was subsequently conducted on a phylogenetic tree with a maximum likelihood of 1000 bootstrap replicates based on the WAG with Freqs. (+F) Model. The Itools online website [32] was used to modify and visualize the tree.

According to the position information of the AP2/EREBP, COX and LTP genes on the chromosome, a karyotype map of the AP2/EREBP, COX and LTP genes was drawn using TBtools [33].The output image was used to show all the AP2/EREBP, COX and LTP genes on the chromosome.

The rates of synonymous (Ks) and nonsynonymous (Ka) substitutions were calculated by TBtools [33] to investigate selection pressure. The divergence time of the gene pairs was estimated using the synonymous mutation rate of substitutions per synonymous site per million years ago (Mya) as follows: “T = Ks/2λ”, with a λ value of 6.05 × 10⁻⁹ [34].

The duplicated genes were identified as paralogous if the alignment covered ≥70% of the longer gene and if the identity of the aligned region was ≥70% [35]; additionally, the genes were identified by the MEGA-11 [31] gene duplication wizard. Paralogous gene pair (tandem and segmental genes) collinearity analysis was visualized as a Circos plot through TBtools [33].

TBtools [33] were used to determine the syntenic relationships of the AP2/EREBP, COX and LTP genes in Z. mays L. against O. sativa, H. vulgare and A. thaliana.

Conserved domain, conserved motif, gene structure and Promoter analyses of the AP2/EREBP, COX and LTP genes

The NCBI conserved domain tool [36] was used to searchagainst the Pfam v34.0–19178 PSSMs database for AP2/EREBP, COX and LTP proteins. The InterPro tool [37] was used to analyze the domains of the AP2/EREBP, COX and LTP proteins.MEME 5.5.2 [38] was used to compute the conserved motifs of the AP2/EREBP, COX and LTP proteins. Pfam [39] was used for motif description.The gene structures obtained from the GFF file were downloaded from the phytozome of the Z. mays L. genome and subsequently illustrated using TBtools [33]_.

The promoter sequences of the AP2/EREBP, COX and LTP genes in Z. mays L.1500 bp upstream of the TSS of each AP2/EREBP, COX and LTP gene were retrieved from the Z. mays L. genome sequence file and downloaded from the Phytozome database [27]. Cis-regulatory elements (CREs) were also analyzed in Plant CARE [40]. A graphical representation of the CRE elements present in the promoter region of the gene was generated via TBTool [33].

Subcellular localization, nuclear localization signal, transmembrane helices, phosphorylation sites and three-dimensional (3-D) structure prediction

Subcellular localization predictor (CELLO) version 2.5 (http://cello.life.nctu.edu.tw/) [41] was used to predict the subcellular localization of the proteins, and TBtools was used to visualize the results [33]. NLSDB [42] was used to search for nuclear localization signal potentials. The TMHMM server version 2.0 [43] confirmed the presence of transmembrane helical domains (TMs) in the AP2/EREBP, COX and LTP proteins. The NetPhos 3.1 server [44] was used to predict the phosphorylation sites of the AP2/EREBP, COX and LTP proteins.TheI-TASSER [45] program predicted the three-dimensional (3-D) structure of the AP2/EREBP, COX and LTP proteins.

Prediction of miRNAs targeting the AP2/EREBP, COX and LTP genes

ThepsRNATarget database [46] and miRBase [47] were used to predict miRNAs. IPknot [48] was used to predict RNA secondary structures with pseudoknots for the AP2/EREBP, COX and LTP proteins.

Gene Ontology enrichment and functional relationship analysis of the AP2/EREBP, COX and LTP genes

ShinyGO 0.77 [49] was used for Gene Ontology enrichment analysis. We performed a gene ontology (GO) annotation analysis by submitting all the AP2/EREBP, COX and LTP gene sequences to the eggNOG database [50]and Phytozome database [27]. The GO annotation data were processed in SRPLOT [51] (http://www.bioinformatics.com.cn/login_en/) to construct the gene ontology chord for the functional relationships of the AP2/EREBP, COX and LTP genes.

Maize plant growth and drought treatment

These experiments were conducted in the Department of Botany and Microbiology, Faculty of Science at Cairo University. The seeds used in this study were obtained from the Agricultural Research Center (ARC), located in Giza, Egypt. The seeds originated from Egypt and were certified to be of the white three-way cross 310 variety. Drought treatment was conducted in 2 groups. The first group consisted of control plants, whereas the other consisted of stressed plants. Thirty seeds were planted in small pots in a growth room for 14 days. The second group of stressed plants was subjected to continuous water withholding for 12 hours, while the first group was treated with Hoagland’s solution as a control. The plants were harvested after 12 hours of drought stress. Three control plants and three stressed plants were subjected to RNA extraction and sequencing.

RNA isolation, qRT‒PCR expression analysis and sequencing

This study identified AP2/EREBP (forward primer: AACCCAAGAACACGCTTCCT and reverse primer: AAGCCACATCCCATCCCAAC), COX (forward primer: TCCGTAGTTGGGATTCGTCG and reverse primer: CTGGATTGGTTTCTAGTTTCTTTGA) and LTP genes (forward primer: ATAGGAACGTACGCACGCAG and reverse primer: ATGCAAGTCGTGATCATGCG). Total RNA was isolated from the leaves of 15-day-old Z. mays L. seedlings using a GeneTireX kit. The residual DNA was removed using RNase-free recombinant DNase I (Thermo Scientific, Litwania). First-strand cDNA was synthesized in a 20 μL reaction mixture using a Grisp reverse transcription kit (https://grisp.pt/) with approximately two micrograms of DNA-free total RNA from each sample. qRT‒PCR was performed to quantify the relative transcription levels of the AP2/EREBP, COX and LTP genes expressed in the leaves. qPCR was performed with a CFX Connect Real-Time PCR System (Bio-Rad, Singapore) under the following conditions: 94 °C for 5 min; 40 cycles of 94 °C for 10 s, 58 °C for 20 s, and 72 °C for 30 s; a plate read; a melt curve of 65–95 °C with an increment of 0.5 °C for 10 s; and subsequent sequencing. The Ct (cycle threshold) value was used as a measure of the starting copy number of the target gene [52]. The relative gene expression level was calculated using the 2^-ΔΔCT method [53]. Actin (Act) was used as an internal reference gene. The forward primer used was CTGAGGTTCTATTCCAGCCATCC, and the reverse primer used was CCACCACTGAGGACAACATTACC.

Identification of the AP2/EREBP, COX and LTP genes in Z. mays L.

A total of 78 AP2/EREBP, 6 COX and 10 LTP candidate genes were retrieved from the Z. mays L. genome and were named according to their chromosomal positions from AP2-EREBP-1 to AP2-EREBP-78, COX-1 to COX-6 and LTP-1 to LTP-10 for the AP2/EREBP, COX and LTP genes, respectively (Table S1 Online Resource SI 1).

Characterization of the AP2/EREBP, COX and LTP proteins in Z. mays L.

The sequence identities of 78 AP2/EREBP, 6 COX and 10 LTP proteins are shown by the color-by-E-value ratio (blue, ≤ 60%; green, ≤ 80%; orange, ≤ 90%), as shown in Fig. 1. Analysis of protein physical and chemical properties revealed that the length of the AP2/EREBP family amino acids in Z. mays L. ranged from 154 (AP2-EREBP-28) to 452 (AP2-EREBP-18). The length of the COX family amino acids ranged from 88 (COX-3) to 483 (COX-1). The length of the LTP family amino acids ranged from 106 (LTP-5) to 247 (LTP-1). The molecular weights (MWs) of AP2/EREBP ranged from 16837.95 (AP2-EREBP-28) to 48127.18 (AP2-EREBP-18). The molecular weights of COX ranged from 9972.12 (COX-3) to 51834.35 (COX-1). The molecular weights of the LTPs ranged from 11062.05 (LTP-5) to 24921.63 (LTP-1). The isoelectric point (PI) of AP2/EREBP ranged from 4.63 (AP2-EREBP-30) to 10.31 (AP2-EREBP-10). The isoelectric point of COX ranged from 4.46 (COX-4) to 10.25 (COX-1). The isoelectric point (p-P) of LTP ranged from 4.69 (LTP-7) to 9.73 (LTP-5). The total number of atoms in AP2/EREBP ranged from 2329 (AP2-EREBP-28) to 6674 (AP2-EREBP-24). The total number of atoms in COX ranged from 1361 (COX-3) to 7348 (COX-1). The total number of atoms in the LTP ranged from 1564 (LTP-5) to 3509 (LTP-1). The average hydropathicity value (GRAVY) of AP2/EREBP ranged from − 0.787 (AP2-EREBP-13) to -0.408 (AP2-EREBP-58). The average hydropathicity value of COX ranged from − 0.559 (COX-6) to 0.231 (COX-1). The average hydropathicity value of LTP ranged from 0.147 (LTP-10) to 0.683 (LTP-6) (Table S1 Online Resource SI 1).

Phylogenetic, chromosomal distribution, evolutionary analysis and synteny analysis of the AP2/EREBP, COX and LTP genes

A phylogenetic tree was constructed using maximum likelihood with 1000 bootstrap replicates, and the AP2/EREBP, COX and LTP protein sequences were used to analyze the possible evolutionary history of Z. mays L.. In the resulting phylogenetic tree, the AP2/EREBP proteins were classified into three distinct clades. The AP2/EREBP protein family (AP2) includes five different domain types according to the Phytozome-13 website [27]: the AP2/ERF domain, EREBP-like factor (EREBP), ethylene responsive factor (ERF), dehydration responsive element binding protein (DREB) and SHN SHINE (Fig. 2 and Table S2 Online Resource SI 1). COX and LTP proteins were classified into three distinct clades (Fig. S1. Online Resource SI 2).

Based on the information available on the Phytozome-13 website [27], the AP2/EREBP, COX and LTP genes were physically drawn on the chromosomes in the Z. mays L. genome. AP2/EREBP genes were found on 10 chromosomes of Z. mays L.. COX genes were found on chromosomes 1, 3, 4, 5, 7 and 8. LTP genes were found on chromosomes 1, 3, 6, 8, 9 and 10 (Fig. 3).

The selective pressure on the AP2/EREBP, COX and LTP genes was investigated by calculating the nonsynonymous/synonymous ratio (Ka/Ks). A Ka/Ks ratio > 1 suggested positive selection, a Ka/Ks ratio = 1 indicated neutral selection, and a Ka/Ks ratio < 1 suggested purifying selection [54]. In the present study, the Ka/Ks ratios of the AP2/EREBP paralogous pairs were less than 1, which indicates that the AP2/EREBP genes were influenced primarily by purifying selection, which suggests that the AP2/EREBP genes received strong environmental pressure during evolution. The Ka/Ks ratios of the COX-3/COX-4 paralogous pairs were greater than 1, which indicated that the COX-3/COX-4 genes were involved in positive or Darwinian selection (driving change). For the LTP genes, the Ka/Ks ratios of the LTP-1/LTP-10, LTP-5/LTP-3 and LTP-4/LTP-8 paralogous pairs were less than 1, while those of the LTP-2/LTP-6 paralogous pairs were greater than 1 (Table 1).

The duplication time of the AP2/EREBP paralogous gene pairs in Z. mays L. ranged from approximately 9.364 to 100.935 Mya. The duplication time of the COX-3/COX-4 paralogous gene pair was approximately 5.217 Mya. The duplication time of the LTP paralogous gene pairs ranged from approximately 19.064 to 96.477 Mya (Fig. 4 and Table 1).

Table 1

Paralogous pairs of *AP2/EREBP*, *COX* and *LTP* genes and the Ka/Ks ratio.
locus 1	locus 2	Ka	Ks	Ka/Ks	Time
AP2-EREBP-15	AP2-EREBP-16	0.201887685	0.502399279	0.401847083	38.292628
AP2-EREBP-36	AP2-EREBP-7	0.435204658	0.87977765	0.494675737	67.05622333
AP2-EREBP-52	AP2-EREBP-61	0.07647816	0.207878061	0.367899141	15.84436439
AP2-EREBP-8	AP2-EREBP-20	0.385987339	0.570267544	0.676853071	43.46551404
AP2-EREBP-74	AP2-EREBP-18	0.05168891	0.193976752	0.266469614	14.78481342
AP2-EREBP-69	AP2-EREBP-3	0.077309887	0.187624472	0.412045858	14.30064575
AP2-EREBP-44	AP2-EREBP-32	0.057691935	0.122859443	0.469576728	9.364286818
AP2-EREBP-54	AP2-EREBP-59	0.110298946	0.164779455	0.669373168	12.55940971
AP2-EREBP-50	AP2-EREBP-11	0.338025374	0.624790478	0.541021968	47.62122548
AP2-EREBP-21	AP2-EREBP-71	0.334683458	0.545162686	0.61391483	41.55203397
AP2-EREBP-9	AP2-EREBP-23	0.103291176	0.277490994	0.372232534	21.15022823
AP2-EREBP-33	AP2-EREBP-67	0.176957458	1.324271753	0.133626242	100.935347
AP2-EREBP-47	AP2-EREBP-63	0.128995603	0.294776002	0.437605512	22.46768307
AP2-EREBP-4	AP2-EREBP-30	0.449320876	0.611974544	0.734214978	46.64440122
AP2-EREBP-49	AP2-EREBP-64	0.056604591	0.171835289	0.329411909	13.09720188
AP2-EREBP-37	AP2-EREBP-73	0.290799369	0.632410291	0.459827067	48.2020039
AP2-EREBP-34	AP2-EREBP-35	0.187778474	0.553114724	0.339492814	42.15813446
AP2-EREBP-42	AP2-EREBP-26	0.083190211	0.207698132	0.400534227	15.83065032
COX-3	COX-4	0.100244299	0.068451386	1.464459755	5.217331218
LTP-1	LTP-10	0.80611789	1.265784761	0.636852264	96.477497
LTP-2	LTP-6	0.837292901	0.570546713	1.467527341	43.48679218
LTP-5	LTP-3	0.397424829	0.419284855	0.947863543	31.95768715
LTP-4	LTP-8	0.111949387	0.25012764	0.447569039	19.06460671

The AP2/EREBP, COX and LTP genes were analyzed for interspecies collinearity to determine the orthologous relationships of Z. mays L. with O. sativa, H. vulgare and A. thaliana. Collinearity analysis revealed robust orthologs of the AP2/EREBP, COX and LTP genes among Z. mays L. compared with those of the other three plant species (Fig. 5 and Table S3 Online Resource SI 1).

Conserved domain, conserved motif and gene structure and Promoter analyses of the AP2/EREBP, COX and LTP genes

Domain analysis was carried out for all 78 AP2/EREBP, 6 COX and 10 LTP proteins, and domain analysis confirmed the presence of the AP2 domain (Fig. 6), Cyt_c_Oxidase_Vb domain (Fig. S2 Online Resource SI 2) and the nsLTP1 domain (Fig. S3. Online Resource SI 2) on the AP2/EREBP, COX and LTP proteins, respectively. Motif analysis indicated that the phylogenetic relationships were similar to the conserved motif distributions within the clade. For instance, the motif distributions of the AP2/EREBP, COX and LTP proteins exhibited similar motifs within the clade, with few differences. The AP2/EREBP motif distributions for AP2-EREBP-26, AP2-EREBP-42, AP2-EREBP-68, AP2-EREBP-43, AP2-EREBP-27, AP2-EREBP-14, AP2-EREBP-75, AP2-EREBP-41, AP2-EREBP-66, AP2-EREBP-35, AP2-EREBP-34, AP2-EREBP-22, AP2-EREBP-73, AP2-EREBP-37, AP2-EREBP-30, AP2-EREBP-4, AP2-EREBP-46, AP2-EREBP-13, AP2-EREBP-77, AP2-EREBP-64 and AP2-EREBP-49 proteins had conserved motif numbers 1, 2, 3, and 8. The AP2-EREBP-67, AP2-EREBP-33, AP2-EREBP-51, AP2-EREBP-12, AP2-EREBP-78, AP2-EREBP-28, AP2-EREBP-63 and AP2-EREBP-47 proteins carried conserved motif numbers 1, 2, 3, and 9. The remaining AP2/EREBP proteins carried conserved motif numbers 1, 2 and 3 (Fig. 6 and Sheet 2 Online Resource SI 1). The COX motif distributions for the COX-4, COX-3, COX-2 and COX-6 proteins revealed conserved motif numbers 1 and 3 (Fig. 6 and Sheet 3 Online Resource SI 1). Most of the LTP proteins presented conserved motif numbers of 1, 2 and 3 (Fig. 6 and Sheet 4 Online Resource SI 1). The exon‒intron structure is an important source of plant biodiversity and gene family evolution. The gene structure results revealed that 21 of the 78 AP2/EREBP genes had introns (Fig. 6). All the COX genes had introns, while 8 LTP genes had introns (Fig. S2 and Fig. S3 Online Resource SI 2).

The AP2/EREBP, COX and LTP gene sequences (1500 bp upstream of the start codon) (Table S4 Online Resource SI 1) were selected for cis-element analysis using the PlantCARE web tool to identify their biological functions (stress response, growth and development). The promoter regions of the AP2/EREBP, COX and LTP genes in Z. mays L. contain a large number of plant hormone response elements. Most AP2/EREBP, COX and LTP proteins contain defense and stress response elements, abscisic acid-responsive elements, methyl jasmonate (MeJA)-responsive elements, salylic acid and the MYB binding site (MBS) element, which are involved in the drought response (Fig. 7).

Subcellular localization, nuclear localization signal, transmembrane helices, phosphorylation sites and three-dimensional (3-D) structure prediction

Subcellular localization analysis revealed that most of the AP2/EREBP proteins were located in the nucleus. COX proteins were predicted to be expressed in different organelles; for instance, COX-1 was predicted to be expressed in the plasma membrane, whereas COX-2 and COX-6 were predicted to be expressed in chloroplasts. Most of the LTP proteins were located in the extracellular space. A heatmap was constructed to predict the subcellular localization of the AP2/EREBP, COX and LTP proteins, as shown in Fig. S4 Online Resource SI 2 and Table S5 Online Resource SI 1.

Sixteen putative nuclear localization signals (NLSs) were predicted for 21 AP2/EREBP proteins, whereas no nuclear localization signals (NLSs) were predicted for COX or LTP proteins (Table S6 Online Resource SI 1).

The TMHMM results predicted the transmembrane helices in AP2-EREBP-10, AP2-EREBP-21, COX-1 and all 10 LTP proteins (Fig. S5, Fig. S6 and Fig. S7 Online Resource SI 2).

The phosphorylation site prediction results for the AP2/EREBP, COX and LTP proteins for kinases are shown in Table S7 Online Resource SI 1.

To study the putative functions of the AP2/EREBP, COX and LTP proteins in Z. mays L., we selected a protein from each clade. The AP2-EREBP-24, AP2-EREBP-51, AP2-EREBP-53, COX-1, COX-2, COX-5, LTP-1, LTP-3 and LTP-7 proteins were modeled with I-TASSER software to construct 3-D structures. The 3-D structures were constructed according to similar structural templates and crystal structures obtained from the Protein Data Bank (Fig. 8). C-scores were used to estimate the confidence of the constructed protein model for the AP2-EREBP-24, AP2-EREBP-51, AP2-EREBP-53, COX-1, COX-2, COX-5, LTP-1, LTP-3 and LTP-7 proteins. The closest structural similarity protein models were selected as the best-predicted models for the AP2/EREBP, COX and LTP proteins, with C-scores ranging (Table 2). Due to their structural similarity, proteins that are structurally close to the target in the PDB often have similar functions. The C-scores suggested that the structures of the AP2/EREBP, COX and LTP proteins were constructed with high accuracy.

Table 2

Modeling parameters for the AP2/EREBP, COX and LTP proteins.
Protein	C-Score	TM-Score	RMSD (Å)	Best Identified Structural Analogs in PDB
Protein	C-Score	TM-Score	RMSD (Å)	PDB Hit	TM-Score ^a	RMSD ^a	IDEN ^a	Cov
AP2-EREBP-24	-1.98	0.48 ± 0.15	11.8 ± 4.5	5fmwA	0.896	2.59	0.094	0.957
AP2-EREBP-51	-3.82	0.30 ± 0.10	14.9 ± 3.6	6fokA	0.438	5.86	0.053	0.748
AP2-EREBP-53	-2.77	0.40 ± 0.13	11.8 ± 4.5	5wx9A	0.561	2.35	0.315	0.634
COX-1	-1.50	0.53 ± 0.15	10.8 ± 4.6	6a2jA	0.623	1.12	0.154	0.631
COX-2	-2.04	0.47 ± 0.15	9.5 ± 4.6	6t15d	0.690	1.18	0.283	0.727
COX-5	-4.30	0.26 ± 0.08	16.4 ± 3.0	6rc9A1	0.435	6.24	0.063	0.771
LTP-1	-4.31	0.26 ± 0.08	16.5 ± 3.0	5owvC	0.536	4.62	0.044	0.757
LTP-3	-1.19	0.57 ± 0.15	6.8 ± 4.1	4xuwA	0.718	0.88	0.446	0.748
LTP-7	-0.89	0.60 ± 0.14	6.1 ± 3.8	4xuwA	0.720	1.08	0.319	0.765

Prediction of miRNAs targeting the AP2/EREBP, COX and LTP proteins

A total of 187 microRNAs were predicted to target the AP2/EREBP genes, 48 microRNAs were predicted to target COX proteins, and 30 microRNAs were predicted to target LTP genes. The microRNA targeting relationships for the AP2/EREBP, COX and LTP genes are shown in Table S8 Online Resource SI 1.

The results from the prediction of RNA secondary structures with pseudoknots for the AP2/EREBP (AP2-EREBP-24, AP2-EREBP-51 and AP2-EREBP-53); COX (COX-1, COX-2 and COX-5); and LTP (LTP-1, LTP-3 and LTP-7) proteins are shown in Fig. S8: Fig. S16 Online Resource SI 2.

Gene Ontology enrichment and functional relationship analysis

To further determine the functions of the AP2/EREBP, COX and LTP genes, we performed enrichment analysis and gene ontology (GO) analysis based on biological processes and molecular functions. GO terms help us understand the function of genes at the molecular level (Fig. S17, Fig. S18 and Fig. S19 Online Resource SI 2). GO terms for the AP2/EREBP, COX and LTP genes confirmed the functional role of AP2/EREBP, COX and LTP as stress responsive genes (Fig. S20, Fig. S21 and Fig. S22 Online Resource SI 2).

In the present study, qRT‒PCR analysis revealed that the AP2/EREBP, COX and LTP proteins were expressed in leaves, and drought decreased the expression levels of AP2/EREBP, COX and LTP by 0.84, 0.53 and 0.31, respectively, after 12 h of drought stress (Sheet 1 Online Resource SI 1). Domain structure, promoter and gene ontology enrichment analyses confirmed the functional role of the AP2/EREBP, COX and LTP proteins in stress responses.

Genome-wide analysis identified 78 AP2/EREBP genes in Z. mays L.. Phylogenetic classification revealed that the 78 AP2/EREBP proteins could be divided into three distinct clades, which included the AP2/ERF domain, the EREBP-like factor (EREBP), the ethylene responsive factor (ERF), the dehydration responsive element binding protein (DREB) and the SHN SHINE according to the description available on Phytozome-13 [27]. Cheng et al. [55] identified 229 AP2/ERF genes in the maize genome. In addition, phylogenetic analysis revealed that the ZmAP2/ERF family members could be divided into five clades, namely, 27 AP2 (APETALA2), 105 ERF (ethylene responsive factor), 89 DREB (dehydration responsive element binding), 5 RAV (related to ABI3/VP) and a soloist. In this study, we identified 6 COXs in Z. mays L.. To date, no comprehensive investigation of the COX gene in maize has been reported via genome-wide analysis. We also identified 10 LTP genes in Z. mays L.. Wei and Zhong [56] identified 63 LTP genes in maize, which were divided into six types, whereas Fang et al. [57] identified 65 LTP genes in maize. Our qRT‒PCR results indicated that AP2/EREBP COX and LTP were downregulated, with fold changes of 0.84, 0.53 and 0.31, respectively, under drought stress. Sharoni et al. [58] reported the same results for the AP2, DREB, and ERF genes in the IR77298-14-1-2-B-10 line; these genes were highly activated in leaves under severe stress treatment and downregulated under severe stress treatment. Similarly, Trindade et al. [59] reported the same results in Medicago truncatula for COX5b, which strongly downregulated under water deficit conditions. Wei and Zhong [56] reported the same results in maize, in four ZmLTP which were downregulated under drought stress. These results suggest that, under drought stress, ZmLTP genes may exhibit tissue-specific expression because ZmLTP1.2 was significantly downregulated in ovarian tissue but upregulated in the leaf meristem. In the present study, Pfam domain analysis confirmed the presence of the AP2 domain, Cyt_c_Oxidase_Vb and nsLTP1 on the AP2/EREBP, COX and LTP proteins, respectively. Motif and gene structure analyses indicated that genes with closer phylogenetic relationships exhibited more similar genetic structures. The promoter regions of the AP2/EREBP, COX and LTP genes contain defense and stress response elements, abscisic acid-responsive elements, methyl jasmonate (MeJA)-responsive elements, salylic acid and the MYB binding site (MBS) element, which are involved in the drought response. The AP2/EREBP genes were found on 10 chromosomes of Z. mays L., the same results were found by Cheng et al [55]. COX genes were found on chromosomes 1, 3, 4, 5, 7 and 8. LTP genes were found on chromosomes 1, 3, 6, 8, 9 and 10. The Ka/Ks ratios of the AP2/EREBP paralogous pairs were less than 1, which indicates that the AP2/EREBP genes were influenced primarily by purifying selection, which means that the AP2/EREBP genes received strong environmental pressure during evolution. The Ka/Ks ratios of the COX-3/COX-4 paralogous pairs were greater than 1, which indicated that the COX-3/COX-4 genes were involved in positive or Darwinian selection (driving change). For the LTP genes, the Ka/Ks ratios of the LTP-1/LTP-10, LTP-5/LTP-3 and LTP-4/LTP-8 paralogous pairs were less than 1, which indicates that these genes were involved in purifying selection, while the Ka/Ks ratios of the LTP-2/LTP-6 paralogous pairs were greater than 1. Synteny analysis of the AP2/EREBP, COX and LTP proteins revealed collinearity orthologous relationships in O. sativa, H. vulgare and A. thailana. Gene Ontology enrichment analysis confirmed the functional role of stress-responsive AP2/EREBP, COX and LTP.

This is the first study to identify the COX gene in Z. mays L. by genome-wide analysis. Domain structure, promoter and gene ontology enrichment analyses confirmed the functional role of the AP2/EREBP, COX and LTP proteins in stress responses. The results of the present study could improve the understanding of how AP2/EREBP, COX and LTP are mechanistically linked to drought stress responses in maize and could be used for the genetic improvement of maize.

AP2/EREBP

APETALA2/ethylene-responsive element-binding protein

COX

cytochrome c oxidase

LTP

nonspecific lipid transfer protein

Ka/Ks

ratio of nonsynonymous/synonymous

RMSD

root mean square deviation.

Ethical approval:

The authors declare that the experimental research work involving the growth of plants in this study, was conducted in compliance with relevant institutional, national, and international guidelines and legislation.

Data availability:

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Authors contributions

A. M. devised the study, M. A. and A. M. collected the data, conducted the analysis, and discussed the data. A. M. wrote the first draft of the manuscript. Both authors wrote, read, and approved the final version of the manuscript.

Funding

No funding was received.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Author affiliations

[M. A].1 Botany and Microbiology Department, Faculty of Science, Cairo University, Giza, Egypt.

[A. M]. 2 Department of Plant Genetic Transformation, Agricultural Genetic Engineering Research Institute (AGERI), Agricultural Research Center (ARC), Giza, Egypt.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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The authors declare no competing interests.

Genome-wide identification, characterization and evolutionary analysis of the APETALA 2/ethylene responsive element-binding protein (AP2/EREBP), cytochrome c oxidase (COX) and lipid transfer protein (LTP) genes in Zea mays under drought stress

Status:

Version 1

Abstract

Figures

Introduction

METHODS

Results

Gene Ontology enrichment and functional relationship analysis

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1