Chemical characterizations of Neurotransmission Receptors of human and plant to unfold the evolutionary relationships among them

Vertebrates have very well deﬁned nervous system. It is established that plant also has an alternative sort of sensitive nervous system. Researchers ﬁnd a close relationship of the neurotransmission mechanism of animal with the plant and suspects close relation in amino acid transport mechanism among both the organisms. Along with the abilities of determining protein structure and various functions, the chemical properties of amino acids also have great contribution in molecular evolution. Hence, in this present work it is aimed at making comparative study on the distribution of amino acids in the neurotransmission receptors associated to animals and plants, based on the chemical properties the amino acids contain. It is possible to classify 20 amino acids into 8 chemical groups and are identiﬁed by speciﬁc numeric value. The common pattern ﬁnding procedure in numerical representation of protein sequences ﬁnd some conserved regions in the receptor protein sequences of both the species. The comparative study has been made on distributions of chemical properties in protein sequences of ionotropic glutamate receptor protein sequences and GABA receptor protein sequences of two species namely human, a vertebrate and Arabidopsis thaliana, a plant. Experiments have been carried out to calculate proximity between protein sequences based on the distribution of each chemical group (in percentage) in them and phylogenetic trees have been constructed to ﬁnd evolutionary relationships of neurotransmission receptors of both the spices.


Introduction
Neurotransmission refers to a process by which signaling molecules are released by the axon terminal of a neuron, and then react with the receptors on the dendrites of another neuron by binding with them. Thus, vertebrates have very well defined nervous system. Plants grow in silence. Reported research works established the fact that plant also has an alternative sort of sensitive nervous system. In both the species some receptors are there which play very vital role in neurotransmissions. In vertebrates Ionotropic glutamate Receptors are a kind of receptors which have the ability of synapses to modify their synaptic strength in response to activity. It is a fundamental property of the nervous system [1]. There are four classes of ionotropic glutamate receptors in animals, namely NMDA (N-methyl-D-aspartate) receptor, AMPA (-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor, Delta receptor and kainate receptors [2]. According to researchers, similar to ionotropic glutamate receptor (iGluR) gene family of animals, glutamate-like receptor (GLRs) gene family in plants also have some functions in neurotransmission [3]. In human, another type of receptors that respond to the neurotransmission is gamma-aminobutyric acid (GABA) receptor. It is the chief inhibitory neurotransmitter in the vertebrate central nervous system [4]. Two classes of GABA receptors are there, GABA A and GABA B. GABA A receptors are ligand-gated ion channels (also known as ionotropic glutamate receptors), whereas GABA B receptors are G protein-coupled receptors (also known as metabotropic receptors). Ionotropic receptors(IRs) are made up of some protein subunits that together form an ion-conducting pore in the center of the receptor. IRs are more ancient than ORs (Olfactory Receptors). GABA is synthesised in a pathway known as the GABA shunt, which operates not only in the animals, but in plants too. A plant 'GABA receptor' inhibits anion passage through the aluminium-activated malate transporter family of proteins (ALMTs). GABA regulation of aluminium activated malate transporter (ALMT) activity could function as a signal that modulates plant growth, development, and stress response.
Numerous works since Darwin [5] have proved the response of plants to various signals like light, touch, wounding, stimulating etc. Now it has been reported that glutamate receptors of vertebrate nervous system and similar receptors in the plant equivalent of nerve have so close similarities that they must have evolved from a common ancestor [6,7,8]. Authors have tried to establish evolutionary relationships between the iGLuR gene family of mammals and GLRs of plants like Arabidopsis thaliana [3,9]. Characterization of an Arabidopsis GABA transaminase mutant (pop2) revealed that a GABA concentration gradient in the style is required to guide pollen tubes to the ovary [10]. Now, if specific protein sequences are there in plants which make control over such functionalities in neuro-transmission, then the next question arose that do the plants sleep and feel pain? Do they have consciousness? Do they have effects of anesthetics [11]? Numerous research papers are reported on it [12]. Till now reported research works indicate that plants lack the neural anatomy and all behaviors related to pain [13,14]. Amino acids are the building block of a protein. Evidence suggests that along with the role of protein synthesis some of the canonical amino acids play significant roles in modulating neurotransmission in animals [15]. Like, glutamic acid (Glu) works with ionotropic glutamate receptors and acts as excitatory neurotransmitter in Central Nervous System [16,17,18]. Aspertic acid (Asp) plays both the roles of neurotransmitter and neuromodulator [19,20]. Isoleucine, leucine and valine are called as Branched Chain Amino Acids (BCAAs) and act as precursors for Glutamate [21,22]. Glycine (Gly), Serine (Ser) and Alanine play the role of co-agonist for NMDA receptors [23,24,25]. Arginine [26,27], Lycine [28], Glutamine [29,30] and Asparagine (asn) [31] act as precursors. Deficiencies in Asparagine can be responsible for the abnormalities in the brain. Proline (Pro) acts as a neuromodulator at receptores like NMDA, AMPA/Kainate and Glycine [32].
According to Jack C. Schultz [33] plant's behaviour is largely biochemical. Researchers find a close relationship of the neurotransmission mechanism of animal with the plant protein sequences and suspects close relation in AA transport mechanism among both the organism [34]. It is known that each of 20 standard canonical amino acids contain specific chemical properties. Thus, 20 amino acids can be classified into 8 groups based on 8 distinct chemical properties [35]. Hence, it is worth to make comparative study on the distribution of amino acids in the receptor protein sequences associated to animals and plants, based on the chemical properties the amino acids contain. The investigation may unfold information about evolutionary relationships (if any) among the two organisms. Thus, in this present work a comparative study has been made on distributions of chemical properties in protein sequences of ionotropic glutamate receptor protein sequences and GABA receptor protein sequences of two species namely human, a vertebrate and Arabidopsis thaliana, a plant. Experiments have been carried out to investigate evolutionary relationship among the neurotransmission receptors of both the species.

Collection of protein sequences
To carry out the investigation two sets of protein sequences are considered for the species of human from animals and Arabidopsis thaliana from plant. To characterize ionotropic glutamate receptors 19 protein sequences from Arabidopsis thaliana and 18 protein sequences from human are collected from NCBI Genebank. The second set is Gamma-aminobutyric acid (GABA) receptors , where 8 Aluminumactivated Malate Transporters (ALMT) protein sequences are representing the Arabidopsis thaliana and 19 GABA A protein sequences of human . The details about the dataset taken are summarized in the Table 1.

Methods 2.2.1. Classification of Amino Acids with respect to their chemical properties
Along with the abilities of determining protein structure and various functions, the chemical properties of amino acids also have great contribution in molecular evolution. Hence, in this section it is aimed at making comparative study on the distribution of amino acids in the neurotransmission receptors associated to animals and plants, based on the chemical properties the amino acids contain. It is possible to classify 20 amino acids into 8 chemical groups as shown in Table 2.

Numerical Representation of protein sequences according to the chemical classification
Numerical representation of genomic sequences based on some predefined biological facts or properties simplifies the process of quantitative analysis of them [3,35,36]. In this subsection the amino acids of each protein sequences are converted into their corresponding group number of the chemical properties as specified in table 2. Thus here we find the numerical representation of all the protein sequences. Let S = S1, S2, ...Sn is an arbitrary protein sequence, where for any S, Si represents a single amino acid from the set, such that Si ∈ A, C, D, E, F, G, H, I, K, L, M, N, O, P, Q, R, S, T, V, W, Y . Let's consider 20 amino acids are classified into a set of groups C, then it is possible to read each amino acid of a protein sequence by its corresponding chemical group, such that, Ci ∈ 1, 2, 3, 4, 5, 6, 7, 8. As an example, a string of amino acid sequence 'CLICK' will be represented as '64462' after encoding it into the numeric representation.

Percentage-wise analysis of Amino Acid group present in each protein sequences.
All 20 Amino acids can not be equally distributed throughout the entire sequence. Distribution of chemical properties throughout each sequences are calculated. Thus for each set of data a matrix will be generated, from which it is possible to measure the proximity among the protein sequences.

Finding common patterns and identifying conserved regions
Conservation of DNA or protein sequences across species refers to identical or similar pattern of DNA or amino acids occurring in same or different species over the generations. Sometimes for the functional and structural importance of those conserved regions, they carry very essential ingredients to study the evolutionary relationship between species. Here in this subsection it is aimed to search for firstly block of amino acids which are existing in all the protein sequences of ionotropic glutamate receptors and GABA receptors of both the species respectively. Secondly, common patterns length L(where, 2 ≤ L ≤ 8), are searched in numeric representations of protein sequences to find the proximity of the sequences in terms of distribution of chemical properties. Given an input of pattern length L(where, 2 ≤ L ≤ 8), every possible pattern (made up any combination of numeric value 1 to 8) of length L are investigated among the sequences. If the pattern is found in every sequences, then the pattern is stored along with its location, otherwise it is discarded. Thus, by varying pattern length L, it is possible to find patterns of different lengths along with their corresponding location.

Investigating evolutionary relationships between two species with respect to the chemical properties
of the protein sequence Investigating evolutionary relationships between species measuring proximity between genomic sequences based on specific biological properties and investigating evolutionary relationships among them is a quite accepted approach. Here in this section it is intended to calculate proximity between protein sequences based on the distribution of each chemical group (in percentage) in them. Consider, S 1 and S 2 are any two protein sequences and P = (P 1, P 2, ..P 8) and Q = (Q1, Q2, ..Q8) are the percentage of occurrence of a eight chemical group in the given sequences respectively. Then it is possible to construct a distance matrix for a given set of sequences depending upon the distribution of chemical groups of each protein sequences, such that P i ∈{P1, P2, P3, P4, P5, P6, P7, P8} and Q i ∈{ Q1, Q2, Q3, Q4, Q5, Q6, Q7, Q8 }.
The proximity of S1 and S2 with respect to the distribution of chemical properties can be calculated using the following equation,

Percentage-wise measurement of amino acid compositions in all the protein sequences
Amino acid compositions in protein sequences are well conserved from species to species. The aim behind is to reduce the deleterious impact of mutations, to keep the function and structure of the protein relatively unaltered. In this subsection the amino acid composition of each protein sequence is measured (shown in Figure 1). It has been found that in all the protein sequences of ionotropic glutamate receptor protein sequences from both the species have majorly highest count of Leucine (Leu). Other most occurred amino acids are Glycine (Gly), Valine (Val), and Serine (Ser). The amino acid compositions of all GABA A receptor protein sequences of Human and ALMT receptor protein sequences of Arabidopsis thaliana also have some commonalities such that they all have huge amount of Leucine (Leu), Valine (Val), and Serine (Ser). The percentage-wise count of amino acids of all the protein sequences are shown in supplementary Table S7.

Common pattern finding in protein sequences
It has been observed that it is possible to classify 20 amino acids into 8 chemical groups and are identified by specific numeric value. The common pattern finding procedure of numerical representation of protein sequences find some conserved regions in the receptor protein sequences of both the species. The microscopic view of those common patterns are stated in the tables below.It has been found that all the ionotropic glutamate receptor protein sequences of human have a common pattern ′ 3748444 ′ of of length 7 at 872 th position, which encodes the string of amino acid "Y T AN LAA". The table 7 find common patterns (length of 2 AA and 3 AA) based on chemical groups of protein sequences of ionotropic glutamate receptor protein sequences and the table 8 states the common patterns ((length of 1 AA) found in the protein sequences in GABA receptor genes. It is to be noted that as in the protein sequences in GABA receptor protein sequences no common patterns are found more than length of 1 AA, hence here the common patterns length of 1 AA are considered. Table 9 states the conserved regions considering the amino acids individually.    In this section the pictorial representation of the similarity matrices are stated in the form of phylogenetic tree. The similarity matrices are shown in the supplementary tables S1, S2, S3, S4, S5, and S6. As the phylogenetic trees are constructed to get the ideas of the evolutionary relationships among the species, here figure 2 is showing the species wise evolutionary relationships of ionotropic glutamate receptor protein sequences and GABA receptor genes. Figure 2

Discussion
Animals and plants both react to wounding. Both of them have specific protein sequences which make control over such functionalities in neuro-transmission. Animals have well defined central nervous system, whereas, plants have alternative sort of sensitive nervous system, able to respond to various external stimuli. As mentioned, animals and plants both have glutamate and GABA signaling and receptors respectively. Animal versions of these receptors are targets of anesthetics in the animal nervous systems, but the plant versions are questionable. Here in this present work the objective is to make quantitative understanding of plant and animal receptor protein sequences which acts as neurotransmitters. It is also aimed at investigating the evolutionary relationships among them (if any). We have selected human from vertebrates and Arabidopsis thaliana from species plants. Our attempt is based on the distribution of eight types of chemical properties which an amino acid sequence may have. The reason behind is, they play active role in molecular evolution, where chemical properties of the side chain of amino acids can determine the essential amino acids in the core catalytic region. 'YTANLAA', a sequence of 7 amino acids length is the conserved region at 872 th position in all the human ionotropic glutamat receptor genes. The region resides at M3 domain of glutamate receptors [37]. According to reported research evidences, cysteine substitutions in this region indicates state-dependent accessibility and the region moves in response to receptor activation [38,39]. Thus, M3 domain acts as transduction element [40]. The numerical representation of the amino acid sequences based on the chemical properties have made the job of pattern matching easier as the time complexity is directly proportional to the sequence length N, that is O(N).

Conclusion
We detected the commonalities (conserved elements) during the long evolution of Arabidopsis thaliana and human from a common ancestor. Four and eight different conserved regions have been found (based on individual amino acids) in both the species for the GABA and ionotropic glutamat receptor protein sequences taken respectively. Three different conserved regions(length of two to three amino acids) are also found (based on chemical groups of amino acids) in all ionotropic glutamate receptor protein sequences taken of both the species. Fourteen conserved regions are found (based on chemical groups of amino acids) in all GABA receptor protein sequences taken of both the species. The constructed phylogenetic trees depicts higher proximity between certain protein sequences belong to both the species. Plant's behaviour is largely biochemical. The canonical amino acids play significant roles in modulating neurotransmission in animals. Investigations show a close relationship of the neurotransmission mechanism of animal with those of plant. It is the researchers' firm conviction that there exists a close relationship in amino acid transport mechanisms among both the organisms. Thus, there exists a need for a thorough study of plant neurochemicals as some of the compounds that affect human brain function also affect the growth and development of plants [41]. Therefore, the in-silico analysis performed in this study not only claims some common functionalities between human, a vertebrate and Arabidopsis thaliana, a plant in neurotransmission, but also initiates a new horizon to the biologists for better understanding of both human and plant physiology.
The authors declare no competing interests. Table S1. Similarity matrix of Ionotropic Glutamate receptor protein sequences of Arabidopsis thaliana. Table S2. Similarity matrix of human Ionotropic Glutamate receptor protein sequences. Table S3. Similarity matrix of human Ionotropic Glutamate receptor protein sequences. Table S4. Similarity matrix of human GABA A receptor protein sequences. Table S5. Similarity matrix of all GABA A receptor protein sequences of human and ALMT receptors of Arabidopsis thaliana Table S6. Similarity matrix of all Ionotropic Glutamate receptor protein sequences of both the species. Table S7. Percentage-wise measurement of amino acid compositions in all the protein sequences