7.2
CiteScore
3.7
Impact Factor
Generic selectors
Exact matches only
Search in title
Search in content
Post Type Selectors
Search in posts
Search in pages
Filter by Categories
ABUNDANCE ESTIMATION IN AN ARID ENVIRONMENT
Case Study
Correspondence
Corrigendum
Editorial
Full Length Article
Invited review
Letter to the Editor
Original Article
Retraction notice
REVIEW
Review Article
SHORT COMMUNICATION
Short review
7.2
CiteScore
3.7
Impact Factor
Generic selectors
Exact matches only
Search in title
Search in content
Post Type Selectors
Search in posts
Search in pages
Filter by Categories
ABUNDANCE ESTIMATION IN AN ARID ENVIRONMENT
Case Study
Correspondence
Corrigendum
Editorial
Full Length Article
Invited review
Letter to the Editor
Original Article
Retraction notice
REVIEW
Review Article
SHORT COMMUNICATION
Short review
View/Download PDF

Translate this page into:

ar Arabiczh-CN Chinese (Simplified)nl Dutchen Englishfr Frenchde Germanel Greekiw Hebrewit Italianja Japaneseko Koreanfa Persianpt Portugueseru Russianes Spanishtr Turkishur Urdu
en English

Original article
11 2022
:34;
102293
doi:
10.1016/j.jksus.2022.102293

Prediction of RNA editing sites and genome-wide characterization of PERK gene family in maize (Zea mays L.) in response to drought stress

, , , , , , , , ,
a Institute of Plant Breeding and Biotechnology, MNS University of Agriculture, Multan, Pakistan
b Department of Plant Breeding and Genetics, Faculty Agriculture, Islamia University of Bahawalpur, Pakistan
c Department of Plant Breeding and Genetics, Faculty Agriculture, University of Agriculture Faisalabad, Pakistan
d Department of Agricultural Genetic Engineering, Faculty of Agricultural Sciences and Technologies, Nigde Omer Halisdemir University, Niğde, Turkey
e Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CNRS, Gif-sur-Yvette, France
f Zoology Department, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
g Department of Botany, Hindu College Moradabad (Mahatma Jyotiba Phule Rohilkhand University Bareilly), 244001, India

⁎Corresponding authors. ali.sher@mnsuam.edu.pk (Muhammad Ali Sher), Zulfiqar_ali@uaf.edu.pk (Zulfiqar Ali)

Received: , Accepted: ,
© The Author(s) 2022
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

Objectives

Inadvertent climate changes continuously threating the crops production and thus affecting the livelihood of peoples across the world. The maize production at world level has severely been hampered by the drought stress. Proline-rich extensin-like receptor kinases (PERKs) are considered among the sub-class of plants larger protein family, receptor kinases. Member of PERK gene family play significant role in both abiotic and biotic stress and in various plant metabolic activities and pathways.

Methods

As of now, no comprehensive research is reported for PERK genes in maize. We have performed a genome wide in-silico analysis and identify twenty-three PERK genes in maize. We performed phylogenetic analysis, sequence logos, motif analysis, promoter analysis, chromosomal and subcellular localization, synteny and expression analysis using RNA seq data under drought stress. We also predict RNA editing sites in mitochondrial and chloroplast genome.

Results

Phylogenetic study of PERK genes from eight different plant species divided into four distinct clades. Four subclasses group of ZmPERKs were observed based on domain organization, motif pattern, and phylogenetic analysis. The exon–intron arrangement of the ZmPERK were conserved among members of the same subclasses. In the promoter region different cis-elements were found those were involved in the growth and development, as well as light and stress response. Through gene duplication analysis it was observed that segmental duplications in ZmPERKs played major role in maize evolution. The Ka/Ks ratios indicated that most ZmPERK genes during the evolution have experienced strong purifying selection. The conversion of cytosine (C) to uracil (U) was observed in all predicted editing sites (U). These transitions were mostly based on changes in the first and second codon bases. The in-silico expression analysis of transcriptome data revealed the differential expression of ZmPERK genes in response to drought stress and oil content accumulation.

Conclusion

The current study provides base information on the PERK gene family in maize. Our findings can serve as a reference for further functional analysis of ZmPERKs. These genes can be further explored and used in breeding program to develop cultivars resilient to drought stress.

Keywords

PERK
Genome wide analysis
RNA editing sites drought
Oil content
1

1 Introduction

Maize is considered as important crop which is grown for food and feed around the globe. Abiotic stress especially drought stress severely affected the maize production at world level. Plant breeders are considering the drought stress as one of the most important abiotic stress that causing the hindrance in getting the higher grain yield in different crops especially in maize (Liu and Qin, 2021). Hence it is dire need of the time that breeders should tailor their modern varieties with novel traits that have capacity to buffer the drastic effects of the abiotic stresses especially drought stress. The advent of modern genotyping techniques especially the next generation sequencing (NGS) has revolutionized the field of genetics, furthermore the release of genome dataset has made a substantial increment in developing the strategies to quip the plants with novel traits (Leng and Zhao, 2020). In plants the receptor-like kinases (RLKs) having similar structure are considered as large superfamily of proteins. In this group the PERKs (Proline rich extension like receptor kinases) gene family is also included. Plant Species such as Arabidopsis and rice comprises large gene family of receptor kinase. There is 600 members are reported in arabidopsis for the receptor kinase family and their analogous have been found in around 20 different species (Morris and Walker, 2003). These kinase family play have been found playing a crucial role in the growth and development phase of plants and also defensive mechanisms (Shiu and Bleecker, 2001, Morris and Walker, 2003, Shiu et al., 2004). Role of most of the members of receptor kinase is still unknown. Receptors like kinase are the protein comprising extracellular, carboxyl terminal and intercellular domain with putative amino terminal (see Fig. 1).

Sequence logos of PERK gene family between Maize, Rice and Arabidopsis.
Fig. 1
Sequence logos of PERK gene family between Maize, Rice and Arabidopsis.

Depending on their extracellular domain, receptor kinases interact to a wide range of substances for example, carbohydrates and cell wall components. This domain organization have much resemblance to the animal receptor tyrosine kinases (Shiu, 2001). Receptor kinases have specific and extracellular domains, for example, leucine-rich repeat (LRR) and proline-rich extension-like receptor kinases. Gene duplication and functional redundancy also reported among these different classes of receptor kinase (Champion et al., 2004). The CLV1 and ERECTA receptor kinase is the evidence of existence of functional redundancy (Diévart et al., 2003, Shpak et al., 2003, Shpak et al., 2004). PERK gene family of Arabidopsis have maximum sequence identity to Brassica napus and such as PERK1 of Arabidopsis is much like PERK1 of Brassica napus. Researchers have reported fifteen PERK genes in the Arabidopsis yet their functions still need to be characterize (Silva and Goring, 2002, Nakhamchik et al., 2004, Bai et al., 2009). In Arabidopsis the PERK1 is identified which do functions in response to any wound that occur in the plasma membrane (Silva and Goring, 2002). Likewise, PERK4 is predicted as key regulator for Ca2+ signaling that contributes in production of abscisic acid in root (Bai et al., 2009). It is well documented that under abiotic stresses plant accumulate more calcium contents in cells to boost the production of antioxidant enzyme activity, regulate lipid peroxidation of cell membranes and stomatal apertures to mitigate the impact of stresses on plant growth (Mansfield et al., 1990; Abadi and Sepehri, 2016). The production of reactive oxygen species (ROS) decline in the presence of PERKs whereas increasing level of ROS work as a signal for root hair development (Xing et al., 2013). In an organism the first line of defense against superoxide radicals is the production of superoxide dismutase (SODs) which catalyze the superoxide radical to hydrogen peroxide and molecular oxygen. The copper/zinc SOD (Cu/Zn SOD) is catalyzed through the MAPK cascade under high light-induction. The homologous proteins like MPK3 and MPK6 in plants are detected using the anti-PERK antibodies from animals (Samuel and Ellis, 2002; Hwang et al., 2016). Environmental stresses like heat, drought, nutrients, heavy metals, pathogens, keep threating the plants to express its fully genetic potential. It is becoming more important to scientist to reveal how plants response to internal and external stimuli. Plant sense the environmental changes through the use of cell surface receptors and initiate different signaling pathways to trigger the adaptive responses (Zhu, 2016).

Erratic climate change has become a major constraint in achieving the higher crop yield. At crop level it affects plant morphological, anatomical, and physiological attributes which ultimately results in drastic economic yield loss. Maize is a major food and feed crop grown all over the world. It rated as the world's third most significant staple grain crop (Tiwari and Yadav, 2019). Characterization of PERK family in maize can help us to understand the plant molecular mechanism of tolerance against biotic and abiotic stresses. Only a few PERK genes have been characterized, and the functions of most of them is still unknown. High-throughput genome sequencing of the maize provided an excellent opportunity for genome wide analysis of genes families. In our study, we performed in-silico genome wide analysis of PERK genes in maize. We analyzed phylogenetic relationship between 8 species and only maize separately. Furthermore, gene structure Intron/exons, motif distribution, conserved domain analysis, sequence logos, physio-chemical properties. The structural and functional importance of genes were also assessed using Ka/Ks values and synteny analysis. In-silico expression analysis were also performed for these genes to predict their role and function. The present study and their result enabled us to conclude that PERK gene family paly vital role in maize development and stress response (Fig. 2).

PERK gene family phylogenetic tree. The major cluster of orthologous genes is distinguished with various colours (PERKA-D).
Fig. 2
PERK gene family phylogenetic tree. The major cluster of orthologous genes is distinguished with various colours (PERKA-D).

2

2 Materials and methods

2.1

2.1 PERK gene family identification and characterization in maize genome

Whole maize genome sequence, as well as the general feature format file (GFF3), was downloaded from the Maize Genetic and Genomic Database (Maize GDB)(https://gamma.maizegdb.org). For the purposes of finding the probable candidates of PERK family in maize, the online Pfam database (https://www.sanger.ac.uk/Software/Pfam/) was used to download the PERK domain HMM profile and then subjected as a query into Blastp (Finn et al., 2014). For all of the retrieved protein sequences, the SMART tools (https://smart.embl-heidelberg.de/) were used to verify the presence of the PERK domain (Letunic et al., 2015). Maize PERK gene family sequence were downloaded from maize genome database. TAIR 10 (http: /https://www.Arabidopsis.org) was used to retrieved the arabidopsis sequences while all other sequences of studied organisms were retrieved from online plant database Phytozome version 11 (https://phytozome.jgi.doe.gov/pz/portal.html). ExPASyProtParam, (https://us.expasy.org/tools/protparam.html) an online web tools, were used to retrieve the physiochemical properties.

2.2

2.2 Sequence logos and phylogenetic/evolutionary analysis

The MEGA 7 software was used to find out the conserved sequences for amino acids. Sequence are aligned using ClustalW and the structure was constructed using TBtool (https.//github.com/CJ-Chen/TBtools). Furthermore, using this software the Neighbor-Joining method was used to get the phylogenetic tree to deduce the evolutionary history (Chothia et al., 2003). The distances of the number of amino acid sites in units were measured using the poisson correction parameters (Yang et al., 2008). The Bootstrap algorithm employed with 1000 repetitions to estimate the stability of the nodes in the phylogenetic tree. Total 98 amino acid sequences were used for this analysis.

2.3

2.3 Predicted protein motifs, structure of exon/intron and conserved domain analysis

To find preserved motif of PERK protein online web server Multiple Em for Motif Elicitation (MEME) is used (https://meme-suite.org/tools/meme). The TBtool was used to construct the motif structure using the MEME.xml file which is obtained through MEME suite. The default parameters were as follows: motif recurrence was set to 1 per sequence; frequency of motifs was set to 10; motif width was set to 5–50 residues; and the minimum number of motif sites was set to 5. Arrangement of Exon and Intron of PERK genes was investigated by using gff3 file downloaded from maize GDB. Structure is constructed using TBtool software. Afterward, the NCBI CDD tool (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) was used to perform the conserved domain analysis.

2.4

2.4 Chromosomal localization, gene duplication events and synteny analysis

The start and end position for each identified ZmPERK gene were procure from the Maize Genetic and Genomic Database (Maize GDB) and validated against the GFF3 file. Total chromosomal length is retrieved through using FASTA stat in TBtool. Finally, using MapChart v2.32 (Voorrips, 2002), the ZmPERK genes were spatially mapped onto the maize chromosomes. The phylogenetic tree was used to identify the putative paralogous PERK gene pairs. The resulting pairs were subjected to TBtool software to determine synonymous and non-synonymous substitution rates (Ka). To determine the nature of codon selection the Ka/Ks ratio was also calculated that allegedly occurred during evolution. Further, using the formula T = Ks/2 and assuming a clock rate of 6.05 X10 9 substitutions/synonymous site/year for maize, the approximate period of duplication event was calculated (Kong et al., 2013). The genome sequence files, and gene annotation files (GFF3) of sorghum, rice and maize were used for the collinearity analysis. Required files generated using one step MC scan. TBtool software was used to visualize the results, and the parameter filtering genes in the collinearity block was set to 40.

2.5

2.5 RNA editing sites prediction, subcellular localization, and promoter analysis

RNA editing is a method in which certain cytidines in mitochondrial and chloroplast transcripts of plants are converted to uridines. The online web server like PREP-Cp (for chloroplast genes) and PREP-Mt (for mitochondrial genes) software (https://prep.unl.edu/) with the cutoff value to 0.8 were used in predicting the RNA editing sites (Mower, 2009). Location of genes at cellular level were also predicted using online web server softberry (https://www.softberry.com). In order to perform promoter analysis, the 5′ upstream region of each gene of the ZmPERK was downloaded from NCBI (https://www.ncbi.nlm.nih.gov/) and the resulting file was submitted to the online database plantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) for a cis-element scan.

2.6

2.6 In-silco expression analysis

The NCBI Geodataset (https://www.ncbi.nlm.nih.gov/gds) was used to obtain transcriptome data (GSE136087, GSE40070). RNA-sequencing analysis was performed on seed embryos from 15 days after pollination (DAP) to 40 days after pollination (DAP) (Sekhon et al., 2013). We also retrieved drought stress expression data. Inbred line B73 maize (Zea mays) plants grown in the green house under well-watered and drought stress circumstances until they reached the reproductive stage (at the onset of silk emergence). Plants were hand pollinated for drought stress two to three days after irrigation was stopped, and measurements and samples were collected 24 h later for transcriptome analysis. This gene expression data was used to construct heat map.

3

3 Results

3.1

3.1 Identification and characterization of PERK gene family in maize genome

A systematic approach was followed to identify the PERK protein encoding genes. Taking the advantage of publically available genome of maize, after removing redundant genes we excavated 23 PERK genes in maize genome and named them as ZmPERK1 to ZmPERK23. The PERK gene family comprises 15 Arabidopsis thaliana genes, 8 Oryza sativa genes, 14 Populus trichocarpa genes, 14 Sorghum bicolor genes, 9 Theobroma cacao genes, 23 Z. mays genes, 10 Ananas comosus genes, and 5 Physcomiterlla patens genes. The biophysical properties of the ZmPERK family genes were then determined, including genes ID, start and end positions of genes on chromosomes, polarity of strand, length of CDS sequence (bp), length of protein sequence (aa), protein molecular weights (MW), isoelectric points (pl), and predicted subcellular localization of ZmPERK genes. Table 1 presents all additional estimated biophysical properties. ZmPERK proteins had a peptide length ranged from 432 to 958 amino acids, with an average of 695 A.A (Table 1). The PI (Isoelectric point) of maize PERKs varied between 6.2 and 9.134, while the molecular weight ranged between 36.45 and 100.93 kDa, with an average of 68.69 kDa. The length of nucleotide, amino acid sequences varied greatly, indicating that the ZmPERK genes are highly complex, implying a high level of complexity.

Table 1 Physiochemical properties including Gene IDs, Gene name, Chromosome No, Gene start–end position, Strand, CDS and amino acid sequence length, Protein MW, PI, GRAVY and cellular localization of ZmPERK genes.
Transcripts ID Gene name chromosome Position Strand CDS (bp) Protein
length A.A		 Protein Molecular Weight KDA PI Grand
average
of hydropathicity (GRAVY)		 Subcelluar
lcalization
Start End
Zm00001d037811 ZmPERK1 6 138,545,311 138,550,589 - 1299 432 59.97726 6.47 −0.483 Cell membrane/plasma membrane
Zm00001d043480 ZmPERK2 3 201,473,204 201,476,633 – 2208 735 76.90377 8.51 −0.41 Cell membrane/plasma membrane
Zm00001d011908 ZmPERK3 8 164,313,713 164,318,410 – 1479 792 53.98962 9.3 −0.633 Cell membrane/plasma membrane
Zm00001d034257 ZmPERK4 1 288,176,999 288,179,853 – 1761 588 61.80629 9.13 −0.475 Cell membrane/plasma membrane
Zm00001d030218 ZmPERK5 1 113,763,279 113,766,247 + 1488 495 53.51192 6.41 0.37 Cell membrane/plasma membrane
Zm00001d041476 ZmPERK6 3 122,414,822 122,416,911 – 1524 507 56.60236 9.12 −0.039 Cell membrane/plasma membrane
Zm00001d020148 ZmPERK7 7 95,798,411 95,800,410 – 1797 598 63.45431 8.31 −0.228 Cell membrane/plasma membrane
Zm00001d035774 ZmPERK8 6 47,428,910 47,432,469 – 1788 595 63.40166 8.91 −0.311 Cell membrane/plasma membrane
Zm00001d028337 ZmPERK9 1 31,103,531 31,114,482 – 1752 583 61.77299 6.5 −0.461 Cell membrane/plasma membrane
Zm00001d012743 ZmPERK10 8 179,562,397 179,566,026 + 1233 410 45.3461 9.17 −0.463 Cell membrane/plasma membrane
Zm00001d037464 ZmPERK11 6 126,133,881 126,136,630 + 1671 556 58.84482 5.68 −0.401 Cell membrane/plasma membrane
Zm00001d011450 ZmPERK12 8 150,848,228 150,851,704 – 2052 683 71.52957 8.67 −0.488 Cell membrane/plasma membrane
Zm00001d026668 ZmPERK13 10 149,652,422 149,656,130 – 2877 958 100.93417 5.24 −0.048 Cell membrane/plasma membrane
Zm00001d049391 ZmPERK14 4 28,932,486 28,936,031 + 1002 333 36.4539 8.97 −0.07 Cell membrane/plasma membrane
Zm00001d007848 ZmPERK15 2 240,835,869 240,838,408 – 1614 535 56.9581 9.15 −0.383 Cell membrane/plasma membrane
Zm00001d010421 ZmPERK16 8 114,332,739 114,337,964 + 1989 662 69.62817 8.72 −0.523 Cell membrane/plasma membrane
Zm00001d037066 ZmPERK17 6 111,296,289 111,300,781 + 1789 662 75.50927 6.3 −0.515 Cell membrane/plasma membrane
Zm00001d031482 ZmPERK18 1 191,554,665 191,555,908 + 1401 466 51.97133 6.2 0.159 Cytoplasm
Zm00001d042185 ZmPERK19 3 155,343,261 155,349,083 – 1476 491 53.59911 9.15 −0.608 Cell membrane/plasma membrane
Zm00001d039311 ZmPERK20 3 1,451,510 1,456,285 – 1125 374 57.67767 8.41 −0.488 Cell membrane/plasma membrane
Zm00001d040127 ZmPERK21 3 28,623,779 28,627,767 – 2076 691 72.32502 8.44 −0.278 Cell membrane/plasma membrane
Zm00001d038708 ZmPERK22 6 163,090,768 163,092,715 – 2691 896 93.90373 8.61 −0.3 Cell membrane/plasma membrane
Zm00001d039176 ZmPERK23 6 171,824,226 171,828,823 + 1401 466 51.97133 6.2 0.159 Cytoplasm

3.2

3.2 Sequence logos and phylogenetic/evolutionary analysis

Sequence logos analysis provide more comprehensive information for sequence similarities, significant alignment aspects, and sequence conservation patterns. To check PERK family evolution, we generate sequence logos and results showed that this family remained conserved throughout evolution. For comparison study, the protein sequences of maize, rice and Arabidopsis were used. The results reveal that consensus sequence residues were highly conserved, and there was no compositional bias seen across any specie. These results help in discover and analyze and evaluate PERK gene family protein sequence across the species.

Phylogenetic tree serves an important way to understand the evolutionary relationships pathways. In our study we created phylogenetic tree of PERK genes to depict the evolutionary relationships. The phylogenetic or evolutionary analysis revealed the oldest plant lineage, of the PERK gene family as its members were found in Ananas comosus (angiosperm), Physcomitrella patens (bryophytes), dicots (Arabidopsis thaliana, Theobroma cacao, Populus trichocarpa), and monocots (Oryza sativa, Sorghum bicolor and Z. mays). These findings suggested that these genes evolved in ancient land plants, and that probable orthologous genes can be found across the plant kingdom. The PERK genes were characterized by 29 members in the PERK-A clade, 26 members in PERK-B, 19 members in PERK-C, and 22 members in PERK-D in the phylogenetic study. PERK genes were randomly distributed in all four clades from dicot, monocot, and bryophytes plant species, indicating that these genes evolved after the split of bryophytes. This finding showed that PERK genes possibly expanded and diversified after the radiation of these different species. These evolutionary linkages can facilitate the identification of orthologous genes and help to accelerate their functional characterization.

3.3

3.3 Predicted protein motifs, structure of exon/intron and conserved domain analysis

The 23 ZmPERK protein sequences were classified into four subfamilies using a rectangular phylogenetic tree (subfamily I, II, III, IV). 10 members were found in the Subfamily I, followed by subfamily II (5), subfamily III (4), and subfamily IV (4) (Fig. 3A). In addition, we examined the conserved motifs using MEME software to further investigate the diversity of ZmPERK protein family (Fig. 3). In this study, total 10 motifs were found (Table S3). All the gene exhibits same motif pattern. The type, order, and number of motifs were consistent within a subfamily, but varied across subfamilies. The patterns of ZmPERK protein motif distribution revealed that conserved distribution patterns existed for similar motifs. Domains 1, 2 and 3 represent the distinctive protein kinase-binding domain that is found in all 23 ZmPERK proteins (Fig. 3C). Similarly, Fig. 3D depicts the relative lengths of introns and exon sequence conservation within each ZmPERK gene in maize. A gene's biological function is linked to the distribution of exons and introns. All these genes contain exons ranged between 2 and 10. The findings demonstrated obvious conservation, laying the groundwork for functional conservatism and guiding future functional research.

A: Phylogenetic tree-based categorization of ZmPERK genes. An un-rooted phylogenetic tree an un-rooted phylogenetic tree based on full-length peptide sequences (ZmPERK) was generated. Classification is shown based on a phylogenetic tree using differences into groups. 3B: Motif pattern of ZmPERK genes 3C: Conserved domains of maize PERK protein 3D: Exon–intron structure analyses of ZmPERK genes. The purple line represents introns, while the purple boxes represent exons.
Fig. 3
A: Phylogenetic tree-based categorization of ZmPERK genes. An un-rooted phylogenetic tree an un-rooted phylogenetic tree based on full-length peptide sequences (ZmPERK) was generated. Classification is shown based on a phylogenetic tree using differences into groups. 3B: Motif pattern of ZmPERK genes 3C: Conserved domains of maize PERK protein 3D: Exon–intron structure analyses of ZmPERK genes. The purple line represents introns, while the purple boxes represent exons.

3.4

3.4 Chromosomal localization, gene duplication events and synteny analysis

Each ZmPERK gene's genomic DNA sequence was analyzed in the maize genome database using BLASTn to establish its location, and MapChart was used to visualize the position of identified ZmPERK members on their respective chromosomes. The chromosome map revealed that 23 PERK genes were dispersed out over 8 of the 10 chromosomes. (Fig. 4A). The most ZmPERK genes were found on chr06, which had six members, followed by chr01, 03, and 08, which had 4, 5, and 4 members, respectively. While chromosomes 2, 4, 7, and 10 each had only one gene. Zmchr06 had the most PERK genes (26.08%), followed by Zmchr3 (21.73%), Zmchr1, and Zmchr8 (17.39%), while Zmchr02, Zmchr04, Zmchr06, Zmchr07, and Zmchr10 had the lowest percentage (4.34%) (Fig. 4B).

A: Distribution of 23 ZmPERK genes on their respective chromosomes.4B. Pie chart representing percentage of genes present on chromosome.4C: Pictorial representation of paralog gene pairs on chromosome indicating the type of duplication either tandem or segmental.
Fig. 4
A: Distribution of 23 ZmPERK genes on their respective chromosomes.4B. Pie chart representing percentage of genes present on chromosome.4C: Pictorial representation of paralog gene pairs on chromosome indicating the type of duplication either tandem or segmental.

Gene duplications, either whole-genome or segmental, as well as tandem duplications, are critical for gene family evolution. Although it has been proven that segmental and tandem duplications play a key role in gene family evolution in all plants specie (Cannon et al., 2004).To investigate ZmPERK gene duplications and evolutionary processes in maize, we identified 09 pairs of probable paralogous genes using the maize PERK phylogenetic tree. It is well documented facts that tandem duplication observed when paralogous genes are present on the same chromosome, whereas segmental duplication arise when paralogous genes are located on distinct chromosomes (Panchy et al., 2016). All the paralogous gene pair appeared to have evolved by segmental duplication except one (ZmPERK5-ZmPERK9) which evolved through tandem duplication indicating that the evolution of PERK genes appears to have been dominated by segmental duplications in maize. (Fig. 4C) Segmental duplication is the primary force that drives the evolution of a gene family. The estimated time of divergence for paralogous gene pairs was determined using synonymous (Ks) and non-synonymous (Ka) substitution rates. The Ka/Ks ratios for all paralog ZmPERK varied between 0.10 and 0.65 (Table 2). It indicates that purifying selection may have been performed on codons in the development and proliferation of parallel PERK genes in maize.

Table 2 Gene duplication event along with Ka/Ks ratio and time of evolution MYA.
Gene I Gene II Ka Ks Ka/Ks Type of Duplication T = Ks/2λ
ZmPERK3 ZmPERK19 0.022323415 0.212693 0.104956101 Segmental 6.97
ZmPERK6 ZmPERK14 0.241933478 0.521139 0.658737178 Segmental 4.21
ZmPERK13 ZmPERK23 0.05503852 0.043242 0.120303727 Segmental 1.31
ZmPERK2 ZmPERK10 0.146633906 0.370521 0.395750292 Segmental 1.21
ZmPERK12 ZmPERK17 0.45049383 1.189433 0.378746589 Segmental 3.901
ZmPERK8 ZmPERK21 0.222577445 2.176048 0.102285191 Segmental 7.13
ZmPERK4 ZmPERK15 0.291936478 0.54189 0.538737178 Segmental 1.77
ZmPERK5 ZmPERK9 0.070311852 0.062761 0.160303727 Tandem 2.05
ZmPERK1 ZmPERK16 0.028134859 0.206977 0.135932012 Segmental 6.788

Non-synonymous and synonymous substitutions are designated by Ka and Ks, respectively.

Multiple collinearity scan tool was used to find orthologous genes among genomes of maize, Sorghum, and rice to further understand the Synteny links of ZmPERK genes with these plant species. (Fig. 5). 18 pairs of collinearity genes of PERK gene family between maize and rice whereas sixteen pairs in maize and sorghum were observed in the synteny analysis (Table S2). Gene IDs of all collinear genes is given supplemental file. According to these findings, the collinearity between maize and sorghum is significant as compared to the collinearity values between maize and rice furthermore, these PERK genes in maize derived from a common ancestor.

Collinearity analysis of maize, rice, and sorghum. (A) Collinearity analysis of all chromosomes reveals duplicated PERK genes in maize and sorghum. The lines connect the pairs of duplicated genes. (B) The collinearity study of maize and rice chromosomes. The PERK genes are represented by the red flags on distinct chromosomes.
Fig. 5
Collinearity analysis of maize, rice, and sorghum. (A) Collinearity analysis of all chromosomes reveals duplicated PERK genes in maize and sorghum. The lines connect the pairs of duplicated genes. (B) The collinearity study of maize and rice chromosomes. The PERK genes are represented by the red flags on distinct chromosomes.

3.5

3.5 Prediction of RNA editing sites, subcellular localization, and promoter analysis

The Prep-CP and Prep-Mt prediction tools were used to find the RNA editing sites of ZmPERK chloroplast and mitochondrial genes, respectively. In chloroplast genes, 196 RNA editing sites were predicted (Table 3A) and 268 in mitochondrial genes (Table 3B). All predicted editing sites in the chloroplast and mitochondrial genomes were distributed among 23 genes, with an average of 8.5 and 11.26 editing sites per gene, respectively. The chloroplast gene ZmPERK1 contains maximum RNA editing sites (12) while minimum editing sites (5) were predicted in ZmPERK14. Similarly, mitochondrial gene ZmPERK12 contain maximum editing sites (21) while minimum sites (7) were predicted in ZmPERK14. The position of RNA editing sites was further explored, and it was observed that all the predicted sites were based on first and second codon base changes. At the third codon base, we couldn't find any site for RNA editing. The transition of cytosine → uracil (C-U) seems to be present in all editing sites, resulting in amino acid substitutions. Eleven type of amino acid change found in chloroplast and mitochondrial genes (Fig. 6A). Amino acid conservation caused by RNA editing including A (Alanine) → V (Valine), T (Threonine) → I (Isoleucine), H (Histidine) → Y (Tyrosine), P (Proline) → S (Serine, P (Proline) → L (Leucine), R (Arginine) → C (Cysteine), S (Serine) → F (Phenylalanine), R (Arginine) → W (Tryptophan), P (Proline) → F (Phenylalanine), S (Serine) → L (Leucine), T (Threonine) → M (Methionine), L (Leucine)F (Phenylalanine).

Table 3A Predicted RNA editing sites in chloroplast genome.
Genes Nucleotide Position Amino acid Position Amino acid Conservation Genes Nucleotide Position Amino acid Position Amino acid Conservation Genes Nucleotide Position Amino acid Position Amino acid Conservation Genes Nucleotide Position Amino acid Position Amino acid Conservation
ZmPERK1 14 5 TCC (S) => CTC (F) ZmPERK7 134 45 CCG (P) => CTG (L) ZmPERK13 319 107 CTC (L) => TTC (F) ZmPERK19 4 2 CCC (P) => TCC (S)
175 59 CCG (P) => TCG (S) 485 162 TCT (S) => TTT (F) 850 284 CCG (P) => TCG (S) 65 22 TCG (S) => TTG (L)
203 68 CCG (P) => CTG (L) 514 172 CCG (P) => TCG (S) 853 285 CCC (P) => TCC (S) 77 26 ACG (T) => ATG (M)
280 94 CCA (P) => TCA (S) 521 174 CCA (P) => CTA (L) 1187 396 ACC (T) => ATC (I) 187 63 CAC (H) => TAC (Y)
331 111 CCT (P) => TCT (S) 856 286 CAC (H) => TAC (Y) 1246 416 CTC (L) => TTC (F) 209 70 CCG (P) => CTG (L)
338 113 CCG (P) => CTG (L) 949 317 CAC (H) => TAC (Y) 1379 460 CCA (P) => CTA (L) 250 84 CCG (P) => TCG (S)
340 114 CCG (P) => TCG (S) 1034 345 GCA (A) => GTA (V) 1556 519 TCC (S) => TTC (F) 292 98 CAT (H) => TAT (Y)
347 116 CCA (P) => CTA (L) 1225 409 CCC (P) => TTC (F) 1744 582 CAC (H) => TAC (Y) 349 117 CTT (L) => TTT (F)
544 182 CAC (H) => TAC (Y) 1226 409 CCC (P) => TTC (F) 1877 626 ACC (T) => ATC (I) 353 118 CCG (P) => CTG (L)
655 219 CAT (H) => TAT (Y) 355 119 CCG (P) => TCG (S)
ZmPERK8 236 79 CCC (P) => CTC (L) ZmPERK14 379 127 CGG (R) => TGG (W) 362 121 CCG (P) => CTG (L)
268 90 CCG (P) => TCG (S) 424 142 CTT (L) => TTT (F)
415 139 CCC (P) => TTC (F) 313 105 CCT (P) => TCT (S) 442 148 CCA (P) => TCA (S) ZmPERK20 211 71 CCA (P) => TCA (S)
ZmPERK2 416 139 CCC (P) => TTC (F) 812 271 TCG (S) => TTG (L) 881 294 GCC (A) => GTC (V) 236 79 CCT (P) => CTT (L)
461 154 TCA (S) => TTA (L) 1229 410 GCT (A) => GTT (V) 896 299 CCG (P) => CTG (L) 277 93 CCA (P) => TCA (S)
577 193 CCC (P) => TCC (S) 1277 426 GCT (A) => GTT (V) 322 108 CCG (P) => TCG (S)
742 248 CCG (P) => TCG (S) 1307 436 ACT (T) => ATT (I) ZmPERK15 326 109 CCG (P) => CTG (L) 373 125 CCT (P) => TCT (S)
778 260 CAC (H) => TAC (Y) 421 141 CCG (P) => TCG (S) 389 130 CCC (P) => CTC (L)
841 281 CAC (H) => TAC (Y) ZmPERK9 604 202 CCG (P) => TCG (S) 676 226 CAC (H) => TAC (Y) 406 136 CCA (P) => TCA (S)
979 327 CAT (H) => TAT (Y) 865 289 CAC (H) => TAC (Y) 1034 345 GCG (A) => GTG (V) 475 159 CCT (P) => TCT (S)
1093 365 CAC (H) => TAC (Y) 958 320 CAC (H) => TAC (Y) 1187 396 GCC (A) => GTC (V) 512 171 ACA (T) => ATA (I)
997 333 CCG (P) => TCG (S) 1241 414 CCG (P) => CTG (L)
863 288 ACC (T) => ATC (I) 1063 355 CCC (P) => TCC (S) 1244 415 ACG (T) => ATG (M) ZmPERK21 826 276 CCG (P) => TCG (S)
ZmPERK3 1036 346 CCT (P) => TCT (S) 1457 486 TCC (S) => TTC (F) 1306 436 CCC (P) => TTC (F) 851 284 ACG (T) => ATG (M)
1103 368 CCT (P) => CTT (L) 1307 436 CCC (P) => TTC (F) 1000 334 CCG (P) => TCG (S)
1151 384 CCT (P) => CTT (L) ZmPERK10 19 7 CTT (L) => TTT (F) 1520 507 CCC (P) => CTC (L) 1003 335 CCT (P) => TCT (S)
1187 396 TCG (S) => TTG (L) 241 81 CAC (H) => TAC (Y) 1040 347 CCG (P) => CTG (L)
1195 399 CAT (H) => TAT (Y) 556 186 CCT (P) => TCT (S) ZmPERK16 529 177 CCG (P) => TCG (S) 1072 358 CCG (P) => TCG (S)
1214 405 CCG (P) => CTG (L) 737 246 GCT (A) => GTT (V) 586 196 CCA (P) => TCA (S) 1117 373 CCA (P) => TCA (S)
842 281 GCG (A) => GTG (V) 628 210 CCT (P) => TCT (S) 1148 383 TCC (S) => TTC (F)
289 97 CCC (P) => TCC (S) 883 295 CCG (P) => TCG (S) 1021 341 CAC (H) => TAC (Y) 1312 438 CCT (P) => TCT (S)
ZmPERK4 482 161 CCG (P) => CTG (L) 1132 378 CTC (L) => TTC (F) 1327 443 CAT (H) => TAT (Y)
491 164 CCG (P) => CTG (L) 1172 391 CCG (P) => CTG (L) 1379 460 GCA (A) => GTA (V) ZmPERK22 205 69 CCC (P) => TCC (S)
565 189 CAT (H) => TAT (Y) 1435 479 CTT (L) => TTT (F) 286 96 CAC (H) => TAC (Y)
577 193 CCC (P) => TCC (S) ZmPERK11 178 60 CCG (P) => TCG (S) 1441 481 CTT (L) => TTT (F) 611 204 CCA (P) => CTA (L)
619 207 CCG (P) => TCG (S) 181 61 CCA (P) => TCA (S) 715 239 CCT (P) => TCT (S)
626 209 ACA (T) => ATA (I) 211 71 CCG (P) => TCG (S) ZmPERK17 106 36 CCG (P) => TCG (S) 965 322 GCA (A) => GTA (V)
859 287 CAC (H) => TAC (Y) 259 87 CCT (P) => TTT (F) 121 41 CCT (P) => TTT (F) 1307 436 CCA (P) => CTA (L)
260 87 CCT (P) => TTT (F) 122 41 CCT (P) => TTT (F) 1319 440 ACC (T) => ATC (I)
143 48 TCG (S) => TTG (L) 302 101 TCT (S) => TTT (F) 167 56 TCA (S) => TTA (L) 1358 453 CCA (P) => CTA (L)
ZmPERK5 238 80 CCG (P) => TCG (S) 352 118 CCG (P) => TCG (S) 329 110 CCG (P) => CTG (L) 1385 462 CCA (P) => CTA (L)
752 251 TCA (S) => TTA (L) 359 120 CCG (P) => CTG (L) 338 113 CCA (P) => CTA (L)
1010 337 GCC (A) => GTC (V) 376 126 CCC (P) => TCC (S) 382 128 CCT (P) => TTT (F) ZmPERK23 649 217 CCT (P) => TCT (S)
1031 344 CCG (P) => CTG (L) 388 130 CCG (P) => TCG (S) 383 128 CCT (P) => TTT (F) 652 218 CCC (P) => TCC (S)
1226 409 GCC (A) => GTC (V) 425 142 TCT (S) => TTT (F) 793 265 CTT (L) => TTT (F)
1367 456 TCC (S) => TTC (F) ZmPERK12 389 130 CCG (P) => CTG (L) 821 274 TCG (S) => TTG (L)
1394 465 GCG (A) => GTG (V) 394 132 CCG (P) => TCG (S) ZmPERK18 863 288 ACT (T) => ATT (I) 842 281 TCC (S) => TTC (F)
430 144 CCC (P) => TCC (S) 1036 346 CCT (P) => TCT (S) 847 283 CCA (P) => TCA (S)
275 92 ACT (T) => ATT (I) 535 179 CCA (P) => TCA (S) 1150 384 CCT (P) => TCT (S) 859 287 CCG (P) => TCG (S)
ZmPERK6 400 134 CCT (P) => TTT (F) 542 181 CCG (P) => CTG (L) 1190 397 CCT (P) => CTT (L) 916 306 CCA (P) => TCA (S)
401 134 CCT (P) => TTT (F) 571 191 CCG (P) => TCG (S) 1198 400 CAT (H) => TAT (Y) 1252 418 CTC (L) => TTC (F)
512 171 ACA (T) => ATA (I) 920 307 TCG (S) => TTG (L) 1274 425 ACC (T) => ATC (I)
940 314 CCC (P) => TCC (S) 1105 369 CTC (L) => TTC (F) 1319 440 ACC (T) => ATC (I)
1319 440 ACG (T) => ATG (M) 1369 457 CCT (P) => TCT (S) 1343 448 GCG (A) => GTG (V)
1439 480 TCC (S) => TTC (F) 1490 497 ACG (T) => ATG (M)
Table 3B Predicted RNA editing sites in mitochondrial genome.
Genes Nucleotide Position Amino acid Position Amino acid Conservation Genes Nucleotide Position Amino acid Position Amino acid Conservation Genes Nucleotide Position Amino acid Position Amino acid Conservation Genes Nucleotide Position Amino acid Position Amino acid Conservation
ZmPERK1 5 2 ACG (T) => ATG (M) ZmPERK8 8 3 TCC (S) => TTC (F) ZmPERK13 319 107 CTC (L) => TTC (F) ZmPERK20 104 35 CCG (P) => CTG (L)
38 13 TCG (S) => TTG (L) 31 11 CCG (P) => TCG (S) 850 284 CCG (P) => TCG (S) 121 41 CCC (P) => TCC (S)
40 14 CCC (P) => TCC (S) 38 13 CCG (P) => CTG (L) 853 285 CCC (P) => TCC (S) 149 50 TCG (S) => TTG (L)
82 28 CTC (L) => TTC (F) 80 27 GCC (A) => GTC (V) 1187 396 ACC (T) => ATC (I) 182 61 GCA (A) => GTA (V)
185 62 CCT (P) => CTT (L) 103 35 CCC (P) => TCC (S) 1246 416 CTC (L) => TTC (F) 239 80 CCA (P) => CTA (L)
215 72 CCC (P) => CTC (L) 107 36 GCA (A) => GTA (V) 1379 460 CCA (P) => CTA (L) 272 91 TCT (S) => TTT (F)
284 95 CCC (P) => CTC (L) 139 47 CAC (H) => TAC (Y) 1556 519 TCC (S) => TTC (F) 277 93 CCA (P) => TCA (S)
305 102 ACC (T) => ATC (I) 215 72 CCA (P) => CTA (L) 1744 582 CAC (H) => TAC (Y) 284 95 GCC (A) => GTC (V)
314 105 GCA (A) => GTA (V) 250 84 CCG (P) => TCG (S) 1877 626 ACC (T) => ATC (I) 305 102 CCA (P) => CTA (L)
328 110 CCT (P) => TCT (S) 278 93 TCG (S) => TTG (L) 1925 642 GCC (A) => GTC (V) 320 107 GCG (A) => GTG (V)
332 111 CCT (P) => CTT (L) 302 101 GCC (A) => GTC (V) 2045 682 CCC (P) => CTC (L) 329 110 GCA (A) => GTA (V)
362 121 GCG (A) => GTG (V) 308 103 GCA (A) => GTA (V) 2054 685 GCT (A) => GTT (V)
329 110 CCT (P) => CTT (L) ZmPERK21 535 179 CCT (P) => TCT (S)
ZmPERK2 389 130 CCT (P) => CTT (L) 341 114 GCA (A) => GTA (V) ZmPERK14 92 31 ACG (T) => ATG (M) 545 182 CCG (P) => CTG (L)
391 131 CCA (P) => TCA (S) 428 143 CCG (P) => CTG (L) 170 57 TCA (S) => TTA (L) 550 184 CCA (P) => TCA (S)
406 136 CCG (P) => TCG (S) 386 129 CCT (P) => CTT (L) 566 189 TCT (S) => TTT (F)
440 147 CCA (P) => CTA (L) ZmPERK9 164 55 CCG (P) => CTG (L) 394 132 CAT (H) => TAT (Y) 641 214 CCT (P) => CTT (L)
469 157 CCG (P) => TCG (S) 166 56 CCA (P) => TCA (S) 752 251 GCA (A) => GTA (V) 749 250 GCG (A) => GTG (V)
512 171 TCA (S) => TTA (L) 230 77 TCG (S) => TTG (L) 923 308 GCA (A) => GTA (V) 815 272 GCG (A) => GTG (V)
566 189 ACG (T) => ATG (M) 254 85 GCT (A) => GTT (V) 950 317 GCC (A) => GTC (V) 829 277 CCG (P) => TCG (S)
572 191 TCA (S) => TTA (L) 389 130 GCA (A) => GTA (V) 836 279 CCT (P) => CTT (L)
578 193 CCC (P) => CTC (L) 457 153 CCG (P) => TCG (S) ZmPERK15 32 11 CCG (P) => CTG (L) 857 286 CCG (P) => CTG (L)
808 270 CCG (P) => TCG (S) 671 224 TCG (S) => TTG (L) 106 36 CCC (P) => TCC (S) 935 312 CCG (P) => CTG (L)
877 293 CCT (P) => TCT (S) 803 268 GCC (A) => GTC (V) 230 77 GCG (A) => GTG (V)
881 294 TCG (S) => TTG (L) 922 308 CTT (L) => TTT (F) 233 78 GCG (A) => GTG (V) ZmPERK22 34 12 CAC (H) => TAC (Y)
958 320 CAC (H) => TAC (Y) 281 94 GCC (A) => GTC (V) 76 26 CCA (P) => TCA (S)
ZmPERK3 223 75 CTT (L) => TTT (F) 1358 453 CCC (P) => CTC (L) 326 109 CCG (P) => CTG (L) 205 69 CCC (P) => TCC (S)
317 106 GCT (A) => GTT (V) 1741 581 CAA (Q) => TAA (X) 404 135 GCG (A) => GTG (V) 667 223 CCT (P) => TCT (S)
931 311 CCT (P) => TCT (S) 428 143 CCG (P) => CTG (L) 836 279 CCA (P) => CTA (L)
1198 400 CAC (H) => TAC (Y) ZmPERK10 5 2 ACG (T) => ATG (M) 430 144 CCG (P) => TCG (S) 890 297 CCA (P) => CTA (L)
1277 426 GCC (A) => GTC (V) 8 3 CCG (P) => CTG (L) 439 147 CCG (P) => TCG (S) 1000 334 CAT (H) => TAT (Y)
1322 441 ACC (T) => ATC (I) 10 4 CCG (P) => TCG (S) 488 163 GCG (A) => GTG (V) 1033 345 CCC (P) => TCC (S)
1373 458 CCA (P) => CTA (L) 31 11 CCG (P) => TCG (S) 571 191 CCC (P) => TCC (S) 1040 347 GCT (A) => GTT (V)
1382 461 ACC (T) => ATC (I) 98 33 GCG (A) => GTG (V) 614 205 GCC (A) => GTC (V) 1181 394 TCG (S) => TTG (L)
476 159 GCG (A) => GTG (V) 659 220 ACC (T) => ATC (I)
ZmPERK4 8 3 TCT (S) => TTT (F) 608 203 GCT (A) => GTT (V) 748 250 CCC (P) => TCC (S) ZmPERK23 125 42 CCG (P) => CTG (L)
31 11 CCA (P) => TCA (S) 614 205 ACA (T) => ATA (I) 155 52 CCG (P) => CTG (L)
38 13 CCG (P) => CTG (L) 884 295 CCG (P) => CTG (L) ZmPERK16 235 79 CCC (P) => TTC (F) 191 64 TCG (S) => TTG (L)
41 14 TCT (S) => TTT (F) 236 79 CCC (P) => TTC (F) 218 73 GCA (A) => GTA (V)
74 25 TCT (S) => TTT (F) ZmPERK11 34 12 CCT (P) => TCT (S) 302 101 CCA (P) => CTA (L) 395 132 GCC (A) => GTC (V)
89 30 ACT (T) => ATT (I) 77 26 ACG (T) => ATG (M) 331 111 CCG (P) => TCG (S) 944 315 CCA (P) => CTA (L)
107 36 GCG (A) => GTG (V) 176 59 ACT (T) => ATT (I) 425 142 ACG (T) => ATG (M) 1165 389 CCT (P) => TTT (F)
137 46 CCC (P) => CTC (L) 194 65 CCG (P) => CTG (L) 571 191 CCG (P) => TCG (S) 1166 389 CCT (P) => TTT (F)
164 55 GCG (A) => GTG (V) 224 75 CCC (P) => CTC (L) 626 209 CCT (P) => CTT (L) 1405 469 CCG (P) => TCG (S)
182 61 GCT (A) => GTT (V) 232 78 CCA (P) => TCA (S) 632 211 GCT (A) => GTT (V) 1739 580 GCT (A) => GTT (V)
197 66 CCA (P) => CTA (L) 239 80 GCT (A) => GTT (V) 815 272 TCG (S) => TTG (L)
272 91 CCC (P) => CTC (L) 254 85 CCT (P) => CTT (L) 1021 341 CAC (H) => TAC (Y)
260 87 CCT (P) => CTT (L) 1124 375 GCG (A) => GTG (V)
ZmPERK5 208 70 CCC (P) => TCC (S) 289 97 CCT (P) => TTT (F) 1228 410 CAT (H) => TAT (Y)
236 79 CCG (P) => CTG (L) 290 97 CCT (P) => TTT (F) 1277 426 GCT (A) => GTT (V)
257 86 ACC (T) => ATC (I) 340 114 CCA (P) => TCA (S)
577 193 CCG (P) => TCG (S) 347 116 GCA (A) => GTA (V) ZmPERK17 34 12 CAC (H) => TAC (Y)
686 229 GCG (A) => GTG (V) 371 124 CCG (P) => CTG (L) 76 26 CCA (P) => TCA (S)
943 315 CCA (P) => TCA (S) 383 128 GCC (A) => GTC (V) 205 69 CCC (P) => TCC (S)
1010 337 GCC (A) => GTC (V) 395 132 CCT (P) => CTT (L) 667 223 CCT (P) => TCT (S)
1094 365 GCG (A) => GTG (V) 836 279 CCA (P) => CTA (L)
ZmPERK12 266 89 TCG (S) => TTG (L) 890 297 CCA (P) => CTA (L)
ZmPERK6 49 17 CCA (P) => TCA (S) 268 90 CCG (P) => TCG (S) 1000 334 CAT (H) => TAT (Y)
64 22 CCT (P) => TTT (F) 296 99 CCT (P) => CTT (L) 1033 345 CCC (P) => TCC (S)
65 22 CCT (P) => TTT (F) 299 100 CCT (P) => CTT (L) 1040 347 GCT (A) => GTT (V)
92 31 ACA (T) => ATA (I) 304 102 CCG (P) => TCG (S) 1181 394 TCG (S) => TTG (L)
775 259 CTT (L) => TTT (F) 332 111 CCG (P) => CTG (L)
1316 439 TCA (S) => TTA (L) 356 119 GCG (A) => GTG (V) ZmPERK18 55 19 CCC (P) => TTC (F)
1412 471 ACA (T) => ATA (I) 359 120 GCG (A) => GTG (V) 56 19 CCC (P) => TTC (F)
1420 474 CCG (P) => TCG (S) 386 129 GCA (A) => GTA (V) 62 21 GCC (A) => GTC (V)
1441 481 CCG (P) => TCG (S) 395 132 CCG (P) => CTG (L) 83 28 GCC (A) => GTC (V)
440 147 GCC (A) => GTC (V) 223 75 CTC (L) => TTC (F)
ZmPERK7 65 22 CCG (P) => CTG (L) 449 150 ACG (T) => ATG (M) 317 106 GCT (A) => GTT (V)
109 37 CTT (L) => TTT (F) 461 154 TCA (S) => TTA (L) 931 311 CCT (P) => TCT (S)
137 46 GCC (A) => GTC (V) 476 159 GCG (A) => GTG (V) 1127 376 TCA (S) => TTA (L)
143 48 GCG (A) => GTG (V) 521 174 ACC (T) => ATC (I) 1226 409 GCT (A) => GTT (V)
257 86 GCT (A) => GTT (V) 524 175 GCC (A) => GTC (V) 1370 457 CCA (P) => CTA (L)
260 87 CCA (P) => CTA (L) 535 179 CCA (P) => TCA (S) 1379 460 ACC (T) => ATC (I)
275 92 GCG (A) => GTG (V) 616 206 CCA (P) => TCA (S)
290 97 TCA (S) => TTA (L) 620 207 CCT (P) => CTT (L) ZmPERK19 4 2 CCC (P) => TTC (F)
311 104 GCG (A) => GTG (V) 644 215 CCG (P) => CTG (L) 5 2 CCC (P) => TTC (F)
320 107 GCT (A) => GTT (V) 674 225 TCG (S) => TTG (L) 26 9 CCG (P) => CTG (L)
323 108 GCT (A) => GTT (V) 38 13 TCG (S) => TTG (L)
524 175 ACA (T) => ATA (I) 43 15 CCG (P) => TCG (S)
614 205 CCC (P) => CTC (L) 59 20 TCT (S) => TTT (F)
770 257 GCG (A) => GTG (V) 70 24 CTT (L) => TTT (F)
98 33 GCG (A) => GTG (V)
194 65 CCG (P) => CTG (L)
200 67 CCG (P) => CTG (L)
299 100 GCC (A) => GTC (V)
(A) RNA editing of the PERK genes results in amino acid conservation. (B) Identified cis-acting elements in ZmPERK gene family promoters.
Fig. 6
(A) RNA editing of the PERK genes results in amino acid conservation. (B) Identified cis-acting elements in ZmPERK gene family promoters.

Location of gene at cellular level was also determined. Results indicated that 21 of the 23 ZmPERK proteins were localized to the plasma membrane, while two (ZmPERK18 and ZmPERK23) were localized to the cytoplasm, Table1 contains the details of these parameters. The promoter region, which is located upstream of the start codon area, controls gene transcription. Understanding gene regulation and function requires a thorough examination of cis-elements (Higo et al., 1999). We discovered and classified cis-acting factors in the upstream region of the ZmPERK genes. (See Table S4) The cis-elements were categorized based on their roles in growth and development, as well as light and stress actions. The upstream region of ZmPERK genes contained cis-acting factors like MeJA responsive, MYB-binding sites associated with light responsive elements, ABA responsive elements, defense, stress, low temperature, gibberellin acid (GA), and salicylic acid (SA)responsive elements, as per the promoter analysis results (Fig. 6B). The promoters of the ZmPERK gene have the most MeJA responsiveness elements. where they were found in 296 promoters. There were 166 light responsive elements, 88 ABA responsive elements, 27 GA responsive elements, 25 MYB light responsive elements, and 4 auxin responsive elements. The cis-element analysis showed that during abiotic stress and plant development phase the ZmPERK genes could respond.

3.6

3.6 In-silico expressions analysis

Expression patterns give information regarding the biological activities of genes because gene expression is required for optimal regulation of plant growth and development. We looked examined the expression patterns of the ZmPERK under drought stress and at different stages of seed embryo development from 15 days after pollination (DAP) to 40 days after pollination (DAP) in two distinct varieties (High oil content and low oil content) (Fig. 7A).Under drought stress 9 genes were upregulated in both tissues leave and cob (ZmPERK2, ZmPERK 3, ZmPERK 8, ZmPERK 10, ZmPERK 14, ZmPERK 16, ZmPERK 19, ZmPERK 20) 4 genes (ZmPERK 1, ZmPERK 7, ZmPERK 18, ZmPERK 21) only upregulated in leave tissue and 1 gene ZmPERK 18 upregulated in cob (Fig. 7B). While Seven genes downregulated (ZmPERK 4, ZmPERK 5, ZmPERK 11, ZmPERK 12, ZmPERK 15, ZmPERK 17, ZmPERK 22) under drought stress.For oil content accumulation in embryo 9 genes shows upregulated expression pattern (ZmPERK 1, ZmPERK 3, ZmPERK 6, ZmPERK 8, ZmPERK 13, ZmPERK 14, ZmPERK 16, ZmPERK 19 ZmPERK,20) while 12 genes show downregualted trend (ZmPERK 2, ZmPERK 5, ZmPERK 6, ZmPERK 7, ZmPERK 9, ZmPERK 10, ZmPERK 11, ZmPERK 12, ZmPERK 17, ZmPERK 18, ZmPERK 22, ZmPERK 23). Interestingly, some genes show similar expression pattern under both conditions. For example, ZmPERK 3 ZmPERK 8 ZmPERK 14 ZmPERK 16 ZmPERK 19 ZmPERK 20 regardless of the tissues or stresses applied, they were always upregulated. We may conclude from these findings that ZmPERK gene expression is involved in drought stress and oil content accumulation in the embryo.

A: ZmPERK gene expression patterns at different developmental stages of seed embryos from 15 days after pollination (DAP) to 40 days after pollination (DAP). H-represent expression in high oil content varaiety while L represent low oil content varaiety B: Expression pattern of ZmPERKgene under drought stress DS-L(Drought stress leave sample) DS-C (Drought stress cob sample) CL(Controled leave sample) CC (Control Cob sample.
Fig. 7
A: ZmPERK gene expression patterns at different developmental stages of seed embryos from 15 days after pollination (DAP) to 40 days after pollination (DAP). H-represent expression in high oil content varaiety while L represent low oil content varaiety B: Expression pattern of ZmPERKgene under drought stress DS-L(Drought stress leave sample) DS-C (Drought stress cob sample) CL(Controled leave sample) CC (Control Cob sample.

4

4 Discussion

Many ancient land plants evolved over the time also possess PERKs genes (Nakhamchik et al., 2004, Qanmber et al., 2019, Chen et al., 2020) which depict that these gene families are present from centuries in the plants. Now a day’s modern techniques of DNA sequences have revolutionized the field of DNA sequencing especially the advent of next-generation sequencing technologies, have shorten the time of sequencing with more accuracy in the results. The availability of maize genome assemblies has open the new avenues for studying various functions of genes at genome-wide level. There has been no systematic study of maize to date. However, in our study we discovered 23 ZmPERK genes in the maize genome. In phylogenetic analysis, we divided ZmPERK genes into four groups. The findings demonstrated that PERK genes were initially originated in ancient land plants and their orthologous genes may be found throughout the plant kingdom. Plant PERK genes from dicot, monocot, lycophytes, and chlorophytes were assigned to each of the four clades randomly. Our study showed that ZmPERK genes remained evolutionarily conserved, as these were found in each of the species which was used in this study. Furthermore, the expansion of these gene into higher plants was occurred with the passage of time. According to sequence logos for PERK genes, the protein sequence residues were highly conserved, and no compositional bias was seen across the studied species. To study the evolutionary history of multiple gene families (Ohta, 2010). It is essential to know the structure of the genes. The length of nucleotide and amino acid sequences varied considerably, indicating that the ZmPERK genes are diverse. It is quite worth to study the exon–intron structure as insertion/deletion events play important role in determine the structure of exon–intron. The introns gain or lose have been witnessed throughout eukaryotic diversification. The exon–intron pattern of duplicated genes is similar, whereas more diversification observed in the intron length suggesting that the intron length may be significant in ZmPERK functional diversification. In ZmPERK proteins, different combinations of conserved motifs were identified. Protein motif analysis revealed that protein from same species were tend to fall in the same cluster together. Arrangements of motif were comparable among members of the same subfamily.

The study of gene duplication events is critical as these play crucial role in the genome expansions and alignments (Tamura et al., 2011). The gene duplication events have been witnessed in various transcription factor families of plants (Liu et al., 2011; Shan et al., 2013). To differentiate whether the gene duplication was the result of tandem or segmental if the duplications are the result of the presence two or more genes the same chromosome, it will be considered as tandem duplication whereas segmental or WGD duplications are when two or more genes are duplicated on different chromosomes. The intron expansion is mainly the result of the tandem duplications and thus give rise to the formation of the new genes (Yang et al., 2008), but we only find evidence of one tandem and eight segmental duplications in this study. In Plants the environmental and selection factors have expanded multiple gene families more than other eukaryotes organisms. The Ka/Ks ratios demonstrated that maize PERK genes have been subjected to extensive selection, with relatively minimal functional variations due to whole genome and segmental duplication.

ZmPERK genes possess cis-elements associated with stress responses in their promoter regions. ZmPERK contains cis-elements such as MeJA responsive, MYB-binding sites associated with light responsiveness elements, ABA responsive, defense, and stress responsive, low temperature and gibberellin acid (GA) responsive elements. The presence of these cis elements with specified characteristics demonstrated the putative role in plant growth, development, as well as in biotic and abiotic stress response. Synteny is a framework for assessing homologous gene and gene order conservation across genomes of different species. The collinearity between maize and sorghum was shown to be more significant than the collinearity between maize and rice.

Plant growth and development are assisted by RNA editing, which is an effective strategy for regulating gene expression at the post-transcriptional level in higher plant organelle genomes. The discovery and identification of RNA editing sites is critical for a better knowledge of their biological activities and establishing the framework for future research and comprehension of their molecular processes. In this work, the RNA editing sites of chloroplast and mitochondrial genes in maize were predicted. Table 3A lists 196 RNA editing sites predicted in chloroplast genes and 268 in mitochondrial genes (Table 3B). In the chloroplast and mitochondrial genomes, these sites were detected on 23 genes, with an average of 8.5 and 11.26 editing sites per gene, respectively. The transition and conservation of cytosine (C) to uracil (U) was observed in all of the editing sites. Changes in the first and second codon nucleotides were mostly involved for these transitions. The current study laid the groundwork for future research into the biological functions of chloroplast and mitochondrial RNA editing in maize. The expression patterns of genes are closely related to their biological functions. According to expression analyses ZmPERK genes were shown to be important in drought tolerance and oil content accumulation in embryos.

5

5 Conclusion

The current study found 23 non-redundant ZmPERK encoding genes in maize. The PERK gene family is conserved among the analyzed plant species, according to their classification, characterization in terms of gene structure, motif, conserved domains, and comparative phylogenetic analyses. Furthermore, gene duplication analysis and syntenic relationship studies reveal that the maize paralogous genes proliferate through segmental duplications, whereas codons went under purifying selection, resulting in a significant expansion of the ZmPERK gene family. The existence of putative cis-elements in the ZmPERK gene promoter regions suggests that they have a functional role in growth, development, and stress resilience. Most of the genes were found to be up regulated in response to stress and oil content, accumulation showing that they may play a role in stress modulation and development process in maize. Overall, these findings will assist in the functional characterization of maize PERK genes. The candidate ZmPERK genes can be employed in a breeding program.

Acknowledgement

This project was supported by Researchers Supporting Project Number (RSP-2023R7) King Saud University, Riyadh, Saudi Arabia.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References

  1. , , . Effect of Piriformospora indica and Azotobacter chroococcum on mitigation of zinc deficiency stress in wheat (Triticum aestivum L.) Symbiosis. 2016;69(1):9-19.
    [Google Scholar]
  2. , , , . Plasma membrane-associated proline-rich extensin-like receptor kinase 4, a novel regulator of Ca2+ signalling, is required for abscisic acid responses in Arabidopsis thaliana. Plant J.. 2009;60(2):314-327.
    [Google Scholar]
  3. , , , . The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biol.. 2004;4(1):1-21.
    [Google Scholar]
  4. , , , . Arabidopsis kinome: after the casting. Funct. Integr. Genomics. 2004;4(3):163-187.
    [Google Scholar]
  5. , , , . Genome-wide analysis of proline-rich extension-like receptor protein kinase (PERK) in Brassica rapa and its association with the pollen development. BMC Genomics. 2020;21(1):1-13.
    [Google Scholar]
  6. , , , . Evolution of the protein repertoire. Science. 2003;300(5626):1701-1703.
    [Google Scholar]
  7. , , , . CLAVATA1 dominant-negative alleles reveal functional overlap between multiple receptor kinases that regulate meristem and organ development. Plant Cell. 2003;15(5):1198-1211.
    [Google Scholar]
  8. , , , . Pfam: the protein families database. Nucleic Acids Res.. 2014;42(D1):D222-D230.
    [Google Scholar]
  9. , , , . Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res.. 1999;27(1):297-300.
    [Google Scholar]
  10. , , , . Cell wall-associated ROOT HAIR SPECIFIC 10, a proline-rich receptor-like kinase, is a negative modulator of Arabidopsis root hair growth. J. Exp. Bot.. 2016;67(6):2007-2022.
    [Google Scholar]
  11. , , , . Genome-wide identification and expression analysis of calcium-dependent protein kinase in maize. BMC Genomics. 2013;14(1):1-15.
    [Google Scholar]
  12. , , . Transcription factors as molecular switches to regulate drought adaptation in maize. Theor. Appl. Genet.. 2020;133(5):1455-1465.
    [Google Scholar]
  13. , , , . SMART: recent updates, new developments and status in 2015. Nucleic Acids Res.. 2015;43(D1):D257-D260.
    [Google Scholar]
  14. , , , . Genome-wide analysis of the auxin response factor (ARF) gene family in maize (Zea mays) Plant Growth Regul.. 2011;63(3):225-234.
    [Google Scholar]
  15. , , . Genetic dissection of maize drought tolerance for trait improvement. Mol. Breed.. 2021;41(2):1-13.
    [Google Scholar]
  16. , , , . Some current aspects of stomatal physiology. Ann. Rev. Plant Physiol. Plant Mol. Biol.. 1990;41:55-75.
    [Google Scholar]
  17. , , . Receptor-like protein kinases: the keys to response. Curr. Opin. Plant Biol.. 2003;6(4):339-342.
    [Google Scholar]
  18. , . The PREP suite: predictive RNA editors for plant mitochondrial genes, chloroplast genes and user-defined alignments. Nucleic Acids Res.. 2009;37(suppl_2):W253-W259.
    [Google Scholar]
  19. , , , . A comprehensive expression analysis of the Arabidopsis proline-rich extensin-like receptor kinase gene family using bioinformatic and experimental approaches. Plant Cell Physiol.. 2004;45(12):1875-1881.
    [Google Scholar]
  20. , . Gene conversion and evolution of gene families: an overview. Genes.. 2010;1(3):349-356.
    [Google Scholar]
  21. , , , . Evolution of gene duplication in plants. Plant Physiol.. 2016;171(4):2294-2316.
    [Google Scholar]
  22. , , , . Genome-wide identification and characterization of the PERK gene family in Gossypium hirsutum reveals gene duplication and functional divergence. Int. J. Mol. Sci.. 2019;20(7):1750.
    [Google Scholar]
  23. , , . Double jeopardy: both overexpression and suppression of a redox-activated plant mitogen-activated protein kinase render tobacco plants ozone sensitive. Plant Cell. 2002;14(9):2059-2069.
    [Google Scholar]
  24. , , , . Maize gene atlas developed by RNA sequencing and comparative evaluation of transcriptomes based on RNA sequencing and microarrays. PLoS ONE. 2013;8(4):e61005.
    [Google Scholar]
  25. , , , . Targeted genome modification of crop plants using a CRISPR-Cas system. Nat. Biotechnol.. 2013;31(8):686-688.
    [Google Scholar]
  26. , . Plant receptor-like kinase gene family: diversity, functions, and signaling. Sci. STKE.. 2001;18:113-122.
    [Google Scholar]
  27. , , . Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases. Proc. Natl. Acad. Sci.. 2001;98(19):10763-10768.
    [Google Scholar]
  28. , , , . Comparative analysis of the receptor-like kinase family in Arabidopsis and rice. Plant Cell. 2004;16(5):1220-1234.
    [Google Scholar]
  29. , , , . Synergistic interaction of three ERECTA-family receptor-like kinases controls Arabidopsis organ growth and flower development by promoting cell proliferation. Develop. 2004;131(7):1491-1501.
    [Google Scholar]
  30. , , , . Dominant-negative receptor uncovers redundancy in the Arabidopsis ERECTA leucine-rich repeat receptor–like kinase signaling pathway that regulates organ shape. Plant Cell. 2003;15(5):1095-1110.
    [Google Scholar]
  31. , , . The proline-rich, extensin-like receptor kinase-1 (PERK1) gene is rapidly induced by wounding. Plant Mol. Biol.. 2002;50(4):667-685.
    [Google Scholar]
  32. , , , . MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol.. 2011;28(10):2731-2739.
    [Google Scholar]
  33. , , . High temperature stress tolerance in maize (Zea mays L.): Physiological and molecular mechanisms. Journal of. Plant Biology.. 2019;62(2):93-102.
    [Google Scholar]
  34. , . MapChart: software for the graphical presentation of linkage maps and QTLs. J. Hered.. 2002;93(1):77-78.
    [Google Scholar]
  35. , , , . MKK5 regulates high light-induced gene expression of Cu/Zn superoxide dismutase 1 and 2 in Arabidopsis. Plant Cell Physiol.. 2013;54(7):1217-1227.
    [Google Scholar]
  36. , , , . Recent duplications dominate NBS-encoding gene expansion in two woody species. Mol. Genet. Genomics. 2008;280(3):187-198.
    [Google Scholar]
  37. , . Abiotic stress signaling and responses in plants. Cell. 2016;167(2):313-324.
    [Google Scholar]

Appendix A

Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jksus.2022.102293.

Appendix A

Supplementary data

The following are the Supplementary data to this article:

Supplementary data 1


Fulltext Views
56

PDF downloads
45
View/Download PDF
Download Citations
BibTeX
RIS
Show Sections

Suggested read for related articles:

© Copyright 2025 – Journal of King Saud University – Science.

Published by Scientific Scholar on behalf of King Saud University.

ISSN (Print): 1018-3647
ISSN (Online): 2213-686X

Scientific Scholar CrossRef Creative Commons Open Acess Portico