L- Myo-Inositol 1-Phosphate Synthase (MIPS) in Chickpea: Gene Duplication and Functional Divergence

Gene duplication is one of the key driving forces in the evolution of genes and important features of genomic architecture of living organisms including plants. Moreover, much of the plant diversity may have arisen largely due to duplication, followed by divergence and adaptive specialization of the pre existing genes (Ohno,1970; Zhang, 2003; Flagel & Wendel,2009). Current impetus on genomic sequence data provides substantial evidence for the profusion of duplicated genes in all organisms surveyed. Functional divergence after gene duplication can possibly result in two alternative evolutionary fates: i) neofunctionalization where one copy acquires an entirely new function whereas the other copy maintains the original function. ii) Subfunctionalization, in which each copy adopts part of the task of their parental gene (Ohno,1970; Nowak et al., 1997; Jenesen,1976; Orgel,1977;Hughes,1994). However, subfunctionalization is reported as a more prevalent outcome than neofunctionalization in nature. In any case, functional divergence of such paralogous proteins is found to be the key force shaping molecular network in organisms (Ohno, 1970). Recent studies also suggest that duplicate genes diverge mostly through the partitioning of gene expression as in subfunctionalization (Force et al.,1999; Wagner,2000; Gu et al.,2002). In addition, subfunctionalization can also take place at the protein function level leading to functional specialization, when one of the duplicated genes becomes better at performing one of the original functions of the progenitor gene (Hughes, 1994; Gu et al.,2002; Conant &Wolfe, 2008; Hughes, 1999; Zhang et al., 2002). Myo-inositol-1-phosphate synthase (MIPS;EC5.5.1.4) is an evolutionary conserved enzyme which catalyzes the rate limiting step in well conserved inositol biosynthetic pathway and is extremely widespread in living organisms including plants (Loewus & Murthy, 2000; Majumder et al., 2003). The evolution of MIPS gene/ protein among the prokaryotes seems to be more divergent and complex than amongst the eukaryotes, however they preserve a conserve core catalytic domain among the MIPS proteins (Majumder et al., 2003). Many of the plant species are known to contain more than one copy of gene encoding MIPS and are hypothesized to arise through gene duplication. Expression studies of multiple gene encoding MIPS have revealed the possibility of specialized role for individual enzyme isoforms. Previously, two genes encoding MIPS have been identified and characterized from chickpea by Kaur et al. A comparative study of two divergent genes (CaMIPS1 & CaMIPS2)


Introduction
Gene duplication is one of the key driving forces in the evolution of genes and important features of genomic architecture of living organisms including plants.Moreover, much of the plant diversity may have arisen largely due to duplication, followed by divergence and adaptive specialization of the pre existing genes (Ohno,1970;Zhang, 2003;Flagel & Wendel,2009).Current impetus on genomic sequence data provides substantial evidence for the profusion of duplicated genes in all organisms surveyed.Functional divergence after gene duplication can possibly result in two alternative evolutionary fates: i) neofunctionalization where one copy acquires an entirely new function whereas the other copy maintains the original function.ii) Subfunctionalization, in which each copy adopts part of the task of their parental gene (Ohno,1970;Nowak et al., 1997;Jenesen,1976;Orgel,1977;Hughes,1994).However, subfunctionalization is reported as a more prevalent outcome than neofunctionalization in nature.In any case, functional divergence of such paralogous proteins is found to be the key force shaping molecular network in organisms (Ohno, 1970).Recent studies also suggest that duplicate genes diverge mostly through the partitioning of gene expression as in subfunctionalization (Force et al.,1999;Wagner,2000;Gu et al.,2002).In addition, subfunctionalization can also take place at the protein function level leading to functional specialization, when one of the duplicated genes becomes better at performing one of the original functions of the progenitor gene (Hughes, 1994;Gu et al.,2002;Conant &Wolfe, 2008;Hughes, 1999;Zhang et al., 2002).Myo-inositol-1-phosphate synthase (MIPS;EC5.5.1.4)is an evolutionary conserved enzyme which catalyzes the rate limiting step in well conserved inositol biosynthetic pathway and is extremely widespread in living organisms including plants (Loewus & Murthy, 2000;Majumder et al., 2003).The evolution of MIPS gene/ protein among the prokaryotes seems to be more divergent and complex than amongst the eukaryotes, however they preserve a conserve core catalytic domain among the MIPS proteins (Majumder et al., 2003).Many of the plant species are known to contain more than one copy of gene encoding MIPS and are hypothesized to arise through gene duplication.Expression studies of multiple gene encoding MIPS have revealed the possibility of specialized role for individual enzyme isoforms.Previously, two genes encoding MIPS have been identified and characterized from chickpea by Kaur et al. A comparative study of two divergent genes (CaMIPS1 & CaMIPS2) reveals features of both functional redundancy and diversification (Kaur et al., 2008).This chapter explores how a possible gene duplication of MIPS gene in chickpea lead to a functional diversification that perhaps contributed adaptive evolution to the plant.

Evolution and diversification of MIPS
The inositols are the nine isomeric forms of cyclohexane hexitols and myo-inositol is the most abundant and physiologically favored molecule in the biological system.The biosynthesis of myo-inositol has been acknowledged as an evolutionary conserved pathway and its importance across biological organisms from different domains of life has been recognized for long time.The first and rate limiting step of this pathway is catalyzed by an evolutionary conserved enzyme named as MIPS.MIPS particularly catalyzes the conversion of glucose 6-phosphate (Glc6P) to myo inositol 1phosphate (Ins1P) through an internal oxidoreduction reaction involving NAD + .Subsequently inositol 1-phosphate is dephosphorylated to produce free inositol by Mg 2+ dependent myo-inositol 1-phosphate phosphatase (IMP: EC 3.1.3.25)(Loewus & Loewus,1983;Loewus,1990).This free myo inositol occupies the central position in inositol metabolism since this free inositol can be chanellized to various metabolic routes and produce different inositol derivatives (Fig- 1) (Loewus & Murthy, 2000;Loewus,1990).This free inositol and its derivatives have acquired diverse functions over the course of evolution.As for example, inositol containing phospholipids are the important constituents of many archaea.Few thermophilic archaea also use inositol phosphodiester as thermo protective solutes.Then with the emergence and diversification of eukaryotes, function of inositol and its derivatives proliferated dramatically.So far, inositol and its derivatives have been shown to be involved in growth regulation, membrane biogenesis, hormone regulation, signal transduction, pathogen resistance and stress adaptation in higher plants (Loewus & Murthy, 2000;Stevenson et al., 2000;Michell,2008).

Inositol
Since the usage and distribution of inositol and inositol derivatives are reported in all domains of life, it is imperative to contain MIPS enzyme in diverse organisms such as archaea, eubacteria, parasites, animals, higher plants and many others.Few higher plants and algae are reported to have both cytosolic and chloroplastic isoforms of MIPS.However, the biochemical and enzymatic properties of these two forms do not differ significantly between each other.Recent studies suggest that rice chloroplastic MIPS is coded by OsINO1-1 gene located on chromosome 3 (RayChaudhuri et al.1997;Ray et al., 2010).
The structural gene coding (INO1) for this ancient enzyme was first identified and cloned in Saccharomyces cerevisiae (Donahue & Henry,1981).Subsequently more than 80 INO1 genes were reported from various sources including both prokaryotes and eukaryotes.
Evolution and diversification of MIPS has been highlighted by Majumder et al. (2003) and a clear difference between prokaryotic and eukaryotic MIPS protein sequences was observed when compared among each other.The MIPS protein sequences of prokaryotes are quite divergent among themselves and significantly distinct than any other known eukaryotic sequences.In contrast, the eukaryotic MIPS sequences show remarkable similarities among each other.A phylogenetic tree constructed to include few representative MIPS sequences from diverse organisms present an overall evolutionary divergence of this enzyme in the biological kingdom.The higher plants constitute one close subgroup, while the higher animals, protozoa, fungi form the other subgroups in the eukaryotic cluster (Fig- 2).In Archeoglobus, MIPS shows more sequences similarity to the eukaryotic MIPS than the other known prokaryotic ones and thereby all eukaryotic MIPS seems to have evolved from one common stock, probably from the fusion of an archaebacterial and eubacterial MIPS genes (Fig- 2 & 3).Four stretches of amino acid residues (GWGGNG, LWTANTERY, NGSPQNTFVPGL and SYNHLGNNDG) are found to be conserved in MIPS proteins of all eukaryotes and among them; SYNHLGNNDG is identified as highly conserved.Interestingly among higher plants, MIPS enzyme shows greater conservation in addition to these four domains (Fig- 3).Many of the plant species also possess multiple genes encoding MIPS and are thought to arise through gene duplication in course of time.Subsequent analysis of crystal structures of various MIPS proteins provide ample evidence towards the presence of conserved "core structure" in all MIPS proteins throughout evolution.Moreover, some of the important amino acid residues are identified in the active site of the yeast MIPS and are shown to be highly conserved in all eukaryotic MIPS.These amino acids are considered to be the part of a "eukaryotic core structure" which has remained largely the same during evolution, despite the divergence in rest of the sequences over time (Fig- 3) (Stein & Geiger, 2002;Norman et al.,2002).Crystal structure analysis of MIPS from Saccharomyces cerevisiae also revealed that each monomer of the homo-tetrameric MIPS has three functionally important structural domains namely the NAD binding Rossman fold, the catalytic binding site and the core domain.This study also exemplifies a case of induced fit model for binding of the substrate with the catalytic domain of the enzyme.(Stein & Geiger, 2002) Fig. 2. A phylogenetic tree of few representative MIPS amino acid sequences from various domains of living organisms.Neighbour-Joining algorithm was used to construct tree from the distance matrix using Clustal X. Thousand rounds of bootstrapping were performed to ensure the validity of the tree.

MIPS from chickpea: A case of functional divergence
Chickpea is an annual self-pollinated diploid legume crop which is mostly grown in the arid and semi arid regions of the world.Long term evolution and adaptation to harsh conditions make chickpea rich in resistant genes for environmental stresses including drought and cold.Several classes of genes controlling potential resistance have been identified through genomic and proteomic studies (Ahmaed et al., 2005;Mantri ,2007;Bhusan et al., 2007).
In this particular plant, inositol seems to play an important role in drought tolerance besides growth and development, since inositol content and MIPS transcript was found to be significantly increased under dehydration condition (Boominathan et al.,2004).Subsequently, chickpea is reported to have two MIPS coding genes (CaMIPS1 and CaMIPS2) (Kaur et al. 2008) and both genes are revealed to have overall similar structure consisting of 9 introns and 10 exons (Fig- 4).Sequence analysis of these two genes show high similarity (>85%) in their coding regions but their non-coding or 5' and 3' flanking regions are extremely divergent.Moreover length of each exon is similar between these two genes while the size of introns varies.Such findings suggest that these two MIPS genes most likely arose by ancestral gene duplication and have undergone considerable sequence divergence.
In spite of the remarkable resemblance in their coding sequences, some base substitutions occurs in exons leading to changes in 43 amino acids in protein sequences, however, maintaining four highly conserved functional domains and known active site amino acids of MIPS (Fig- 3) (Majumder et al. 2003).Among these 43 amino acids, 19 amino acids differ considerably between CaMIPS1 and CaMIPS2 while rest of the amino acid substitutions are relatively insignificant, i.e. substitution between amino acids having similar physico chemical properties (Kaur et al., 2008).Kaur et al., 2008] Functional divergence after gene duplication can result in following alternative fates: One copy acquires a novel function (neofunctionalization) or one copy loses its function completely or each copy adopts part of the task of their parental gene (subfunctionalization) (Ohno, 1970;Nowak et al., 1997;Jenesen, 1976;Orgel,1977;Hughes,1994).Functional complementation and in-vitro enzymatic properties were analyzed to check the fate of these two genes.First to check the functional identity of these two divergent genes, a complementation experiment was carried out in natural inositol auxotroph Schizosaccharomyces pombe PR109 which clearly demonstrates that both CaMIPS1 and CaMIPS2 indeed encode functional MIPS enzymes.Subsequently, the enzymatic properties of these two enzymes were examined since CaMIPS1 and CaMIPS2 polypeptides are reported to have some differences in their amino acid sequences.Both enzymes showed nearly same Km values for Glc6P suggesting the similar substrate specificity.For both proteins, the optimum temperature for enzyme activity is at 35°C and optimum pH is 7.0 suggesting the similar biochemical characteristics (table1).
Further the enzymatic activities of each protein under stress environment in invitro conditions were examined and the activities of these two enzyme proteins were shown to differ significantly in response to high temperature and salt concentration (Kaur et al., 2008).CaMIPS1 activity is considerably affected at high temperature or in presence of increasing sodium chloride concentration while the CaMIPS2 activity is less affected in similar conditions and thereby retaining higher activity than CaMIPS1 (Fig- 5).
The amino acid substitutions in protein sequence as analyzed by sequence comparison and the higher enzyme activity in CaMIPS2 under stress condition also indicates that it might be evolved during the course of time to function better under stress conditions.This differential activity towards high temperature and salt of these two enzymes could be supported by the bioinformatics analysis in respect to the available yeast MIPS crystal structure and salt tolerant PcINO1 (MIPS coding gene from Salt tolerant Porteresia coarctata) protein sequence (Majee et al., 2004).Based on the bioinformatics study, CaMIPS2 appears to be more stable towards destabilizing factors such as high temperature, salt, etc, thereby retains better functionality under such conditions (Kaur et al., 2008).Subsequently, growth pattern of CaMIPS1 & CaMIPS2 transformed Schizosaccharomyces pombe under stress conditions were analyzed and CaMIPS2 transformed S. pombe cells were reported to grow or survive better than CaMIPS1 transformants both at high temperature and salt environment (Fig- 6) suggesting CaMIPS2 gene product functions more efficiently under stress conditions due to its stress tolerant property and hence provide sufficient inositol to grow as compared to CaMIPS1.[Modified from Kaur et al., 2008 Kaur et al., 2008] Recent studies suggest that duplicate genes diverge mostly through the partitioning of gene expression as in subfunctionalization and thereby being expressed in a differential manner; redundant genes may acquire functional divergence (Force et al., 1999;Wagner, 2000;Gu et al., 2002).This hypothesis was examined on CaMIPS1 and 2. CaMIPS1 gene was shown to express in root, shoot, leaves, and flower in fairly equal abundance but no transcript was observed in seed, while CaMIPS2 transcript was observed in all examined tissues including seed.This result proposes that CaMIPS1 and CaMIPS2 genes are indeed differentially regulated in different organs to coordinate inositol metabolism with cellular growth as hypothesized previously (Loweus & Murthy,2000).Subsequently, expression pattern of these two genes are examined in various environmental stresses.Interestingly, CaMIPS2 was shown to be induced at different level in various environmental stresses while level of CaMIPS1 transcript was found to be unaltered by such stresses (Fig- 7).This differential expression is also supported by the divergence of their upstream regulatory sequences.Kaur et al., 2008]

Conclusion
Gene duplication, followed by sequence divergence leads to functional divergence of the paralogous proteins, is a major force for adaptation of living organisms.Without gene duplication, the plasticity of genome or organism in adapting to changing environment would be very limited.Chickpea plants are know to be evolved and diversified considerably over time and acquired subsequently various potential genes for their adaptation to environmental stresses.It seems that this drought tolerant legume plant requires more inositol for their adaptation particularly under drought condition and hence acquired CaMIPS2 over time.Collectively, our results exemplified that CaMIPS1 and CaMIPS2 are differentially expressed in chickpea to play discrete though overlapping roles in plant; however CaMIPS2 is likely to be evolved through gene duplication, followed by adaptive changes in its sequences to function better under environmental stresses and thereby play a key role in environmental stress adaptation along with other aspects of inositol metabolism in chickpea.

Fig. 3 .
Fig. 3. Multiple sequence alignment of MIPS from prokaryotes and eukaryotes.Proposed common active site amino acid residues for the Mycobacterium tuberculosis and Saccharomyces cerevisiae MIPS sequence are highlighted in green color and four conserved domains of eukaryotes (GWGGNG, LWTANTERY, NGSPQNTFVPGL and SYNHLGNNDG) are highlighted in yellow color.43 variant positions between CaMIPS1 and 2 have been highlighted in blue color.
This work was supported by a grant from Department of Biotechnology (Next Generation Challenge Programme on Chickpea Genomics), Department of Science and Technology (Fast Track Scheme) , Government of India.We also like to acknowledge the Research support from National Institute of Plant Genome Research, New Delhi.H.K. thanks the Council of Scientific and Industrial Research, Government of India, for Senior Research Fellowship.