**2. Gene duplication and functional divergence of MIPS in chickpea**

### **2.1 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 1 phosphate (Ins1P) through an internal oxidoreduction reaction involving NAD+. Subsequently inositol 1- phosphate is dephosphorylated to produce free inositol by Mg2+ 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).

Fig. 1. Inositol biosynthesis and its consumption in other pathway.

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

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

The inositols are the nine isomeric forms of cyclohexane hexitols and *myo*-inositol is the

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

MIPS particularly catalyzes the conversion of glucose 6- phosphate (Glc6P) to *myo* inositol 1 phosphate (Ins1P) through an internal oxidoreduction reaction involving NAD+. Subsequently inositol 1- phosphate is dephosphorylated to produce free inositol by Mg2+ 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).

Glc6P

Ins1P

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

IMP

MIPS (rate limiting enzyme)

Oxidation pathway

Methylation pathway

functional diversification that perhaps contributed adaptive evolution to the plant.

**2. Gene duplication and functional divergence of MIPS in chickpea** 

most abundant and physiologically favored molecule in the biological system.

**2.1 Evolution and diversification of MIPS** 

by an evolutionary conserved enzyme named as MIPS.

**Inositol** Signaling

Fig. 1. Inositol biosynthesis and its consumption in other pathway.

pathway

Conjugation pathway

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).

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)

L- *Myo*-Inositol 1-Phosphate Synthase

(MIPS) in Chickpea: Gene Duplication and Functional Divergence 229

CaMIPS1 ------MFIENFKVDSPNVKYTETEIQSVYNYETTELVHENRNGTYQWIVKPKTVKYEFK CaMIPS2 ------MFIESFKVESPNVKYTDTEIQSVYSYETTELVHENRNNTYQWVVKPKTIKYEFK OsMIPS ------MFIESFRVESPHVRYGAAEIESDYQYDTTELVHESHDGASRWIVRPKSVRYNFR HsMIPS -----MEAAAQFFVESPDVVYGPEAIEAQYEYRTTRVSREG----GVLKVHPTSTRFTFR ScMIPS MTEDNIAPITSVKVVTDKCTYKDNELLTKYSYENAVVTKTAS---GRFDVTPTVQDYVFK PfMIPS ------------------------------------------------------------ MtMIPS ------------------------------------------------------MSEHQS AfMIPS ------------------------------------------------------------

CaMIPS1 TDTHVP-KLGVMLVGWGGNNGSTLTGGVIANREGISWATKDNIQQANYFGSLTQASATRV CaMIPS2 TQTHVP-KLGVMLVGWGGNNGSTLTGGVIANREGISWATKDKIQQSNYFGSLTQASAIRV OsMIPS TTTTVP-KLGVMLVGWGGNNGSTLTAGVIANREGISWATKDKVQQANYYGSLTQASTIRV HsMIPS TARQVP-RLGVMLVGWGGNNGSTLTAAVLANRLRLSWPTRSGRKEANYYGSLTQAGTVSL ScMIPS LDLKKPEKLGIMLIGLGGNNGSTLVASVLANKHNVEFQTKEGVKQPNYFGSMTQCSTLKL PfMIPS --------MVRVAIIGQGYVASIFAVGLERIKE----------GELGYYG---------- MtMIPS LPAPEASTEVRVAIVGVGNCASSLVQGVEYYYN--------ADDTSTVPG---------- AfMIPS --------MKVWLVGAYGIVSTTAMVGARAIERGIAPKIGLVSELPHFEG----------

CaMIPS1 GSFQ-GEEIYAPFKSLLPMVNPDDIVFGGWDISDMNLADAMARA-RVFDIDLQKQLRPYM CaMIPS2 GSFQ-GEEIYAPFKSLLPMVNPEDIVWGGWDINNMNLADAMGRA-RVFDIDLQKQLRPYM OsMIPS GSYN-GEEIYAPFKSLLPMVNPDDLVFGGWDISNMNLADAMTRA-KVLDIDLQKQLRPYM HsMIPS GLDAEGQEVFVPFSAVLPMVAPNDLVFDGWDISSLNLAEAMRRA-KVLDWGLQEQLWPHM ScMIPS GIDAEGNDVYAPFNSLLPMVSPNDFVVSGWDINNADLYEAMQRS-QVLEYDLQQRLKAKM PfMIPS ----------IPLANELPIKVEDIKIVASYDVDKTKIGLPLSEI-VQRYWKGNVPESLQE MtMIPS ----------LMHVRFGPYHVRDVKFVAAFDVDAKKVGFDLSDA-IFASENNTIKIADVA AfMIPS ------------IEKYAPFSFEFGGHEIRLLSNAYEAAKEHWELNRHFDREILEAVKSDL

CaMIPS1 ESMVPLPGIYDPDFIAANQGDRANNVIKGTKR---------EQINQIIKDIKEFKEANKV CaMIPS2 ESMVPLPGIYDPDFIAANQGDRANNVINGTKK---------EQLQQIIKDIKEFKEASKI OsMIPS ESMVPLPGIYDPDVIAANQGSRANNVIKGTKK---------EQMEQIIKDIREFKEKSKV HsMIPS EALRPRPSVYIPEFIAANQSARADNLIPGSRA---------QQLEQIRRDIRDFRSSAGL ScMIPS SLVKPLPSIYYPDFIAANQDERANNCINLDEKGNVTTRGKWTHLQRIRRDIQNFKEENAL PfMIPS VFVRKGIHLGSLRNLPIEATGLEDEMT---------------LKEAIERLVEEWKEKKVD MtMIPS PTNVIVQRGPTLDGIGK-----------------------------YYADTIELSDAEPV AfMIPS EGIVARKGTALNCGSGIKELGDIKTLEGEGLS-----------LAEMVSRIEEDIKSFAD : .

CaMIPS1 DRVVVLWTANTERYSNLVVGLNDTMENLFAAVDRNE-SEISPSTLFAIACVTENVPFING CaMIPS2 DKVVVLWTANTERYSNVVVGLNDTMENLLASVDKNE-AEISPSTLYALACVLENVPFING OsMIPS DKVVVLWTANTERYSNVCVGLNDTMENLLASVDKNE-AEISPSTLYAIACVMEGIPFING HsMIPS DKVIVLWTANTERFCEVIPGLNDTAENLLRTIELG--LEVSPSTLFAVASILEGCAFLNG ScMIPS DKVIVLWTANTERYVEVSPGVNDTMENLLQSIKNDH-EEIAPSTIFAAASILEGVPYING PfMIPS VIINVPTTEAFTPFGKLEELEKAIKDNNKERLTATQ-AYAYAAAQYAKE--VGGAAFVNA MtMIPS DVVQALKEAKVDVLVSYLPVGSEE-----------------ADKFYAQCAIDAGVAFVNA AfMIPS DETVVINVASTEPLPNYSEEYHGSLEGFERMIDEDRKEYASASMLYAYAALKLGLPYANF . . . :\* . .: \* CaMIPS1 SP-QNTFVPGLIDLAIKRNTLIGGDDFK--SGQTKMKSVLVDFLVGAGIKPTSIVSYNHL CaMIPS2 SP-QNTFVPGLIDLAIQRNSLIGGDDFK--SGQTKMKSVLVDFLVGAGIKPTSIVSYNHL OsMIPS SP-QNTFVPGLIDLAIKNNCLIGGDDFK--SGQTKMKSVLVDFLVGAGIKPTSIVSYNHL HsMIPS SP-QNTLVPGALELAWQHRVFVGGDDFK--SGQTKVKSVLVDFLIGSGLKTMSIVSYNHL ScMIPS SP-QNTFVPGLVQLAEHEGTFIAGDDLK--SGQTKLKSVLAQFLVDAGIKPVSIASYNHL PfMIPS IPTLIANDPAFVELAKESNLVIFGDDGA--TGATPLTADILGHLAQRNRHVLDIVQFNIG MtMIPS LPVFIASDPVWAKKFTDAGVPIVGDDIKSQVGATITHRVLAKLFEDRGVQLDRTMQLNVG

: . \*

\* . .

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.

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.


L- *Myo*-Inositol 1-Phosphate Synthase

chemical properties (Kaur et al.*,* 2008).

Bhusan et al., 2007).

(MIPS) in Chickpea: Gene Duplication and Functional Divergence 231

identified through genomic and proteomic studies (Ahmaed et al., 2005; Mantri ,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

188 69 136 248 227 116 177 189 63 120

109 316 1345 141 156 103 134 103 167

Intron

Exon *CaMIPS1*

(4107 bp)

Fig. 4. Diagrammatic representation of *CaMIPS1* and *CaMIPS2* genomic structure*.* Length of

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)

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

(2979 bp)

188 69 136 248 227 116 177 189 63 120

96 111 325 143 91 250 89 88 253

*CaMIPS2*

exon and intron indicated in bp. [Modified from Kaur et al., 2008]

(Ohno, 1970; Nowak et al., 1997; Jenesen, 1976; Orgel,1977;Hughes,1994).


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.

### **2.2 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

AfMIPS TPSPGSAIPALKELAEKKGVPHAGNDGK--TGETLVKTTLAPMFAYRNMEVVGWMSYNIL \* : \* . \*:\* \* \* : : . . \*

CaMIPS1 GNNDGMNLSAPQTFRSKEISKSNVVDDMVNSNGILY--APGEHPDHVVVIKYVPYVGDSK CaMIPS2 GNNDGMNLSAPQTFRSKEISKSNVVDDMVNSNAILY--QPGEHPDHVVVIKYVPYVADSK OsMIPS GNNDGMNLSAPQTFRSKEISKSNVVDDMVSSNAILY--ELGEHPDHVVVIKYVPYVGDSK HsMIPS GNNDGENLSAPLQFRSKEVSKSNVVDDMVQSNPVLY--TPGEEPDHCVVIKYVPYVGDSK ScMIPS GNNDGYNLSAPKQFRSKEISKSSVIDDIIASNDILYNDKLGKKVDHCIVIKYMKPVGDSK PfMIPS GNTDFLALTDKERNKSKEYTKSSVVEDILG----------YDAPHFIKPTGYLEPLGDKK MtMIPS GNMDFLNMLERERLESKKISKTQAVTSNLKR-------EFKTKDVHIGPSDHVGWLDDRK AfMIPS GDYDGKVLSARDNKESKVLSKDKVLEKMLG-----------YSPYSITEIQYFPSLVDNK \*: \* : .\*\* :\* . : . : :. : \* \* CaMIPS1 RAMDEYTSEIFMGGKSTIVLHNTCEDSLLAAPIILDLVLLAELSTRIQFKSEA-----EN CaMIPS2 RAMDEYISEIFMGGKNTIVLHNTCEDSLLAAPIILDLVLLAELSTRIQFKSQH-----ED OsMIPS RAMDEYTSEIFMGGKSTIVLHNTCEDSLLAAPIILDLVLLAELSTRIQLKAEG-----EE HsMIPS RALDEYTSELMLGGTNTLVLHNTCEDSLLAAPIMLDLALLTELCQRVSFCTDM-----DP ScMIPS VAMDEYYSELMLGGHNRISIHNVCEDSLLATPLIIDLLVMTEFCTRVSYKKVDPVKEDAG PfMIPS FIAMHIEYISFNGARDELIIAGRINDSPALAGLLVDLARLGKIAVDK------------K MtMIPS WAYVRLEGRAFGDVPLNLEYKLEVWDSPNSAGVIIDAVRAAKIAKDRGIG----------

AfMIPS TAFDFVHFKGFLGKLMKFYFIWDAIDAIVAAPLILDIARFLLFAKKKGVKG

CaMIPS1 -------------------------------------------------- CaMIPS2 -------------------------------------------------- OsMIPS -------------------------------------------------- HsMIPS ERPGPSLKRVGPVAATYPMLNKKGPVPAATNGCTGDANGHLQEEPPMPTT ScMIPS L------------------------------------------------- PfMIPS -------------------------------------------------- MtMIPS -------------------------------------------------- AfMIPS --------------------------------------------------

Fig. 3. Multiple sequence alignment of MIPS from prokaryotes and eukaryotes. Proposed

*Saccharomyces cerevisiae* MIPS sequence are highlighted in green color and four conserved

SYNHLGNNDG) are highlighted in yellow color. 43 variant positions between CaMIPS1

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

common active site amino acid residues for the *Mycobacterium tuberculosis* and

domains of eukaryotes (GWGGNG, LWTANTERY, NGSPQNTFVPGL and

**2.2 MIPS from chickpea: A case of functional divergence** 

and 2 have been highlighted in blue color.

CaMIPS1 KFHTFHPVATILSYLTKAPLVPPGTPVVNALSKQRAMLENIMRACVGLAPENNMILEYK-CaMIPS2 KFHSFHPVATILSYLTKAPLVPPGTPVVNALSKQRAMLENILRACVGLAPENNMILEYK-OsMIPS KFHSFHPVATILSYLTKAPLVPPGTPVVNALAKQRAMLENIMRACVGLAPENNMILEYK-HsMIPS EPQTFHPVLSLLSFLFKAPLVPPGSPVVNALFRQRSCIENILRACVGLPPQNHMLLEHKM ScMIPS KFENFYPVLTFLSYWLKAPLTRPGFHPVNGLNKQRTALENFLRLLIGLPSQNELRFEERL PfMIPS ---EFGTVYPVNAFYMKNPGPKEAKNIPRIIAYEKLRQWAGLPPRYL------------- MtMIPS -----GPVIPASAYLMKSPPEQLPDDIARAQLEEFIIG---------------------- AfMIPS -------VVKEMAFFFKSPMDTNVINTHEQFVVLKEWYSNLK------------------

: . : \*: :::\* :.

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).

Fig. 4. Diagrammatic representation of *CaMIPS1* and *CaMIPS2* genomic structure*.* Length of exon and intron indicated in bp. [Modified from 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

L- *Myo*-Inositol 1-Phosphate Synthase

Km

 Gluc 6-P NAD+ Vmax Gluc 6-P NAD+

pH optima Temp. optima

[Modified from Kaur et al., 2008]

Empty

vector

(MIPS) in Chickpea: Gene Duplication and Functional Divergence 233

2.70 mM 0.192 mM

7.5 35°C

0.075 μmole min-1 0.070 μmole min-1

**Characters CaMIPS1 CaMIPS2** 

2.63 mM 0.181 mM

7.5 35°C

CaMIPS1

0.074 μmole min-1 0.069 μmole min-1

Table 1. Biochemical characterization of recombinant CaMIPS1 and CaMIPS2 enzymes.

Fig. 6. Growth pattern of *Schizosaccharomyces pombe* transformed with CaMIPS1 and CaMIPS 2 at high temperature and salt environment. [Modified from Kaur et al., 2008]

al., 2002). This hypothesis was examined on *CaMIPS1* and *2*.

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

*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

CaMIPS2

Control

40°C

250mM

200mM

NaCl

*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*.

Fig. 5. Effect of salt (A) & temperature (B) on CaMIPS1 and CaMIPS2 enzyme activity. [Modified from Kaur et al., 2008]


[Modified from Kaur et al., 2008]

232 Gene Duplication

*CaMIPS2* indeed encode functional MIPS enzymes. Subsequently, the enzymatic properties of these two enzymes were examined since CaMIPS1 and CaMIPS2 polypeptides are

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

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

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

reported to have some differences in their amino acid sequences.

optimum pH is 7.0 suggesting the similar biochemical characteristics (table1).

conditions and thereby retaining higher activity than CaMIPS1 (Fig-5).

property and hence provide sufficient inositol to grow as compared to *CaMIPS1*.

CaMIPS1 CaMIPS2

Fig. 5. Effect of salt (A) & temperature (B) on CaMIPS1 and CaMIPS2 enzyme activity.

**A B**

Enzyme activity (%)

0

20

40

60

80

100

35°C 45°C 55°C

Temperature

0 100 200 300 400

NaCl (mM)

Enzyme activity (%)

0

[Modified from Kaur et al., 2008]

20

40

60

80

100

Table 1. Biochemical characterization of recombinant CaMIPS1 and CaMIPS2 enzymes.

Fig. 6. Growth pattern of *Schizosaccharomyces pombe* transformed with CaMIPS1 and CaMIPS 2 at high temperature and salt environment. [Modified from 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

L- *Myo*-Inositol 1-Phosphate Synthase

in chickpea.

**5. References** 

**4. Acknowledgement** 

(Fast Track Scheme) , Government of India.

*Proteomics,*vol 6:1868-1884.

Chemistry,vol 256: 7077-7085

*Phytologist,* vol 183: 557-564.

*Microbiology,* vol 30, 409-425

135: 1608-1620

1531-1545

Government of India, for Senior Research Fellowship.

R, Jauhar P. vol1,pp 187-217, CRC Press. USA

new functions. *Nature Review Genetics,* vol 9: 938-950

*Proceedings of the Royal Society*, Lond Ser. B 256: 119- 124

Hughes, A.L. (1999) *Adaptive Evolution of genes and genomes*, Oxford University press.

(MIPS) in Chickpea: Gene Duplication and Functional Divergence 235

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

This work was supported by a grant from Department of Biotechnology (Next Generation Challenge Programme on Chickpea Genomics), Department of Science and Technology

We also like to acknowledge the Research support from National Institute of Plant Genome

Ahmad, F.; Gaur, P. M. & Croser, J. (2005) Chickpea (*Cicer arietinum* L.) In genetic resources,

Bhusan, D.; Pandey, A.; Choudhary, M.K.; Datta, A.; Chakraborty, S. & Chakraborty, N.

Boominathan, P.; Shukla, R.; Kumar, A.; Manna, D.; Negi, D.; Verma, P.K. &

Conant, G.C. & Wolfe, K.H. (2008) Turning a hobby into a job: How duplicated genes find

Donahue, T.F. & Henry, S.A. (1981) *Myo*-inositol 1- phosphate synthase. Characteristics of

Flagel, L.E. & Wendel, J.F. (2009) Gene duplication and evolutionary novelty in plants. *New* 

Force, A.; Lynch, M.; Pickett,F.B.; Amroes A.; Yan,Y-L. & Postlethwait, J. (1999) Preservation

Gu, Z.; Nicolae, Lu, H-S. & Li, H.W. (2002) Rapid divergence in expression between duplicate genes inferred from microarray data. *Trends in Genetics,* vol 18,609-613 Hughes, A.L. (1994) The evolution of functionally novel proteins after gene duplication.

Jensen, R.A. (1976) Enzyme recruitment in the evolution of new function. *Annual Review of* 

Kaur, H.; Shukla, R.; Yadav, G.; Chattopadhyay, D. & Majee, M. (2008) Two divergent genes

encoding L -*myo* inositol 1 phosphate synthase1 (CaMIPS1) and 2 (CaMIPS2) are differentially expressed in chickpea. *Plant, Cell &Environment*, vol31, 1701-1716

Chromosome Engineering and Crop Improvement. Grain legumes Edited by Singh

(2007) Comparative proteomics analysis of differentially expressed proteins in chickpea extracellular matrix during dehydration stress. *Molecular & Cellular* 

Chattopadhyay, D. (2004) Long term transcript accumulation during the development of dehydration adaptation in *Cicer arietinum*. *Plant Physiology,* vol

the enzyme and identification of its structural gene in yeast. Journal of Biological

of duplicate genes by complementary, degenerative mutations. *Genetics,* vol 151,

Research, New Delhi. H.K. thanks the Council of Scientific and Industrial Research,

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.

Fig. 7. Expression analysis of CaMIPS1 and CaMIPS2 through real time PCR analysis under various stresses. [Modified from Kaur et al., 2008]
