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http://journals.tubitak.gov.tr/biology/

Turkish Journal of Biology Turk J Biol(2014) 38: 140-150© TÜBİTAKdoi:10.3906/biy-1307-14

Comprehensive analysis of beta-galactosidase protein in plants based on Arabidopsis thaliana

Samin SEDDIGH1,*, Maryam DARABI2

1Department of Plant Protection, Varamin-Pishva Branch, Islamic Azad University, Varamin, Iran2Department of Agronomy and Plant Breeding Sciences, College of Aboureihan, University of Tehran, Tehran, Iran

* Correspondence: seddigh@iauvaramin.ac.ir

1. IntroductionBeta-galactosidases (βgals) (EC 3.2.1.23) have a broad distribution, which encompasses plants, animals, and microorganisms. βgals catalyze the hydrolysis of the glycosidic bonds of terminal nonreducing β-D-galactosyl residues of oligosaccharides and β-D-galactopyranosides (Figure 1).

The biological roles of these enzymes include degradation of structural polysaccharides in plant cell walls (McCartney et al., 2000; Sorensen et al., 2000), thereby controlling fruit softening during ripening (Moctezuma et al., 2003); hydrolysis of dietary lactose (Cuatrecasas et al., 1965; Bayless et al., 1966); degradation of glycolipids and proteoglycans in mammals (Conzelmann et al., 1987; O’Brien, 1989); and metabolism of lactose and other galactosides in microorganisms (Campbell et al., 1973; Akasaki et al., 1976).

The 1024 amino acids of Escherichia coli βgal were first sequenced in 1970 by Fowler and Zabin. Dimri et al. (1995) described a new isoform for βgal with optimum activity at pH 6.0. Data composed at the Photon Factory have indicated that the βgal monomer is constructed from 5 different domains designed around a central alpha/beta barrel (Jacobson et al., 1994). Domain 1 is a jelly roll type

barrel, domains 2 and 4 are fibronectin type III-like barrels, domain 5 is a β-sandwich, and the central domain 3 is a TIM-type barrel. The third domain includes the active site (Matthews, 2005).

βgal is widely distributed and assessed in many creatures, such as insects (Seddigh and Bandani, 2012), and different plant tissues, like leaves (Hirano et al., 1994), seedlings (Li et al., 2001), hypocotyls (Kotake et al., 2005), and meristem zones of roots, cotyledons, vascular tissues, trichomes, and pollens (Hruba et al., 2005; Wu and Liu, 2006). The enzyme has been implicated in a number of biological processes, including plant growth and fruit ripening. Softening-related βgals have been purified from the fruits of tomato (Pressy, 1983; Carey et al., 1995; Carrington and Pressy, 1996), apple (Knee, 1973; Dick et al., 1990; Ross et al., 1994; Yoshioka et al., 1994), muskmelon (Cucumis melo) (Ranwala et al., 1992), avocado (Persea americana) (De Veau et al., 1993), kiwifruit (Ross et al., 1993), coffee (Coffea arabica) (Golden et al., 1993), persimmon (Diospyros kaki) (Kang et al., 1994), mango (Mangifeera indica) (Ali et al., 1995), and Japanese pear (Kitagawa et al., 1995) plants and their biochemical properties have been identified.

Abstract: Beta-galactosidases (βgals) (EC 3.2.1.23) have been detected in a wide range of plant organs and tissues and are described by their ability to hydrolyze terminal nonreducing β-D-galactosyl residues from β-D-galactosides. In this study, 92 βgal protein sequences from different plants, 7 animal samples including human and mouse, 3 samples from bacteria including Escherichia coli, and 4 samples from insects including Drosophila melanogaster were aligned. Sequences were analyzed by computational tools to predict the protein properties, such as molecular mass, isoelectric point, signal peptide, motifs, transmembrane domain, and secondary and spatial structure. Protein structure analysis revealed there is a high identity between plants and other organisms. The modeled βgal has a typical spatial structure with catalytic regions. The 3-dimensional model of Arabidopsis thaliana βgal (Accession Number: NP_001154292) was further checked by PROCHECK algorithm, and showed the majority of the amino acid residues were located in the most-favored regions in a Ramachandran plot. This result suggested that the simulated 3-dimensional structure was reliable. Phylogenetic analysis indicated that A. thaliana βgal has a close relationship with some plants’ βgal from different families such as Malvaceae, Solanaceae, and Poaceae. According to these results, βgals should be derived from a common ancestor.

Key words: Arabidopsis thaliana, β-galactosidase, catalytic domain, phylogenetic tree

Received: 09.07.2013 Accepted: 03.10.2013 Published Online: 02.01.2014 Printed: 24.01.2014

Research Article

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The increase in βgal activity during seed germination was also reported in plants such as pinto beans (Agrawal and Bahl, 1968), castor beans (Harley and Beevers, 1985), nasturtium (Edwards et al., 1988), barley (Giannakouros et al., 1991), and lupin (Buckeridge and Reid, 1994). The increase in βgal activity during the germination of barley seeds is very moderate when compared with the many-fold increase in the activity of the same enzyme observed in the seeds of dicotyledon plants (Giannakouros et al., 1991). Thus, the developmental regulation of plant βgal during germination and growth has become obvious.

Despite the fact that some functions of βgals have been investigated, limited information is available on the evolution of this protein in creatures. Recently, bioinformatics and genomic tools have revolutionized the studies of organisms’ metabolism (Darabi et al., 2012; Altay et al., 2013; Darabi and Seddigh, 2013a, 2013b; Kesici et al., 2013). In this study, we applied bioinformatics analysis on this key gene in plants for elucidation of structural, functional, and phylogenetic relationships. Analyses of molecular mass, isoelectric point, signal peptide, motifs, transmembrane domain, secondary and spatial structure, and evolution were performed to provide insights into the evolutionary mechanisms of the βgal protein family.

2. Materials and methods2.1. Collection of βgal sequencesβgal protein sequences belonging to plant species, animal species (including Homo sapiens [Hominidae] and Mus musculus [Muridae]), a bacterium species (Escherichia coli [Enterobacteriaceae]), and an insect species (Drosophila melanogaster), were downloaded from NCBI (http://www.ncbi.nlm.nih.gov) (date received: May 2013) in FASTA and GenBank format. The data consisted of 92 plant protein data, 7 animals, 4 insects, and 3 bacterial protein data, which were related to βgal enzyme. They are listed in Table 1.2.2. Bioinformatics analysisSeveral online web services and software were used for analyses of βgals in plants and other organisms. Comparative and bioinformatics analyses of βgals were carried out online at 2 websites (http://www.ncbi.nih.gov and http://expasy.org/tools). The phylogenetic analysis of βgals was

done with DNASTAR (MegAlign) using CLUSTALW with default parameters. While there are many tools for defining the presence or absence of recognizable domains, they are unable to recognize smaller individual motifs and more divergent patterns. Thus, the motifs of protein sequences were created using the program Multiple Em for Motif Elicitation (MEME; version 4.9.0) at a website (http://meme.nbcr.net/meme/cgi-bin/meme.cgi) (Bailey et al., 2006) to study the diversification of βgal protein in plants and other samples. The parameters of MEME analysis were applied as follows: minimum width for each motif, 6; maximum width for each motif, 20; maximum number of motifs to find, 4; and number of repetitions, 0 or 1 per sequence. The phylogenetic tree was constructed from aligned sequences using the software Molecular Evolutionary Genetic Analysis (MEGA; version 5). The maximum parsimony (MP) method was used to construct the phylogenetic tree as follows: MP search method, close-neighbor-interchange (CNI) on random trees; number of initial trees (random addition), 10; MP search level, 1. The homology-based tertiary structural modeling of At-βgals (Accession Number: NP_001154292) was done by ESYPred3D (http://www.unamur.be/sciences/biologie/urbm/bioinfo/esypred/) using neural networks. The 3D model of protein was created using the 3D structure (PDB Accession Code: 3t2o) chain A as a model. This model shared 38.9% of its identities with our query sequence (using the ALIGN program). WebLab ViewerLite 4 was used for 3D structure visualization. In order to evaluate the stereochemical qualities of the 3D model, PROCHECK analysis (http://www.ebi.ac.uk/pdbsum/procheck) (Laskowski et al., 1993) was performed and a Ramachandran plot was drawn. Other bioinformatics tools that were used for analysis are shown in Table 2.

3. ResultsThe results of the sequence alignment showed that βgals in plants and other organisms have high homology with each other. βgals contained 4 motifs: DAPDPVINTCNGFYC, HLRIGPYVCAEWNFGGFPVW, WAAQMAVGLNTGVPWIMCKQ, and LFESQGGPIILSQ IENEYGP (Figure 2).

In this study, βgal protein sequences from 54 different plant species, and 4 samples of other organisms,

Figure 1. Beta galactosidase reaction (http://en.wikipedia.org/wiki/Beta-galactosidase).

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Table 1. The 58 species analyzed in this study.

Index Abbreviation Scientific name Family Numbers of sequences

1 Ad Actinidia deliciosa var. deliciosa Actinidiaceae 12 At Arabidopsis thaliana Brassicaceae 33 Ao Asparagus officinalis Asparagaceae 24 Bo Brassica oleracea Brassicaceae 15 Ca Coffea arabica Rubiaceae 16 Can Capsicum annum Solanaceae 27 Car Cicer arietinum Fabaceae 48 Cme Citrus medica Rutaceae 19 Cm Cucumis melo var. cantalopensis Cucurbitaceae 110 Cp Carica papaya Caricaceae 411 Cs Citrus sinensis Rutaceae 212 Dc Dianthus caryophillus caryophyllaceae 213 Dk Diospyros kaki Ebenaceae 114 Dm Drosophila melanogaster Drosophilidae 415 Ec Escherichia coli Entrobacteriaceae 316 Fa Fragaria X ananassa Rosaceae 117 Fc Ficus carica Moraceae 118 Fch Fragaria chiloensis Rosaceae 119 Gb Gossypium barbadense Malvaceae 120 Ghe Gossypium herbaceum subsp. africanum Malvaceae 121 Gh Gossypium hirsutum Malvaceae 122 Gm Glycine max Fabaceae 223 Gr Gossypium raimondii Malvaceae 124 Hc Hymenaea courbari Fabaceae 125 Hs Homo sapiens Hominidae 326 Hv Hordeum vulgare Poaceae 127 La Lupinus angustifolius Fabaceae 128 Lu Linum usitatissimum Linaceae 129 Ma Musa acuminata Musaceae 130 Md Malus domestica Rosaceae 231 Mi Magnifera indica Anacardiaceae 132 Mm Mus musculus Muridae 433 Mt Medicago truncatula Fabaceae 134 Np Narcissus pseudonarcissus Amaryllidaceae 135 Nt Nicotiana tabacum Solanaceae 136 Os Oryza sativa japonica group Poaceae 237 Pam Persea americana Lauraceae 139 Pa Prunus armeniaca Rosaceae 139 Pc Pyrus communis Rosaceae 140 Ph Petunia X hybrida Solanaceae 141 Ppa Physcomitrella patens Funariaceae 242 Pp Prunus persica Rosaceae 4

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including fruit fly (Drosophila melanogaster), human (Homo sapiens), mouse (Mus musculus), and a bacterium (Escherischia coli) were aligned by CLUSTALW. The alignment of protein sequences showed high homology among various organisms. As shown in Figure 3, motif 4 (LFESQGGPIILSQIENEYGP) was the most conserved motif in plants and other organisms; it is the most distributed motif among different organism species. Table 3 shows some of the percentage identity between proteins in the multiple alignment of βgal.

The highest homology in βgal protein sequences of E. coli with plants belonged to Ricinus communis (XP-002513059) with a 39.1% identity. The lowest identity belonged to Rhaphanus sativus (BAD20774) with a 4.1% identity.

In order to investigate the protein structures of βgal in plants, Arabidopsis thaliana βgal (GenBank Accession Number: NP_001154292) was selected and analyzed by bioinformatics tools. The At-βgal protein (length: 1052 aa) had a molecular mass of 119544.2 KDa and a theoretical isoelectric point value of 7.14. SignalP analysis showed At-

43 Psa Prunus salicina Rosaceae 144 Ps Picea sitchensis Pinaceae 345 Pt Populus trichocarpa Salicaceae 446 Rc Ricinus communis Euphorbiaceae 447 Rs Rhaphanus sativus Brassicaceae 148 Sa Sandersonia aurantiaca Colchicaceae 149 Sb Sorghum bicolor Poaceae 250 Sl Solanum lycopersicum Solanaceae 551 Sm Selaginella moellendorffii Selaginellaceae 252 Th Thellungiella halophila Brassicaceae 153 Tm Triticum monococcum Poaceae 154 Tp Trifolium pratense Fabaceae 155 Vr Vigna radiata Fabaceae 156 Vv Vitis vinifera Vitaceae 457 Zj Ziziphus jujuba Rhamnaceae 158 Zm Zea mays Poaceae 3

Table 1. (continued).

Table 2. Servers used for bioinformatics analysis.

Software Application Website

ProtParam Predicting primary structure http://web.expasy.org/protparam/SOPMA Predicting secondary structure http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.htmlTMHMM Prediction of transmembrane helices http://www.cbs.dtu.dk/services/TMHMM-2.0

SignalP Prediction of the presence and location of signal peptide cleavage sites http://www.cbs.dtu.dk/services/SignalP

Figure 2. Sequence logo of each beta galactosidase motif created by MEME.

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Figure 3. Motifs for βgal proteins. The MEME motifs are shown as different-colored boxes. Motif 4 is the most distributed motif among different organisms, as shown in purple.

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βgal had no signal peptide that synthesized in the cytoplasm. No transmembrane region of At-βgal-NP_001154292 was recognized by TMHMM 2.0 analysis.

The secondary structure of At-βgal was analyzed by SOPMA (Geourjon and Deleage, 1995). Based on hierarchical neural network analysis (Combet et al., 2000), this protein was composed of 25.57% α-helix, 23.76% extended strand, 6.56% beta turn, and 44.11% random coil.

An important indicator of the stereochemical quality of the model is a distribution of the RMS distances from different planar groups in the structure in a Ramachandran plot (Figure 4), which shows the best-fit plane. Therefore, the structure of At-βgal has good chain stereochemistry.

The 3D structure of At-βgal-NP_001154292 was predicted by ESYPred3D (automated homology modeling program using neural networks) (Figure 5). Molecular modeling results revealed that At-βgal had an intricate spatial architecture very similar to the E. coli βgal whose catalytic portion and activity were determined (Jacobson et al., 1994).

The phylogenetic tree was constructed based on the amino acid sequences of βgals in plants and other βgals from different organisms, including human, mouse, bacteria, and insect, to investigate the evolutionary relationships among them. The MP method was used to design the phylogenetic tree (Figure 6). The principle of MP searches for a tree that requires the smallest number of changes to explain the differences observed among the taxa

under study. In practical terms, the MP tree is the shortest: the one with the fewest changes, which, by definition, is also the one with the fewest parallel changes. Results of the phylogenetic tree showed that because all βgal proteins separated in different groups, they have a relationship with each other and they may derive from a common ancestor.

Motif analyses of the βgal protein in plants revealed that At-βgal had 4 motifs, which are shown in Figure 7.

4. DiscussionThe βgal assay is used frequently in genetics, molecular biology, and other life sciences for a blue-white screen. The βgal reporter gene is extensively used in biological research. In spite of the fact that it was originally identified in E. coli, this reporter is functional in many other organisms, such as yeast, Caenorhabditis elegans, Drosophila, plants, and mammals. Other advantages of the βgal system concern the stability of βgal enzyme, easy activity assay procedure, and availability of a broad range of substrates (Serebriiskii and Golemis, 2000). βgal, the product of the lacZ gene of E. coli, is one of the most extensively used reporters of gene expression in molecular biology. In E. coli, the biologically active enzyme exists as a tetramer of 4 identical subunits (Kalnins et al., 1983).

In this study, primary and secondary structures of At-βgal-NP_001154292 were detected. Present through the majority of the parts of the secondary structure,

Table 3. The percentage identity between βgal protein sequences (above 90%) in the multiple alignments of different plant species.

Index Characteristics Identity (%)

1 Fch-βgal-ADW19770, Car-βgal-CAA07236 90.9

2 Fch-βgal-ADW19770, Cp-βgal-ACP18874 90.0

3 Fch-βgal-ADW19770, Cp-βgal-ACP18875 90.0

4 Fch-βgal-ADW19770, Pp-βgal-ABV32545 94.1

5 Fch-βgal-ADW19770, Mt-βgal-ABN08398 90.4

6 Psa-βgal-ABY71826, Pp-βgal-ABV32546 98.4

7 Cp-βgal-ACP18874, Cp-βgal-ACP18875 98.8

8 Gb-βgal-ADZ16125, Ghe-bgal-ADZ16126 98.6

9 Vr-βgal-AAF67341, Gm-βgal-ACF22882 93.6

10 Sl-βgal-AAC25984, Can-βgal-AAK40304 90.9

11 Sl-βgal-AAC25984, Can-βgal-BAC10578 90.9

12 Gr-βgal-ADZ16127, Fa-βgal-CAC44501 98.7

13 Pp-βgal-ADD62393, Pa-βgal-AAG12249 97.1

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random coils and α-helix are the most plentiful structural elements of At-βgal; extended strand and beta turns were intermittently distributed in the protein.

A secretory protein is a protein that is secreted by a cell, such as many hormones, enzymes, toxins, and antimicrobial

peptides. They are synthesized in endoplasmic reticulum and are identified with their signal peptides.

A signal peptide is a short peptide (5–30 amino acids long) that exists at the N-terminal of most of the newly synthesized proteins, which are destined towards

RMS distances from planarityfile

PROCHECK

0.00 0.01 0.02 0.03 0.04

ARG

Freq

uenc

y

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ASN

Freq

uenc

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0.00 0.01 0.02 0.03 0.04

ASP

Freq

uenc

y

0.00 0.01 0.02 0.03 0.04

GLN

Freq

uenc

y

0.00 0.01 0.02 0.03 0.04

GLU

Freq

uenc

y

0.00 0.01 0.02 0.03 0.04

HISFr

eque

ncy

0.00 0.01 0.02 0.03 0.04

PHE

Freq

uenc

y

0.00 0.01 0.02 0.03 0.04

TRP

Freq

uenc

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0.00 0.01 0.02 0.03 0.04

TYR

Freq

uenc

y

Histograms showing RMS distances of planar atoms from best-fit plane.Black bars indicate large deviations from planarity: RMS dist > 0.03 for rings, and > 0.02 otherwise.

Figure 4. These histograms show the RMS distances from planarity for the different planar groups in the structure. The dashed lines indicate different ideal values for aromatic rings (Phe, Tyr, Trp, and His) and for planar end-groups (Arg, Asn, Asp, Gln, and Glu). The default values are 0.03Å and 0.02Å, respectively. Histogram bars beyond the dashed lines are highlighted.

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Figure 5. The 3D structures of At-βgal (Arabidopsis thaliana βgal; NP_001154292) (left) and Ec-βgal (Escherischia coli βgal; EGH38654) (right) established by homology-based modeling (PDB Accession Code: 3t2o) using EsyPred 3D. The α-helix is shown helix-shaped in red, the beta sheet wide ribbon-shaped in green, and the random coil line-shaped in gray. The 3D structure was shown as a solid ribbon model by WebLab ViewerLite 4. Domains and active sites are shown in the E. coli 3D structure.

Figure 6. The phylogenetic tree of βgals from plants and other organisms using the CLUSTALW (MEGA 5) program. The MP method was used to construct the tree. Animal sequences are shown with red branches, insect sequences with violet, bacteria with blue, and plants with green.

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Figure 7. Motifs in protein sequence of Arabidopsis thaliana βgal (GenBank Accession Number: NP_001154292).

the secretory pathways (Blobel and Dobbertein, 1975). Signal peptides are extremely heterogeneous and many prokaryotic and eukaryotic signal peptides are functionally interchangeable even between different species; however, the efficiency of protein secretion is strongly determined by the signal peptide (Kober et al., 2013; Von Heijne, 1985). SignalP analysis showed that At-βgal-NP_001154292 is not a secretory protein, which is the characterization of βgal.

Proteins can end up as integral membrane components. These proteins are said to be transmembrane. In the other words, a transmembrane protein is a protein that goes from one side of a membrane to the other. βgal is not a transmembrane protein. Bioinformatics analysis also revealed that At-βgal-NP_001154292 has no transmembrane region, which describes βgal.

Recently, many studies have been carried out on conserved regions of different sequences to investigate their relationship and their role in biology (Darabi et al., 2012, Darabi and Seddigh, 2013a, 2013b). We performed motif analyses of these proteins in order to find patterns of conserved motifs, and ran the MEME program to detect sequence motifs in plants, bacteria, and 3 animal samples. At-βgal had 4 motifs, which are shown in Figure 7.

The 3D structure of Ec-βgal is known, and the structural basis for its reaction mechanism has been reported. Three-dimensional modeling of At-βgal-NP_001154292, which has a simple spatial architecture, was similar to that of the E. coli βgal whose catalytic portion and activity were previously determined (Fowler and Zabin, 1970; Matthews, 2005).

As phylogenetic analyses can be the basis for molecular and biochemical analyses of protein families, we performed protein research on the βgal protein family in different organisms based on Arabidopsis thaliana βgal (Accession Number: NP_001154292). Phylogenetic analysis showed that A. thaliana βgal had a close relationship with the βgal in plants from different families, such as Gossypium hirsutum (Malvaceae), Nicotiana tabacum (Solanaceae), Hordeum vulgare, and Triticum monococcum (Poaceae). Although similarities are clear in a family, there is not enough knowledge among different families’ relationships and similarities. However, these results suggested that, in plants, βgals are inherited from a common ancestor. Furthermore, A. thaliana has a close relationship with some of the animals’ βgals, like Mus musculus and Drosophila melanogaster.

The results of MEME showed that all βgals from different creatures have similar conserved sites. This result is supported by the phylogenetic tree. Although animals are separated in a group, they have a close relationship with other organisms, like plants. These results can lead us to understand new things about evolutionary relationships among different creatures.

AcknowledgmentsWe sincerely appreciate the support for this research from Islamic Azad University, Varamin-Pishva Branch.

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