Phycology International 2019; volume 2:62

biology. It is required, either in its free form

or in different conjugated forms, as a major
Comparative characteristics of
Correspondence: Sautrik Basu, Post Graduate
cytosolic and chloroplastidial component of reproductive units; precur- Department of Botany, Barasat Government
D/L- myo-Inositol-1-phosphate sors of storage phosphates in seeds, pollen College, 10, K. N. C Road, Kolkata 700 124,
wall polysaccharides.1,2 and signal India.
molecules.3 In addition to these it is also a Tel.: 9433045535.
phosphatase partially purified
from Enteromorpha intestinalis precursor and substrate of many crucial E-mail: [email protected]
(L.) Nees (a marine macro alga) metabolites in plants.4 The biosynthesis of
Key words: Cytosol, Chloroplast,
grown under high salinity niche this indispensable sugar alcohol is essential- Enteromorpha intestinalis, myo-inositol-1-
ly reliant on two enzymes, out of which the phosphate phosphatase, salinity tolerance.
Sautrik Basu,1 Anusuya Basak,2 first one is a rate limiting NAD+-dependent
Dibyendu Sekhar Mahanty,1 oxidoreductase (L-myo-Inositol-1-phos- Contributions: SBa carried out the responsi-
Sayani Bhattacharjee,1 phate synthase) and the second one is a bility of plant material collection, identifica-
Jukta Adhikari1,2 Mg+2 dependent phosphatase (D/L- myo- tion of the plant sample, took part in some
Inositol-1-phosphate phosphatase). characterization experiments, determination
1Post Graduate Department of Botany, D/L- myo-Inositol-1-phosphate phos- of PAGE profile and preparation of the manu-
Barasat Government College; phatase (MIPP; EC 3.1.3.25) dephosphory- script. AB carried out the experiments of
2Biochemistry Laboratory, Department MIPP purification, partial characterization of
lates myo-Inositol-1-phosphate (MIP) in
of Botany, Presidency College the enzyme and estimated free myo-inositol
order to maintain the cellular inositol pool
contents. DSM carried out some experiments
(Currently Presidency University), which is essential for many metabolic and
of partial characterization. SBh carried out
Kolkata, India signalling pathways in plants.4 It has been some experiments of partial characterization.
reported earlier that many plants accumu- JA designed the entire project, isolated chloro-

ly
late organic osmoprotectants/osmolytes in plasts, performed Kinetic data analysis and
response to salinity stress and evidence of assisted in manuscript preparation.

on
Abstract high level of osmolyte accumulation in
response to salinity stress are available for Conflict of interest: the authors declare no
The present investigation describes a many plant species.5,6 Thus myo- Inositol in potential conflict of interest.

e
protocol for partial purification and basic its free form has been recognized to play a
characterization of one of the key enzymes us
crucial role in salt tolerance of plants.5 Funding: the authors are grateful to the
of myo-inositol biosynthesis, D/L- myo- Taking these facts collectively in considera- University Grants Commission, Govt. of
Inositol-1-phosphate phosphatase (MIPP), tion, attempts have been made in the present
India, for partial financial assistance [Project
from a marine macro alga (Enteromorpha No: F-PSW-240/15-16 (ERO)].
al
work for a comparative study of purifica-
intestinalis) growing under varying salinity tion and characterization between cytosolic Received for publication: 9 November 2018.
ci

conditions of Chilika lagoon (Odisha, and plastidial forms of MIPP after its basic Revision received: 4 February 2019.
India). Cytosolic and chloroplastidial forms identification from a Chlorophycean marine
er

Accepted for publication: 25 February 2019.

of MIPP were partially purified to about 55- macro alga Enteromorpha intestinalis
and 16- folds respectively following low- which is found to grow profusely in Chilika This work is licensed under a Creative
m

speed centrifugation, high-speed centrifu- lagoon throughout all seasons under differ- Commons Attribution NonCommercial 4.0
gation, 0-80% ammonium sulfate precipita- License (CC BY-NC 4.0).
om

ent salinity regimes.

tion, successive chromatography through The MIPP reaction has been reported
CM-cellulose, Sephadex G-200 and ©Copyright S. Basu et al., 2019
from several prokaryotic as well as eukary- Licensee PAGEPress, Italy
UltrogelAcA 34 columns. The apparent
-c

otic organisms such as Escherichia coli,7 Phycology International 2019; 2:62

molecular weights of the native cytosolic Synechocystis sp,8 yeast,9 vascular cryp- doi:10.4081/phycol.2019.62
and plastidial forms of MIPP were deter-
on

togams,10,11 higher plants12,13 and ani-

mined to be 146 and 148 kDa respectively. mals.14-16 It is noteworthy to mention here
Cytosolic and plastidial MIPP were remark- that although the inositol biosynthetic path-
N

ably active within the temperature range of

way is necessarily functional in several
20-40°C and function within a narrow pH
plant groups, supporting evidence are pri- Materials and Methods
range (7.0-7.5). Using nonlinear regression
marily based only on purification and char-
kinetics, the Km value for D-MIP of the
acterization of MIPS but extensive informa- Plant material
cytosolic MIPP and its plastidial iso-form Experimental macro alga
tion regarding MIPP is still very meagre.
were 0.07277 mM and 0.07332 mM respec- Enteromorpha intestinalis (L.) Nees, was
Perusal of available literature clearly
tively. Different monovalent as well as collected from Rambha, Chilika lagoon,
reveals that although MIPP activity has
divalent cations exhibited variable effects Odisha (geographic location: 19° 50’ N and
been previously reported from some flower-
on enzyme activity of either preparation. 85 °30’ E), India. The samples were kept
ing plant species12,13 and also from some
The activity of MIPP was remarkably found frozen under -20°C until use.
vascular cryptogams10,11 so far no work has
to increase proportionately with the salinity
been carried out regarding its activity in
of Chilika lagoon. Isolation of chloroplasts
non-vascular cryptogams, particularly its
role in salt-stress physiology. Thus the pre- Chloroplasts of Enteromorpha intesti-
sent investigation attempts to bridge the nalis were isolated following the method of
Hachtel (1976)17 with minor modifications
Introduction existing gap towards understanding the
basic regulatory principles underlying inos- as suggested by Adhikari et al. (1987).18 The
Myo-Inositol is a 6 carbon cyclohexane itol biosynthesis in the marine macro algae authenticity of the isolated chloroplasts was
hexitol which has a diverse role in plant E. intestinalis as a model. established as described by Adhikari et al.18

[Phycology International 2019; 2:62] [page 1]

 Article

Purification and characterization of pooled together and the subsequent column supernatants were eventually transferred to
D/L- myo-Inositol-1-phosphate phos- chromatography was carried out through respective test tubes for enzyme assay. The
phatase Sephadex G-200 (obtained from Amersham MIPP activity was assayed following the
In order to purify the MIPP from the Pharmacia Biotech, UK) (0.8×14.0 cm). assay method described earlier.
experimental sample separately, all opera- The MIPP active Sephadex G-200 fractions
tions were carried out at 0-4°C following were pooled together and finally molecular Molecular weight determination of
identical methods for both the soluble sieve chromatography was completed native myo-inositol-1-phosphate
(cytosolic) and particulate (chloroplastidial) through UltrogelAcA 34 column (obtained phosphatase
enzyme forms. About 75-100 g of plant tis- from Bio Rad, USA) (0.8×8.0 cm). The
Approximate molecular weight of the
sues (for cytosolic enzyme) / 10-15 ml MIPP active fractions obtained from
native MIPP obtained from the respective
chloroplast pellet (obtained from 80-100 g Ultrogel AcA 34 were pooled together and
samples was determined by gel-filtration
tissues for plastidial enzyme) were thor- used as the enzyme preparation for bio-
through Sephadex G-200. The Sephadex G-
oughly washed with sterile cold distilled chemical characterization of MIPP(s). All
200 was suspended in 50 mM Tris- acetate
water twice. The tissues were then homoge- experiments were repeated twice with mul-
(pH 7.5) and packed in a column of suitable
nized in a mortar and pestle with 2 volumes tiple replicates.
size and calibrated with 1 ml each of marker
of extraction buffer [50 mMTris-HCl (pH proteins e.g., catalase (221.6 kDa); bovine
7.2), containing 0.2 mM of 2- Assay of myo-inositol-1-phosphate
serum albumin dimer (66.5kDa); phospho-
Mercaptoethanol (standard buffer)] in pres- phosphatase rylase-b (97.4 kDa); ovalbumin (43 kDa),
ence of neutral sand. The slurry was cen- MIPP was assayed by the method of carbonic anhydrase (29 kDa) and lysozyme
trifuged at 800 g for 5 minutes in a Remi C- Eisenberg Jr. (1967)14 with slight modifica- (14.3 kDa). The void volume was deter-
24 BL cold centrifuge. The homogenate was tions.10 Inorganic phosphate was estimated mined with blue dextran 2000 (1 mg / ml).
spun again at 11,400 g for 30 minutes in a following the method of Chen et al. All standards were loaded in the column

ly
refrigerated centrifuge (Remi C-24 BL) and (1956).19 The specific activity was defined separately and fractions (0.75 ml) were col-
the supernatant was collected (Low-speed either as n mol myo-inositol- 1-phosphate

on
lected at a flow rate of 0.75 ml / 6 min. Each
supernatant). The resultant low-speed released (mg)-1 protein h-1 or n mol Pi
individual protein peak was located by
supernatant was then centrifuged at 1, released mg-1 protein h-1.
spectrophotometric scanning at 280 nm in a
10,000 g in an ultracentrifuge (Hitachi GX

e
Jasco V-5 UV-Vis Spectrophotometer. A
Series, Model: CS 120G X 2 Micro Estimation of protein standard curve was prepared by plotting rel-
Ultracentrifuge, Rotor No. 26) for 2 hours.
On completion of centrifugation, the clear
us
Protein was determined according to the
method of Bradford (1976)20 with BSA as a
ative elution volume of proteins against
their respective log molecular weights.
supernatant (High-speed supernatant) was standard. Polyacrylamide gel electrophore-
al
collected from the centrifuge tubes and sis of the active Ultrogel AcA 34 fractions
Kinetic analysis
were kept ready for the next step.The high- were performed under non denaturing con-
ci

As far as the evaluation of the kinetic

speed supernatant was fractionated with ditions following the method of Bollag et
parameters of MIPP is concerned, the
er

ammonium sulfate [(NH4)2SO4]: 20-80% al. (1996)21 with necessary modifications as

Michaelis–Menten constant (Km) was anal-
(for cytosolic) / 0-80% (for chloroplastidial) elucidated by Banerjee et al. (2007).10 For
ysed. In order to establish relation between the
m

saturation]. The resultant (NH4)2SO4 pellet the MIPP assay from the native PAGE,
was dissolved in minimal volume of stan- replicate gels were run. One of the gels was substrate (D/L-MIP) and the rate of the enzyme
catalysed reaction, all the parameters were cal-
om

dard buffer and dialyzed overnight against stained after completion of the run by
the same (at least against 700 volumes). Coomassie Brilliant Blue (R-250) to visual- culated by nonlinear regression method using
Dialyzed fraction was chromatographed ize the protein bands and the other was the software Prism 5 (Graph Pad).
-c

using a cation-exchange matrix CM-cellu- sliced successively to 5 mm fragments

lose (obtained from GENEI, Bangalore, using a gel slicer. The enzyme from each of Isolation and quantification of free
on

India) in a glass column (1.2×15.0 cm). The the slice was then extracted in relevantly Inositol
effluent was collected and the column was marked eppendorf tube with 250 µl of 50 Free myo-inositol was isolated by the
washed with one bed volume of the stan- mM Tris-acetate buffer (pH 7.5) and kept method of Charalampous and Chen
N

dard buffer. The elution of adsorbed pro- overnight at 0°C to 4°C. The eppendorf (1966)22 with minor modifications. It was
teins was made by a linear gradient of 0 to tubes were centrifuged at 11,400 g for 30 quantified spectrophotometrically follow-
0.5 M KCl prepared in standard buffer. The minutes using Remi RM- 02 Plus mini cen- ing the method of Gaitonde and Griffiths
MIPP active CM-cellulose fractions were trifuge. The pellets were discarded and the (1966).23

Table 1. Screening of cytosolic and plastidial MIPP activity and free myo-inositol content in the experimental macro alga Enteromorpha
intestinalis grown under different salinity conditions.
Experimental sample Salinity of water Free myo-inositol content Specific Activity of MIPP
(PSU) (mg)/(g) FW ± S.E (n mole Pi released (mg)-1
Protein) h-1
Cytosol Chloroplast Cytosolic Chloroplastidial
Enteromorpha intestinalis 6.41 to 7.21 0.83±0.36 1.34±0.27 12.78±2.66 145.23±15.48
(sample collected in rainy season)
Enteromorpha intestinalis 12.4 to 13.7 1.43±0.52 4.16±0.77 17.14±4.16 270.65±21.43
(sample collected in winter season)

[page 2] [Phycology International 2019; 2:62]

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Determination of salinity sive substrate and dephosphorylated almost was noted to increase with respect to D-
The measurement of practical salinity with equal efficiency by MIPPs. Other hex- MIP concentration of 0.1 to 0.2 mM. The
units values of water, collected from ose monophosphates viz., D-glucose- 6- Km value for D-MIP of cytosolic MIPP was
Chilika lagoon (in separate sterile contain- phosphate, D-fructose- 6- phosphate, D- 0.07277 mM and that of the chloroplastidial
ers) in rainy and winter seasons were mea- galactose- 6- phosphate under identical con- MIPP was 0.07332 mM (Figure 2E, F).
sured using Systronics Water Analyzer centration (0.2 mM) was completely inef- MIPPs function remarkably active between
(Model no: 571). fective as substrates. Under standard assay the temperature ranges of 20-40°C. The
conditions it was observed that the MIPPs temperature maxima for cytosolic MIPP
reaction proceeded linearly with time up to were found to be 40°C, while that of the
a maximum of 60-75 min in both the cases plastidial one was 35°C (Figure 3A, B). The
Results (Figure 2A, B). algal MIPPs exhibited optimum activity at a
The linearity of MIPP reaction under pH range of 7.0 (cytosolic) and 7.5 (plas-
The results obtained in the present study increasing protein concentration (0-80µg) tidial) when 50mM Tris-HCl buffer was
clearly reveal the existence of two forms of indicated 30 and 60 µg respectively for used at a pH range of 6.0-9.0 (Figure 3C,
MIPP (cytosolic and plastidial) in the exper- cytosolic and plastidial enzymes (Figure D). Among the monovalent cations tested,
imental alga E. intestinalis. Although appre- 2C, D). The effect of substrate concentra- K+ had slight stimulatory role and Li+ was
ciable activity has been detected in both tion and kinetic analyses of MIPPs were strongly inhibitory (41-56%). Using the
cytosolic as well as plastidial MIPP, the carried out using D-MIP in the concentra- analogous concentrations of other divalent
plastidial isoform exhibited greater activity tion range of 0.0-1.0 mM. The reaction rate cations, it was revealed that Mg2+ played
in comparison to its cytosolic counterpart
(Table 1). A marked increase in both cytoso-
lic and plastidial MIPP activity (from now
on termed as MIPPs) could be recorded in

ly
thalli collected in the winter season (under

on
higher salinity of lagoon water) and this
high plastidial MIPP activity detected in the
winter thalli of E. intestinalis was also cor-

e
related with a substantial increase in the
content of free myo- inositol in the chloro- us
plasts of E. intestinalis collected in the post
monsoon season (Table 1).
al
Summary of the purification of MIPPs
has been provided in Table 2. The overall
ci

purification of cytosolic MIPP was recorded

to be around 55- fold with about 32% yield
er

while that of the plastidial MIPP was found

to be 16- fold with 26% recovery. Column
m

chromatography using Ultrogel AcA 34

revealed that MIPPs from E. intestinalis
om

were retained and eluted effectively with

the extraction buffer.
The apparent molecular weight (Mr) of
-c

the native MIPPs, determined by gel filtra-

on

tion on a Sephadex G-200 column was 146

k Da (cytosolic) and 148 k Da (plastidial).
By means of native PAGE gel slice
N

assay, the major band of MIPPs was found

to coincide with the enzymatic activity
(Figure 1) as described earlier in the materi-
als and in the method section.
In order to determine the requirements
of MIPP for its catalysis, it was revealed
that in presence of all the assay components
the experimental E. intestinalis MIPP
recorded a maximum activity of 100%.
However, notable reduction of the activity
of MIPPs could be recorded when Tris
buffer and KCl were omitted from the reac-
tion mixture. The MIPP activity dropped to
79% and 96% in absence of the buffer, 91%
and 94% in absence of KCl for cytosolic
and chloroplastidial enzymes respectively.
Regarding substrate specificity, either L- Figure 1. Native PAGE profile showing the myo-inositol 1-phosphate phosphatase
MIP or D-MIP was found to be the exclu- (MIPP) activity of the corresponding band: (A) cytosolic and (B) plastidial.

[Phycology International 2019; 2:62] [page 3]

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appreciable stimulatory role and Ca2+ level of free inositol detected in the thalli of in this work the apparent molecular weight
exhibited inhibitory effect at higher concen- E. intestinalis housed at higher salinity of cytosolic MIPP has been found to be 146
trations (Figure 3E, F). When used between seems logical. However, it must be taken k Da while that of its plastidial isoform has
the concentration ranges of 0-50mM EDTA, into account that although salt stress has been recorded to be 148 k Da. MIPP
it was also revealed that EDTA had signifi- been associated with a number of intercon- obtained from eukaryotic and bacterial
cant inhibitory role on MIPPs. nected physiological responses, the under- sources have been reported to exhibit a
lying mechanisms of salt tolerance in plants broad range of substrate specificity.
are still poorly understood. Since algae are Nevertheless in case of this experimental
inhabitants of biotopes characterized by alga, D-or L- MIP was found to be the
Discussion and Conclusions changing salinities they can serve as model exclusive substrate. The plastidial iso-form
organisms for a better understanding of the on the other hand exhibited feeble activity
Perusal of available literature clearly
salt acclimation phenomenon in higher when D-myo inositol -3- phosphate
reveals that although purification of MIPP
plants.28,29 replaced the principal substrate. This is in
has been carried out dominantly in animal
The characteristics of MIPP recorded in complete agreement with the results
systems,14-16 reports describing preparation
the present investigation are in partial con- obtained in case of Dryopteris filix mas.10
of cytosolic/plastitidial forms of MIPP from
currence with all the major MIPP(s) record- In the present study the temperature maxi-
plant systems is still very inadequate. Only
ed earlier from plant systems.The molecular ma for cytosolic and plastidial MIPP has
a few reports describing purification and
weight of native cytosolic MIPP from the been found to be 40°C and 35°C respective-
characterization of MIPP are available pri-
common fern Dryopteris filix mas was ly (Figure 3A, B). A quick glance on the
marily from angiosperms12,13 and few from
reported to be around 94 k Da.10 However reports published earlier clearly reveals a
vascular cryptogams.10,11 Therefore, the
presence of MIPPs in E. intestinalis adds
further strength to the ubiquitous

ly
metabolism of myo-inositol biosynthesis
in non-vascular cryptogams too. The partial

on
purification of MIPPs from E.
intestinalis and its preliminary characteriza-
tion paves the way towards understanding

e
its role in non- vascular cryptogams.
The results obtained in this work clearly
reveals that the activity of MIPPs gets
us
enhanced in response to high salinity levels
al
of Chilika lagoon and this enhanced MIPPs
activity is also correlated with a proportion-
ci

al increase in the content of free myo- inos-

er

itol. It has been reported earlier that salt

stress results in alterations in plant
m

metabolism, reduced water potential, ion

imbalance and toxicity.24 Moreover, it has
om

also been reported earlier that in addition to

bringing about disturbances in ionic home-
ostasis salt stress also causes considerable
-c

damage to the membrane architecture due

to enhanced accumulation of ROS.
on

Disorganization of the thylakoidal structure

of chloroplasts is also brought about by salt
stress.25 Thus taking these facts into con-
N

templation the enhanced activity of the

organelle- bound MIPP specifically in
response to salt stress seems logical. Work
done by previous workers are indicative of
the fact that the overproduction of cyclitols
in transgenic tobacco, Arabidopsis and
wheat plants have resulted in maintenance
of optimal ionic homeostasis and cellular
integrity.26 It has been reported earlier that
Chilika lagoon undergoes a cyclical varia-
tion in salinity throughout the year and the
distribution of flora and fauna in Chilika is
also influenced greatly by salinity fluctua-
tions.27 Since inositol and its derivatives act
as osmolytes/osmoprotectants and are able
to protect cells from the effects of osmotic
Figure 2. Time course of E. intestinalis MIPP: (A) cytosolic and (B) plastidial. Effect of

imbalance imposed by high salinity levels,

protein concentrations on E. intestinalis MIPP: (C) cytosolic and (D) plastidial.
Determination of Km value for D-MIP of E. intestinalis MIPP by Nonlinear Regression
higher activity of MIPPs along with higher
Kinetics: (E) cytosolic and (F) plastidial.

[page 4] [Phycology International 2019; 2:62]

 Article

marked variation in the temperature maxi-

ma of MIPP obtained from different
sources. In case of the fern pteridophyte
Dryopteris filix mas temperature maxima
for cytosolic MIPP has been recorded to be
about 40°C while in case of non- fern pteri-
dophytes (Lycopsida members) the temper-
ature maxima for cytosolic MIPP has been
recorded to be about 35°C. MIPP(s)
obtained from microbial and mammalian
sources have exhibited a temperature maxi-
ma of 37°C while MIPP obtained from the
cyanobacteria Synechocystis has shown a
slightly higher temperature maxima
(42°C).8 Most MIPP(s) isolated from vari-
ous sources have been found to be optimal-
ly active in the pH range of 7.8-8.5.8
However Enteromorpha MIPP has been
found to be optimally active at pH 7.0
(cytosolic) and 7.5 (Plastidial) (Figure 3C,
D). In Dryopteris filix mas, cytosolic MIPP
has been found to exhibit a pH range of 7.5-

ly
8.510 while in case of non- fern pterido-
phytes the pH range has been recorded

on
between 7.0 and 7.5.11 As far as the kinetic
parameters are concerned, the Km value for
D-MIP of cytosolic MIPP was recorded to

e
be 0.07277 mM and that of the chloroplas- us
tidial MIPP was 0.07332 mM (Figure 2E,
F). It is a sharp contrast of Km values
detected in prokaryotic as well as in mam-
al
malian systems.8,30 In case of mammalian
system Km value for D-MIP has been found
ci

to be much higher while prokaryotic sys-

er

tems have exhibited a much lower Km

value for D-MIP. Among the monovalent
m

cations tested Li+ was found to exert a

strong inhibitory effect on MIPPs. It has
om

been reported earlier that inhibition by Li+

is a general characteristic feature of the
Figure 3. Effect of incubation temperature on E. intestinalis MIPP: (A) cytosolic and (B)

inositol monophosphatase family enzymes.1

plastidial. pH dependence of MIPP: (C) cytosolic and (D) plastidial. Effect of different
-c

monovalent and divalent cations on E. intestinalis MIPP activity: (E) cytosolic and (F)
The results obtained in this study also indi- plastidial.
on

Table 2. Outline of partial purification of (A) cytosolic and (B) chloroplastidial myo-inositol-1-phosphate phosphatase from
N

Enteromorpha intestinalis.(Values are mean±SE, n=3).

(A) CYTOSOLIC MIPP (B) CHLOROPLASTIDIAL MIPP
Purification step Specific activity Total activity Purification Specific activity Total activity Purification
[n mol Pi released [n mol Pi (fold) [n mol Pi released [n mol Pi (fold)
(mg)-1 protein h-1] released h-1] (mg)-1 protein h-1] released h-1]
Homogenate fraction 14.50±4.22 871.88±88.69 1.00±0.07 278.44±17.90 4995.21 ±132.16 1.00±0.14
11,400 g 16.42±6.90 843.65±89.16 1.13±0.04 281.06±14.22 4154.06±89.16 1.00±0.09
supernatant
fraction
1, 10,000 g supernatant 17.55±7.04 837.13±75.11 1.21±0.11 283.66±19.41 3806.71±75.11 1.01±0.07
fraction
20-80 % / 0-80% 23.48±9.92 535.34±82.52 1.61±0.09 286.04±25.63 3195.06±82.52 1.02±0.13
Ammonium sulfate
fraction
CM-cellulose 136.90±16.82 436.71 ±42.80 9.44±0.36 747.23±36.34 2914.19 ±42.80 2.68±0.46
fraction
Sephadex G-200 fraction 188.22±37.11 365.14±41.93 12.98±0.63 1828.77±49.27 2395.68±51.93 6.56±0.61
Ultrogel AcA 34 803.68±56.13 281.28±53.31 55.42±6.34 4611.27±72.34 1291.15±73.31 16.56±2.06
fraction

[Phycology International 2019; 2:62] [page 5]

 Article

cate that MIPPs activity is sensitive to metal 6. Joshi R, Ramanarao MV, Baisakh N. plast membranes in Vicia faba. In:
chelators like EDTA. This is in complete Arabidopsis plants constitutively overex- Bucher T, Neupert W, Sebalt W, Warner
agreement with the results obtained in case pressing a myo-inositol 1-phosphate syn- S, eds. Genetics and Biogenesis of
of vascular cryptogams by previous work- thase gene (SaINO1) from the halophyte Chloroplasts and mitochondria.
ers.10,11 The possible reason behind this sen- smooth cord grass exhibits enhanced Amsterdam: Elsevier/North Holland
sitivity towards a metal chelator like EDTA level of tolerance to salt stress. Plant Biomedical Press; 1976. pp 49-56.
may be attributed to the presence of some Physiol Biochem 2013;65: 61-6. 18. Adhikari J, Majumdar AL, Bhaduri TJ, et
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Thus a comparative analysis of cytoso- Inositol monophosphatase activity from Inositol–1-phosphate synthase. Plant
lic and plastidial MIPP obtained from a nat- the Escherichia coli suhB gene product. J
Physiol 1987;85:611-4.
urally lodged algal source depicted in this Bacteriol 1995;177:200-5.
19. Chen RS, Toribara TY, Warner H.
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Microdetermination of phosphorous.
metabolic features and widespread distribu- Functional identification of sll1383 from
Anal Biochem 1956;28:1756-8.
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inositol 1-phosphate phosphatase (EC 20. Bradford MM. A rapid and sensitive
reinstates its primordial protein nature. The
3.1.3.25): molecular cloning, expression method for the quantitation of microgram
results obtained in this study thus unveil
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stand the phylogeny of these proteins in and sodium sensitive enzyme encoded by Wiley-Liss; 1996.

ly
cryptogams, a thorough search for immuno- a non-essential gene pair. Mol Microbiol 22. Charalampous F, Chen IW. Inositol-1-
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