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. 2022 Dec 6;8(12):e12070. doi: 10.1016/j.heliyon.2022.e12070

Genome-wide comparison and identification of myosin gene family in Arabidopsis thaliana and Helianthusannuus

Hafiz Muhammad Ahmad a,∗∗, Hayat Ali Alafari b, Sajid Fiaz c,∗, Dalal S Alshaya b, Sidra Toor d, Munazza Ijaz e, Nouman Rasool c, Kotb A Attia f,g, Madiha Zaynab h, Saira Azmat i, Asmaa M Abushady j,k, Yinglong Chen l
PMCID: PMC9763749  PMID: 36561675

Abstract

Myosins are essential components of organelle trafficking in all the eukaryotic cells. Myosin driven movement plays a vital role in the development of pollen tubes, root hairs and root tips of flowering plants. The present research characterized the myosin genes in Arabidopsis thaliana and Helianthus annuus by using different computational tools. We discovered a total of 50 myosin genes and their splice variants in both pant species. Phylogenetic analysis indicated that myosin genes were divided into four subclasses. Chromosomal location revealed that myosin genes were located on all five chromosomes in A. thaliana, whereas they were present on nine chromosomes in H. annuus. Conserved motifs showed that conserved regions were closely similar within subgroups. Gene structure analysis showed that Atmyosin2.2 and Atmyosin2.3 had the highest number of introns/exons. Gene ontology analysis indicated that myosin genes were involved in vesicle transport along actin filament and cytoskeleton trafficking. Expression analysis showed that expression of myosin genes was higher during the flowering stage as compared to the seedling and budding stages. Tissue specific expression indicated that HanMYOSIN11.2, HanMYOSIN16.2 were highly expressed in stamen, whereas HanMYOSIN 2.2, HanMYOSIN 12.1 and HanMYOSIN 17.1 showed higher expression in nectary. This study enhance our understanding the function of myosins in plant development, and forms the basis for future research about the comparative genomics of plant myosin in other crop plants.

Keywords: Plant motility, Molecular motors, Actin binding, Fluidic movement

Plant motility; Molecular motors; Actin binding; Fluidic movement.

1. Introduction

The movement of organelles and other cellular components within a cell is termed as cytoplasmic streaming (Wang et al., 2022). Cytoplasmic streaming assists the plants in nutrient delivery, chloroplast movement, metabolites, and other materials throughout the cell (Nebenführ, 2020; Nebenführ and Dixit, 2018a). Various biological processes of plants, such as pollen tubes, cell growth, and root hair development, are associated with cytoplasmic streaming. Organelles movement along the cellular components depends on actin filaments or microtubules (Ouyang et al., 2021). Motor proteins help to move the organelles by binding with specific lipids in the cell membrane (Nebenführ, 2020). In plants, molecular motor proteins comprise three major families: kinesins, dynein, and myosin (Nebenführ and Dixit, 2018a). The first two families play a role as motors on microtubule filaments, while myosin works on actin filaments (Nebenführ and Dixit, 2018a). Plants possess two unique myosin groups, i.e., myosin proteins of class VIII and XI (Ma et al., 2020). Myosin proteins of class VIII are known as slow motor proteins, whereas myosin proteins of XI are the fastest motor proteins. According to their velocity, plant myosin can be categorized into three groups: low-, medium-, and high-velocity groups (Haraguchi et al., 2016, 2018a). In Arabidopsis, 13 genes of myosin XI (XI A, XI B, XI C, XI D, XI E, XI F, XI G, XI H, XI I, XI J, XI K, XI 1, and XI 2) have been identified. Low-velocity group is comprised of only one myosin member, i.e. XI I, whereas there are six myosin genes each in the medium – (XI B, XI H, XI J, XI K, XI 1, XI 2) and high- (XI A, XI C, XI E, XI F and XI G) velocity groups. Further, it has been observed that myosin groups categorized on velocity bases perform specific functions, i.e. low-velocity myosin XI I is known as the nuclear membrane. Medium velocity group members generate motive forces for cytoplasmic streaming. High-velocity group myosin proteins were called pollen-specific (Haraguchi et al., 2018a). Myosin proteins in plants are divided into two main classes, Myosin XI and Myosin V, which act as molecular motors to move fluids across the filaments of actin (Haraguchi et al., 2018a). Myosin proteins control the organization of actin, fluidic dynamics and play a key role in the locomotion of vesicles and organelles (Haraguchi et al., 2016).

Eukaryotes have 35 different types of myosin genes. However, not all of these genes have been discovered in a single organism. Myosin performs various functions in plants; for example, myosin XI B controls pollen tube development in Oryza sativa (Jiang et al., 2007). Myosin XI F strengthens plant organs through tension generation to make them straight (Ueda et al., 2015). Myosin XI I is associated with nuclear shape and movement and is called a nucleocytoplasmic linker (Ueda et al., 2015). Another study proved that Myosin XI I generates the tension and regulates organelle velocity; however, it doesn't participate in cytoplasmic streaming (Haraguchi et al., 2016). Myosin XI K is involved in remodeling and movement of endoplasmic reticulum (Sparkes, 2010). However, the class XI Myosin functional analysis has largely been hindered due to the redundancy of different paralogous genes. Recently, plant myosin have been involved in sugar-induced hypocotyl elongation under dark conditions (Olatunji and Kelley, 2020).

Sunflower (Helianthus annuus) is the world's fourth most important oilseed crop, flowing the palm, soybean and rapeseed (Ahmad et al., 2021; Li et al., 2021). Increasing demand for sunflower cooking oil has further boosted the growing trend of this crop (Ahmad et al., 2020). Recent sequencing and release of the H. annuus genome have opened new horizons to characterize this crop's various traits (Badouin et al., 2017). Improved breeding for cytoplasmic streaming and characterization of molecular motor proteins may be useful to enhance sunflower production to meet the cooking oil demand of the globe. Myosin proteins have been identified and characterized in Arabidopsis thaliana, Gossypium hirsutism, Zea maize, and Glycine max (Ma et al., 2020; Olatunji and Kelley, 2020; Wang et al., 2013). Since the release of H. annuus genome, the myosin gene family has not yet characterized in this specie. This research disparity provoked us to comprehensively explore the myosin family in H. annuus by identifying and comparing protein properties, phylogenetic analysis, intron and exon organization, cis-acting elements, chromosomal distribution, and gene duplication. This study also aimed to discover the expression of myosin genes in various subcellular components in H. annuus.

2. Materials and methods

2.1. Retrieval of protein sequences and characteristics of myosin protein properties

Sequences of myosin proteins of A. thaliana and H. annuus species were downloaded from Phytozome V.13 (https://phytozome-next.jgi.doe.gov) database. Motif search tool (https://www.genome.jp/tools/motif/) was used to confirm the myosin head domains (PF00063), myosin N-terminal SH3-like domains (PF02736) and IQ calmodulin-binding motifs (PF00612) in these proteins. An Online webtool ProtPram (https://web.expasy.org/protparam/) and plant ensemble (https://plants.ensembl.org/) was used to determine the, gene start and endpoint, DNA strand, number of exons, genomic length, transcriptional length (a.a), molecular weight (M.W) and isoelectric point (P.I) of myosins in H. annuus and A. thaliana species.

2.2. Sequence alignment, phylogenetic analysis

Fifty full-length amino acid sequences of A. thaliana and H. annuus myosin proteins were aligned by ClustalX (Thompson et al., 1997). These aligned sequences were used to construct the un-rooted phylogenetic tree at 1000 bootstrap values using CLC sequence viewer software flowing the neighbor-joining methods (Saitou and Nei, 1987).

2.3. Discovery of conserved motifs and gene structure

An online tool MEME suite (https://meme-suite.org/meme/index.html) was used to determine the conserved regions of myosin proteins in A. thaliana and H. annuus according to the method described by (Bailey et al., 2009). Using the default parameters, maximum number of motifs and maximum sites per motif were fixed 10 and 5 respectively whereas motif width was in rang of 5–50 amino acids. Gene structure display server (GDS 2.0) (http://gsds.gao-lab.org./) was used to display the intron and exon organization of myosin genes, using genomic and CDS sequences as input files, and the intron and exon organization was illustrated to the corresponding branch of the tree.

2.4. Chromosomal mapping and gene ontology (GO)

To determine the position of myosin genes on A. thaliana and H. annuus chromosomes, we used MapGene2Chrom web service (http://mg2c.iask.in/mg2c_v2.0/) using the gene start point, gene endpoint, chromosomal length as input files.

To explore myosin proteins' biological functions and molecular and cellular components, we used SHINAYGO VO.741 (http://bioinformatics.sdstate.edu/go/) program with their default parameters and data was presented in a graphical format showing the percentage value of each function/component.

2.5. Synteny and gene duplication analysis

Comparative synteny analysis was carried out using the Circoletto Tool (http://tools.bat.infspire.org/circoletto/) with the goal of visualizing genome conservation among A. thaliana and H. annuus myosin genes. The coding sequences of duplicated genes were structured in MEGA7 using the Muscle (codon) method. The KaKs Calculator 2.0 tool was used to calculate the synonymous and nonsynonymous substitution rates, with the MYN approach. Furthermore, the divergence time ’t = Ks/2λ, where λ = 1.5 × 10−8 for dicots.

2.6. Prediction of subcellular location and expression of myosin genes

We used an online tool WoLFSPORT (https://wolfpsort.hgc.jp) to assess the positions of myosin genes in various subcellular components of A. thaliana and H. annuus and a heat map was generated by using TBtool. The expression of myosin XI genes in different plant tissues of A. thaliana and H. annuus at the different growth stages and tissues were predicted using GENEVESTIGATOR V3 (https://genevestigator.com) tool, and results were represented in a heat map.

3. Results

3.1. Physio-chemical properties of A. thaliana and H. annuus myosin proteins

Physio-chemical properties of A. thaliana and H. annuus myosin proteins are shown in Tables 1 and 2, respectively. Protein AtMYOSIN7.1 showed the highest amino acid length (1730), while AtMYOSIN16.1 exhibited the minimum (1030) number of amino acids. Each protein has an isoelectric point (pI) fixed value at which it will move in an electrical field. The pI value indicates that which protein is basic or acidic in nature. The results in Table 1 showed that almost all myosin proteins are basic in nature having pI value in range of 7.42–9.39 except AtMYOSIN7.1 which is basic possessing pI value 5.45.

Table 1.

Physio chemical properties of A. thaliana myosin proteins.

Transcript ID Common Name Chr. Strand Protein length pI M.W.
AT3G19960.1 AtMYOSIN1.1 3 Forward 1166 8.16 131090.98
AT3G19960.2 AtMYOSIN1.2 3 Forward 1176 8.41 132454.48
AT5G43900.1 AtMYOSIN2.1 5 Reverse 1505 7.69 169930.72
AT5G43900.2 AtMYOSIN2.2 5 Reverse 1562 7.93 176522.26
AT5G43900.3 AtMYOSIN2.3 5 Reverse 1565 7.87 176863.67
AT1G17580.1 AtMYOSIN5.1 1 Forward 1520 8.39 172907.86
AT1G04600.1 AtMYOSIN7.1 1 Forward 1730 5.45 194815.33
AT1G04160.1 AtMYOSIN8.1 1 Forward 1500 7.91 169380.28
AT1G08730.1 AtMYOSIN9.1 1 Forward 1538 9.39 174625.59
AT1G54560.1 AtMYOSIN11.1 1 Reverse 1529 9.36 173610.11
AT2G31900.1 AtMYOSIN12.1 2 Reverse 1556 8.36 176970.04
AT2G31900.2 AtMYOSIN12.2 2 Reverse 1557 8.26 177,098.21
AT2G31900.3 AtMYOSIN12.3 2 Reverse 1299 8.47 148580.84
AT2G31900.4 AtMYOSIN12.4 2 Reverse 1556 8.2 176970.04
AT2G31900.5 AtMYOSIN12.5 2 Reverse 1442 8.57 164557.9
AT2G20290.1 AtMYOSIN13.1 2 Reverse 1493 7.42 169072.88
AT4G28710.1 AtMYOSIN14.1 4 Forward 1516 7.46 172154.64
AT4G33200.1 AtMYOSIN15.1 4 Reverse 1522 8.89 173362.81
AT4G33200.2 AtMYOSIN15.2 4 Reverse 1492 8.8 169,942.95
AT4G33200.3 AtMYOSIN15.3 4 Reverse 1503 8.83 171102.13
AT5G54280.1 AtMYOSIN16.1 5 Forward 1030 8.83 117255.79
AT5G54280.2 AtMYOSIN16.2 5 Forward 1220 8.86 138561.82
AT5G20490.1 AtMYOSIN17.1 5 Reverse 1530 8.4 173245.97
AT5G20490.2 AtMYOSIN17.2 5 Reverse 1531 8.4 173377.16
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Chr., chromosome; M.W, molecular weight pI, isoelectric point.

Table 2.

Physio chemical properties of H. annuus myosin proteins.

Transcript ID Common Name Chr. Strand Protein length PI M.W
HannXRQ_Chr16g0526111 HanMYOSIN1.1 13 Forward 1104 7.56 130875
HannXRQ_Chr13g0407311 HanMYOSIN2.1 13 Reverse 1104 8.45 124476
HannXRQ_Chr08g0234651 HanMYOSIN2.2 8 Forward 900 6.66 101268
HannXRQ_Chr08g0234661 HanMYOSIN2.3 8 Forward 1163 8.66 131606
HannXRQ_Chr12g0366221 HanMYOSIN2.4 12 Reverse 1106 8.48 125302
HannXRQ_Chr05g0139901 HanMYOSIN5.1 5 Forward 1523 9.3 173685
HannXRQ_Chr13g0403781 HanMYOSIN6 .1 14 Forward 1474 8.34 168913
HannXRQ_Chr01g0014821 HanMYOSIN6 .2 1 Reverse 1502 8.20 170036
HannXRQ_Chr11g0342761 HanMYOSIN7.1 11 Reverse 1636 5.7 186421
HannXRQ_Chr11g0336841 HanMYOSIN9.1 11 Forward 472 6.19 53013.6
HannXRQ_Chr11g0339751 HanMYOSIN9.2 11 Forward 466 6.3 52374
HannXRQ_Chr04g0113721 HanMYOSIN9.3 4 Forward 1530 9.04 173687
HannXRQ_Chr11g0321791 HanMYOSIN11.1 11 Reverse 1526 9.11 173596
HannXRQ_Chr13g0404551 HanMYOSIN11.2 13 Forward 305 9.28 34918.6
HannXRQ_Chr11g0339771 HanMYOSIN11.3 11 Forward 1378 9.01 158527
HannXRQ_Chr08g0217201 HanMYOSIN11.4 8 Forward 1626 9.21 185002
HannXRQ_Chr07g0204511 HanMYOSIN11.5 7 Reverse 1523 9.19 173685
HannXRQ_Chr08g0217201 HanMYOSIN11.6 8 Reverse 1578 9.21 185002
HannXRQ_Chr04g0117591 HanMYOSIN12.1 4 Forward 1563 7.09 178440
HannXRQ_Chr07g0185821 HanMYOSIN14.1 7 Forward 1434 6.36 162826
HannXRQ_Chr08g0207711 HanMYOSIN14.2 8 Forward 205 10.27 23533.8
HannXRQ_Chr14g0434251 HanMYOSIN14.3 13 Reverse 167 5.76 18314
HannXRQ_Chr09g0261841 HanMYOSIN15.1 9 Forward 1523 9.09 172946
HanXRQ_Chr11g0336851 HanMYOSIN16.1 11 Forward 180 9.47 20773.9
HanXRQ_Chr09g0237941 HanMYOSIN17.1 9 Forward 1531 8.55 173685
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Chr., chromosome; M.W, molecular weight pI, isoelectric point.

3.2. Sequence alignment and phylogenetic tree

Phylogenetic tree of A. thaliana and H. annuus myosin proteins indicated that myosin proteins were subdivided into 4 clads (Figure 1). The 1st clad (CLAD1) was comprised of 13 proteins. The 2nd clad consisted of six members belonging to myosin12 proteins from both plant species. The 3rd clad was the largest subclass having seventeen myosin members. The 4th clad was possessing 11 myosin members.

Figure 1.

Figure 1

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The phylogenetic relationship among A. thaliana and H. annuus myosin proteins constructed using CLC Sequence Viewer 8.0. The four clads (CLAD1 to CLAD4) were indicated by different colors.

By submitting full-length amino acid sequences to MEME Tool, conserved motifs of myosin proteins were defined. Different colors represented each preserved motif. Ten conserved motif sequences were identified in these proteins (Figure 2). It was observed that pattern of conserved motif within sub clads of phylogenetic tree was conserved. AtMYOSIN12.4 showed the maximum number of motifs conserved, while myosin HanMYOSIN11.2 possessed only one conserved motif. Previous findings on preserved motifs have confirmed that proteins falling in the same clad showed similar conserved region patterns. Proteins that share identical motifs within a subgroup are probably likely to have similar roles, which may help predict these proteins' function.

Figure 2.

Figure 2

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Conserved motif analysis of A. thaliana and H. annuus myosins. Ten distinct motifs were discovered, and each motif was distinguished with different color.

The exon and intron pattern of myosin proteins was created in GSDS.2 using CDS and genomic sequences as input files. These genes were further depicted with their corresponding genes in the phylogenetic tree. CDS indicates red dots, blue upstream/downstream lines, and black line introns. Difference in the exon-intron structure and pattern between A. thaliana and H. annuus myosin genes was identified (Figure 3). The highest number of introns and exons were counted in AtMYOSIN7.1, AtMYOSIN9.1. The number of introns and exons ranged from 25 to 37. The number of exons in rice ranged from 38 to 52, indicating that the number of exons of these motor proteins in A. thaliana and H. annuus are lower than O. sativa. Within the subset, the chromosomal structure of myosin genes was similar. Similarities in the structure of genes within a subclass has been reported by (Azeem et al., 2018; Waqas et al., 2019).

Figure 3.

Figure 3

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Intron exon organization pattern of A. thaliana proteins. The pattern was organized by using GSDS 2.0. Yellow lines show the complementary sequences, whereas black lines represent introns.

To predict the physical location of myosin genes on A. thaliana and H. annuus chromosomes, non-overlapping Arabidopsis myosin orthologs were used. The results of Figure 4(a) showed that myosin genes were distributed on all five chromosomes of Arabidopsis. Nevertheless, the number of genes is variable. Chromosome 1 carries five genes and chromosomes 2 contains three genes. One gene was present on chromosome 3, while chromones 4 consisted of two gens. Chromosome 5 possessed three genes. In H. annuus myosin genes was present on nine different chromosomes with maximum 6 myosin genes present on chromosome 11 (Figure 4(b)).

Figure 4.

Figure 4

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(a) Location of myosin genes on A. thaliana chromosomes predicated by TAIR. (b), Location of myosin genes on H. annuus chromosomes predicted by MapGene2Chrom web service.

Gene Ontology analysis revealed that major biological functions of myosin genes were vesicle transportation along actin filaments, vesicle cytoskeleton, actin filament-based movement, cytoskeleton dependent intracellular movement, vesicle localization, movement of cell and subcellular components, organization of actin filament, action cytoskeleton, cell growth, and development (Figure 5(A)). Molecular functions of myosin genes include microfilament motor activities, actin-based ATPs activity, GTP-dependent protein binding, actin filament binding, actin binding and motor activity (Figure 5(B)). Gene ontology of cellular components indicated that these genes were located in myosin complex, actin cytoskeleton, root hair tips, root tips, cell tips, cell plate, cell vesicle and nuclear membrane (Figure 5(C)).

Figure 5.

Figure 5

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(a) Biological process, (b) Molecular function, (c) Cellular components: ShinyGO v0.741: Gene Ontology Enrichment Analysis was performed to predict the gene ontology of A. thaliana and H. annuus myosins.

Interaction analysis among various functions was carried by using online tool of SHINAYGO VO.741. results indicated that functions are interlinked with each other as shown in Figure 6. Myosin genes were generally interacting with actin filament based transport, cell development, actin cytoskeleton organization, organelle organization, cellular localization, cytoskeleton dependent transportation, cellular components organization, vesicle transport along action filament, cell growth, vesicle cytoskeleton trafficking actin filament organization, vesicle localization and intracellular transport.

Figure 6.

Figure 6

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Interaction network among myosin genes in H. annuus.

The molecular evolution rate was calculated by finding the value of Ka/Ks for each pair of duplicated genes. When Ka/Ks > 1, it was supposed that selection on the duplicated genes was positive selection; however, when Ka/Ks < 1, it was expected that selection on the duplicated genes was purifying selection, while neutral selection was assumed when Ka/Ks = 1. An increasing amount of purifying selection pressure was applied to the MYOSIN genes that were duplicated in our results, indicating that their function will not be significantly altered in further evolutionary process. In our study we estimated deviation time of the duplicated genes pairs. It is predicted that deviation time was 100 million years ago (MYA), if the MYOSIN genes had Ks values greater than 0.52. In our results, the Ks value of HanMYOSIN14.3, HanMYOSIN11.3 was observed 3.57, so the duplication time maybe 119.27 million years ago (MYA) (Table 3).

Table 3.

Ka/Ks value and time of divergence of myosin genes in H. annuus.

Seq_1 Seq_2 Ka Ks Ka_Ks Time (MYA)
HanMYOSIN11.2 HanMYOSIN16.1 1.128058048 1.955175163 0.576960095 65.17251
HanMYOSIN14.2 HanMYOSIN9.1 0.963793624 3.136308564 0.307301914 104.5436
HanMYOSIN14.3 HanMYOSIN11.3 0.876571223 3.578195044 0.244975808 119.2732
HanMYOSIN1.1 HanMYOSIN2.4 3.322480365 2.72824617 1.217808129 90.94154
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The comparative synteny analysis was performed between H. annuus, and A. thaliana that shows a remarkable relationship in the function of genes, duplication, triplication, and evolution expression. Gene sequences of HanMYOSIN1.1, exhibited synteny with the sequence AtMYOSIN16.1 and AtMYOSIN16.2 of A. thaliana. Similarly, AtMYOSIN11.1, AtMYOSIN7.1 were syntenic to HanMYOSIN11.1 and HanMYOSIN71 respectively. Two genes of A. thaliana such as AtMYOSIN1.1, AtMYOSIN1.2 showed synteny with HanMYOSIN1.1. The synteny analysis have exposed the duplication and conservation of outward and inward tangling events in the form of ribbons, respectively as shown in Figure 7.

Figure 7.

Figure 7

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Synteny analysis of myosin genes between A. thaliana and H. annuus. Colored lines which connect two regions indicate syntenic regions between A. thaliana and H. annuus.

Subcellular analysis enables the researchers to know that these proteins are located at cell membrane, nucleus, or another component. Results showed that myosin genes are located in cytoplasm, chloroplast, nucleus, mitochondria, endoplasmic reticulum, plasma, cell vacuoles, extra cellular locations, and Golgi bodies in both plant species. However, their concentration varies among these subcellular locations as shown in Figure 8(a) and (b) for H. annuus and A. thaliana respectively.

Figure 8.

Figure 8

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(a) Subcellular locations of myosin genes in H. annuus, (b) subcellular locations of myosin genes in A. thaliana. Heat map was constructed using TB tool.

The expression analysis results revealed that expression of H. annuus genes varied during three growth stages such as seedling, budding and flowering (Figure 9). HanMYOSIN5.1, HanMYOSIN14.2 and HanMYOSIN17.1 showed their expression in all three development stages of H. annuus plants. HanMYOSIN7.1, and HanYOSIN11.2 dint showed significant expression during any development stage of H. annuus, indicating that this gene can be specific to stress and may express itself during stress conditions. Tissue specific expression analysis indicated that no myosin gene was significantly expressed during ovary and bract however they were expressing in stamen, pollen, flowers, roots and nectary. Expression of genes in these tissues mean that these genes are involved stamen, pollen development similar results were supported by gene ontology analysis.

Figure 9.

Figure 9

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Heat map representation of expression pattern of H. annuus genes at different development stages (A) and in different plant tissues.

4. Discussion

Computational research is used to study the complex biological systems through realistic modelling and theoretical exploration. Further computational biology may lead to understanding the fundamentals of genomics and proteomics (Ahmad et al., 2021). Bioinformatics tools are also helpful to discover the interaction among macromolecules, distribution of protein properties, their biological functions (Ahmad et al., 2020) and identification of new capable compounds (Azeem et al., 2018). Myosin proteins belong to a superfamily of molecular motors that binds and hydrolyzes ATP to generate the force and movement along actin filaments (Bechtel and Bollhagen, 2021; Ma et al., 2020). This force is necessary to drive various cellular functions, cell movement, muscular contraction, cytokinesis, membrane trafficking and signal transduction (Steffens et al., 2014). In eukaryotic cells, various intracellular cargos, like cytoskeleton polymers, mRNA, protein complexes and membranous organelles are transported by the energy produced by ATP (Tominaga and Ito, 2015). Whereas twelve genes of this family have been identified in O. sativa (Fan et al., 2021; Jiang et al., 2007), and 11 genes have been characterized in Z. mays (Huang et al., 2020; Krendel and Mooseker, 2005b). For developing better understanding of myosin XIs, it is essential to study their functions from molecular to tissue level (Haraguchi et al., 2018b). Directional movement of motor proteins is essential for regulating and maintaining various biological phenomena through the generation of motive force (Tominaga and Nakano, 2012; Vale, 2003).

Plentiful information about the biochemical, functional, and structural characteristics of the motor domain of myosin family is available (Nebenführ and Dixit, 2018b; Thompson and Langford, 2002). But phylogenetic and evolutionary processes of myosin family are not fully understood yet and only few studies are available in this dimension (Sebé Pedrós et al., 2014). Some of these studies have focused on the phylogenetic relationships of restricted myosin clades, while others have chosen exemplars of functionally different classes of myosin. Myosin have structurally variable features, such as sequence divergence, gene duplications, length and domain architecture that may explain the ongoing need for a comprehensive phylogenetic analysis of such a prolific family (Allaby and Woodwark, 2004; De Souza et al., 2018). Myosin is usually classified based on their motor domain sequences (Haraguchi et al., 2019; Kijima et al., 2021). As it is well-documented that strongly supported phylogenetic clustering analysis and conserved domain organizations define new classes, a concise nomenclature of multiple members within these classes has not yet been developed. Such a nomenclature should reflect the phylogenetic relationship of different subtypes within classes, and thus must adhere to single gene duplication, genomic region and branch specific whole genome that result in orthologs and paralogs (Mühlhausen and Kollmar, 2013; Ryan and Nebenführ, 2018).

We used the amino acid sequences of Arabidopsis myosin and their orthologous and constructed the un-rooted phylogenetic tree to study the evolutionary relationship between Arabidopsis myosin members. These proteins were split into six subgroups by the phylogenetic tree, indicating great divergence between these members. Various researchers supported our results in other crops such as Nicotiana tobacco (Amari et al., 2014) and Gossypium hirsutum (Ma et al., 2020). Motif analysis showed that most of the motifs were preserved in a group, suggesting that these motifs were of similar evolutionary origin. Expression analysis showed that the expression of myosin gene varies in the H. annuus genome during different growth conditions. Many other myosin genes were created by gene duplication, divergence, and the acquisition of extra domains, encoding proteins specialized for specific biological functions enabled by alterations in the mechano-chemical ATPase cycle and acquiring various tails to interact with cargo. Abundant expression of myosin genes in Arabidopsis has been reported during organ growth and development (Krendel and Mooseker, 2005a; Tian et al., 2021). Similarly, myosin genes contribute to the growth of root tips, elongation of root hair and formation of pollen tubes (Tian et al., 2021).

5. Conclusion

The Myosin family in plants works by interacting with filaments in cells for cytoplasmic streaming. We discovered 15 myosin genes and their splice variants in A. thaliana and H. annuus. These myosin proteins perform various functions regarding the assigned task within plant cells. Their motility, velocity and enzymatic activities varies regarding plant species such as it is higher in higher plants than their motility in smaller plants. Plant myosins play significant role in the movement of fluids among organelles. The amino acid length of these proteins ranged from 1170 to 1493, suggesting physicochemical characteristics. There were only three acidic myosin proteins, and the remaining ten were basic. The molecular weights of these proteins ranged from 35,000 to 70,000 DKA. Myosins are non-membranous proteins, as suggested by their subcellular localization. Synteny analysis indicate that Arabidopsis and sunflower myosin genes are syntenic to each other although their time of divergence varies.

Declarations

Author contribution statement

Hafiz Muhammad Ahmad: Conceived and designed the experiments; Performed the experiments; Contributed reagents, materials, analysis tools or data.

Sidra Toor, Nouman Rasool, Madiha Zaynab: Performed the experiments; Contributed reagents, materials, analysis tools or data.

Munazza Ijaz, Saira Azmat, Asmaa M. Abushady, Sajid Fiaz, Hayat Ali Alafari, Madiha Zaynab, Yinglong Chen: Analyzed and interpreted the data.

Dalal S. Alshaya, Sidra Toor, Kotb A. Attia, Sajid Fiaz, Munazza Ijaz: Contributed reagents, materials, analysis tools or data; Wrote the paper.

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability statement

Data included in article/supp. material/referenced in article.

Declaration of interest's statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.

Acknowledgements

The authors would like to thank Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP 2022R292), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Contributor Information

Hafiz Muhammad Ahmad, Email: hafizahmad90@yahoo.com.

Sajid Fiaz, Email: sfiaz@uoh.edu.pk.

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