Salinity endurance of marine macro Rhodophycean algae with special emphasis on myo-inositol biosynthesis: An enzymological analysis from Halymenia venusta Børgesen

Altered salinity is one the most important perils encountered by marine plants inclusive of algae. Under hyper saline condition, plants accumulate several stress relieving osmolytes including myo-inositol, the most widespread cyclitol in plants. The present communication reports the occurrence of myo-inositol biosynthesis in six different Rhodophycean seaweeds growing under stressful intertidal habitats of the Okha coast (Gujarat, India), on the basis of a study conducted on two marker enzymes of myo-inositol biosysnthesis [L-myo-inositol-1-phosphate synthase (MIPS)and D/L-myoinositol-1-phosphate phosphatase (MIPP)]. Both enzymes were partially purified from Halymenia venusta to about 27 fold (MIPS) and 39 fold (MIPP) over the homogenate following low-speed centrifugation, 30-75% ammonium sulphate fractionation, successive chromatography through DEAE-cellulose / CM-Cellulose, Sephadex G-200 and BioGel 0.5m / Ultrogel AcA 34 columns. The temperature and pH optima for MIPS and MIPP were similar and were recorded to be 35oC and 7.5 respectively. For MIPS, D-glucose-6-phosphate and NAD were the exclusive substrate and coenzyme respectively and D/L-I-1-P was the sole substrate for MIPP. The Km values for D-glucsoe-6-phosphate and β-NAD were recorded to be 3.599 mM and 0.2366 mM respectively, while the Km value for D-I-1-P was found to be 0.4070 mM. K+ exhibited slight stimulatory effect while Li+ was strongly inhibitory for both the enzymes. Ca2+ on the other hand exhibited a mild stimulatory and Cd2+ reduced activities of both MIPS and MIPP. MIPP was especially stimulated by Mg2+. On the other hand Cu2+ and Hg2+ were strong inhibitors for both the enzymes. A steady and proportionate increase in the content of free myo-inositol was observed along with the elevated levels of recorded salinity.


INTRODUCTION
Salinity is a major abiotic factor which is crucial to aquatic ecosystems and is responsible for the local and/or regional distribution of marine macrophytes. Unlike other abiotic factors viz, temperature, irradiance, nutrients and pH, salinity holds a special position and plays a key role in affecting not only the metabolism but also the growth and survival strategies of several marine macrophytes including seaweeds.
Seaweeds normally occupy rocky intertidal zones and are continuously subjected to desiccation, extreme temperatures and altered salinity. Eukaryotic marine algae are known to possess great plasticity and adaptability to most abiotic stresses including the stress imposed as a result of prolonged growth in hyper saline condition [1]. Hyper salinity affects aquatic macrophytes in several ways which includes osmotic stress, ion stress and ionic ratio imbalance [1]. Plants are known to accumulate several inositol derivatives in response to salt stress which act as osmolytes. These accumulated osmolytes include glycerol, betain, proline, trehalose, raffinose and myo-inositol [2]. myo-Inositol is the most widespread cyclitol in plants having a number of functions including amelioration of salinity stress [3]. It has been reported earlier that myo inositol along with its isomers and methylated derivatives are interconnected with lenience to drought and / or salinity tolerance in plants [4]. Taking all these facts into consideration 'salinitydriven' enhanced synthesis of this cyclitol in plants has become an interesting and highly productive area of research in recent years even at the genetic level [5]. The biosynthesis of myo inositol is essentially reliant on two enzymes, out of which the first one is a rate limiting NAD + -dependent oxidoreductase (L-myo-Inositol-1-phosphate synthase) and the second one is a Mg 2+ dependent phosphatase (D/Lmyo-Inositol-1-phosphate phosphatase).
Taking all these facts into consideration the present study was aimed towards understanding the relationship between inositol biosynthesis and salinity stress in marine macrophytes growing under hyper saline conditions.

Plant Material
The experimental red algal samples, viz. Coelarthrum muelleri, Gracilaria corticata, Grateloupia indica, Halymenia venusta, Kappaphycus alvarezii, Solieria robusta were collected in winter (January 2019) season from the Arabian Sea coast of Okha, Gujarat (latitude 22 o 28 / N and longitude 69 o 05 / E ; Temperature range 21.5 to 30 o C), India and [ Table 1; Figure 1(a-f)]. The samples were brought to the laboratory in a portable ice-chamber and were kept frozen under -20 0 C until use. Specimens were identified using standard literature [17] and with direct comparison with herbarium specimens available in the Cryptogamic Section of 'Central National Herbarium (CAL)', Shibpur, Howrah (West Bengal, India). Voucher specimens were deposited in the Departmental herbarium of Barasat Govt. College.

Extraction of Enzymes comparing MIPS and MIPP activities in the Collected Sample
Sequestration of cytosolic MIPS and MIPP from experimental samples of was carried out by homogenization of tissues in two volumes of the extraction buffer [50 mM Tris-acetate, pH 7.5 containing 0.2 mM β-mercaptoethanol (ME)] followed by a centrifugation at 11,400 g for 30 min in a Remi C-24 BL cold centrifuge. Discarding the pellet the supernatant fraction was collected (Low-speed supernatant fraction). These preparations were dialyzed separately for 18 hours against 50 mM Tris-acetate, pH 7.5 containing 0.2 mM ME under 0 to 4 o C. The dialyzed fractions were recovered from the corresponding dialysis bags and used as the enzyme source(s) for the preliminary screening experiments. The alga showing higher myo-inositol and cytosolic MIPS and MIPP activities were selected for further studies.

Partial Purification MIPS and MIPP
Partial purification of cytosolic MIPS and MIPP from thalli of Halymenia venusta was carried out separately at 0 to 4 o C. Bufferwashed Halymenia venusta thalli(40 g for each enzyme separately) was homogenized in 3 volumes of a buffer (50 mMTris-acetate, pH 7.5, containing 0.2 mM ME; hitherto termed as standard buffer). The homogenate was spun at 11,400 g for 30 min and the supernatant was collected (Low-speed supernatant). The low-speed supernatant was then fractionated with ammonium sulfate [(NH 4 ) 2 SO 4 ] at the level of 30-75 % saturation. The consequential (NH 4 ) 2 SO 4 pellet was dissolved in nominal volume of standard buffer and dialyzed for 18 hours against the same standard buffer. Chromatography of the dialyzed fractions of MIPS and MIPP were done individually expending an anionexchange matrix DEAE-cellulose (for MIPS, obtained from GENEI, Bangalore, India) in a glass column (1.2 x12.0 cm) and cation-exchange matrix CM-cellulose (for MIPP, obtained from GENEI, Bangalore, India) in a glass column (1.2 x13.0 cm). The effluent was collected and the column was washed with one bed volume of the standard buffer. The elution of the adsorbed proteins was done by a linear gradient of 0 to 0.5 M KCl prepared in standard buffer. The MIPS active DEAE-cellulose   140 mM NH 4 Cl, 8 mM NAD, 50 mM ME, 50 mM G-6-P and an appropriate protein aliquot (30-100 µg). The reaction was initiated by incorporation of substrate (G-6-P) promptly after the addition of the enzyme. Duplicate tubes were run along with a blank (minus enzyme) and also a zero minute control in which 200 µl of 20 % (w/v) chilled trichloroacetic acid (TCA) was loaded in the incubation mixture prior to addition of the enzyme. The enzymatic incubation was allowed for 60 min at 37 0 C in a thermostatic digital incubator. After 60 min the reaction was stopped by the addition of 200 µl of chilled 20 % (w/v) TCA. Two such sets (set A -periodate and set B -nonperiodate) were run concurrently each having one blank, one zero minute control and two experimental tubes. The quantity of the enzymatic product was estimated by periodate oxidation followed by the estimation of inorganic phosphates.
On completion of the enzyme incubation, this was treated with 0.7 ml of 200 mM sodium metaperiodate (NaIO 4 ) and further incubated for 60 min at 37 0 C. Then, 1.4 ml of 1(M) Na 2 SO 3 (freshly prepared) was added in case of set-A to destroy excess of NaIO 4 . In set-B, distilled water was added instead of NaIO 4 and Na 2 SO 3 to maintain the volume with that of set-A.
Inorganic phosphate was hydrolysed from myo-inositol-1-phosphate during oxidation which could be subtracted considering the blank or zero minute control from the experimental value in set-A. Hydrolysis of phosphate from G-6-P by contaminating phosphatase (if any) was measured by subtracting the value of the blank or zero minute control from the experimental value in set-B. Product dependent cleavage of inorganic phosphate was estimated by subtracting the corrected value of set-B from that of set-A. Inorganic phosphate was estimated by the method of Chen et al. [19].

Assay of Myo-inositol-1-phosphate Phosphatase
The myo-inositol-1-phosphatase activity was assayed by the method of Eisenberg Jr. [7]. In a total volume of 1.0 ml, the incubation mixture contained 500 µl of 0.67 M Tris-HCl (pH 7.4) containing 50 mM MgCl 2 , 400 µl of 154 mM KCl, 50 µl of 100 mM D-myo-inositol-1-phosphate and an appropriate protein aliquot (50 µl / 50 -200 µg). The MIPP reaction was initiated by adding of substrate immediately after the enzyme. The complete assay mixture was maintained along with an appropriate blank (sans enzyme) and a zero-minute control in which 250 µl of 20% (w/v) chilled TCA was added prior to the addition of the enzyme. The enzymatic incubation was carried out for 60 min at 37 0 C. After 60 min of incubation, the reaction was terminated by 250 µl of 20 % (w/v) chilled TCA. Two such sets (set A-experimental and set B-control), where myo-inositol-1-phosphate was replaced by an identical volume and concentration of D-fructose-6-phosphate were carried out simultaneously each having one blank, one zero-minute control and one with complete assay mixture. Inorganic phosphate was estimated by the method of Chen et al. [19]. As 1 mol of myoinositol-1-phosphate contains 1 mol of inorganic phosphate, the total mole number of inorganic phosphate released was equal to the total mole number of myo-inositol-1-phosphate hydrolyzed.

Assay of L-myo-inositol-1-phosphate Synthase
Assay of L-myo-inositol-1-phosphate synthase was performed by the method of Barnett et al. [18]. In a total volume of 0.5 ml, the incubation mixture comprised of 500 mM Tris-acetate (pH 7.5), The specific activity was defined either as n mol myo-inositol-1-phosphate released / hydrolyzed (mg) -1 protein h -1 .

Determination of Protein
Protein was determined according to the method of Bradford (1976) with BSA as a standard [20].

Determination of Molecular Weights of MIPS and MIPP
Approximate molecular weight of the native MIPS and MIPP obtained from the respective samples was determined by gel-filtration through Sephadex G-200. The Sephadex G-200 was suspended in 50 mM Tris-acetate (pH 7.5) and packed in a column of suitable size and calibrated with 1 ml each of marker proteins e.g., catalase (221.6 kDa); bovine serum albumin dimer (133.352 kDa); phosphorylase-b (97.4 kDa); ovalbumin (43 kDa), carbonic anhydrase (29 kDa) and lysozyme (14.3 kDa). The void volume was determined with blue dextran 2000 (1 mg per ml). All standards were loaded in the column separately and fractions (0.75 ml) were collected at a flow rate of 0.75 ml per 6 min. Each individual protein peak was located by spectrophotometric scanning at 280 nm in a Systronics UV-Visible spectrophotometer. A standard curve was prepared by plotting relative elution volume of proteins against their respective log molecular weights.

Gel Electrophoresis and Slice Assay
Polyacrylamide gel electrophoresis of MIPS and MIPP under non-dissociating condition followed by gel-slice assay was performed using 1.5 mm thick gel slab following the method of Bollag et al. [21].
For the MIPS/MIPP assay from the gel, replicate gels were run. One of the gels was stained with CBB (R-250) in order to visualize the protein bands and the other was sliced to 5 mm fragments. The enzyme from each of the slice was then extracted with 250 µl of 50 mM Tris-acetate buffer (pH 7.5) for 30 min at 0 0 C to 4 0 C.

Isolation and Quantification of Free Myo-inositol
Free myo-inositol was isolated by the method of Charalampous and Chen [22] and free myo-inositol was estimated spectrophotometrically according to the method of Gaitonde and Griffiths [23].

Stability of Enzymes under Laboratory Condition
Both MIPS (obtained from 11, 400 g supernatant fraction and BioGel 0.5m fraction) and MIPP (obtained from 11, 400 g supernatant fraction and Ultrogel AcA 34 fraction) preparations were separately stored in properly labelled Eppendorf tubes (12 x 4). Each tube contained 0.3 ml and was stored at -20 o C until further use. Assay of both enzymes were performed from 0 day to day 60 day (maintaining an interval of 5 days) taking protein from each individual vial.

Determination of Salinity
Sea water of different locales (algal sample collection spots) were collected and stored in properly labelled sterile vials and brought to the laboratory under chilled condition. The practical salinity unit (PSU) values were measured in the laboratory using a Systronics Compact Water Analyzer (Model no: 371). This equipment used the measurements of electrical conductivity (EC) and temperature to provide a measurement of salinity of water, i.e., Practical Salinity Units (PSU). All measurements were recorded in triplicate.

Determination of Km and Vmax Values
The effect of substrate concentration and kinetic analyses for partially purified MIPS were carried out using D-glucose

Effect of Monovalent and Divalent Cations
The different monovalent and divalent cations was incubated separately at 37 o C (concentration of 5 mM each)with chloride anions of the respective metals in addition to all assay components in individual experimental set for both the enzymes separately keeping a control set also where no metal ion was added.

Statistical Data Analysis
All experiments were repeated thrice and quantitative data were expressed as means ± SE of three replicates. As far as the evaluation of the kinetic parameters of MIPS and MIPP are concerned, the Michaelis-Menten constant (Km) was analysed.
In order to establish relation between the substrate (D-G-6-P / D-MIP for MIPS and MIPP respectively), coenzyme (NAD for MIPS) and the rate of the enzyme catalysed reaction, all the parameters were calculated by nonlinear regression method using the software Prism 5 (GraphPad).

RESULTS
Marine Rhodophycean macro algal samples were collected from different spots (mid and low intertidal pools) of the Okha coast (within a radius of 10 Km) in the month of January, 2019 when the seaweed growth is at its peak and the salinity levels are also higher. Specimens collected from locations of highest recorded salinity were chosen for experiments. Preliminary experiments were conducted to screen the selected specimens for their inositol synthesizing potential and the two marker enzymes were assayed following the methods described earlier.
The results depicted in Table 1 show that both the enzymes of myo-inisitol biosynthesis were functional with variable specific activities in all representative species tested. The results also elucidate the fact that maximum activities of MIPS and MIPP were recorded in Halymenia venusta and such elevated activity also corresponded with the higher free myo-inositol content ( Table 1). It is noteworthy to mention here that during collection, all specimens were housed in isolated intertidal pools and/or puddles under varying salinity regimes (PSU value ranged from 29.23 to 37.68) of the near shore environment.
Starting from the crude homogenate both MIPS and MIPP were gradually purified using low speed centrifugation, ammonium sulphate fractionation (30-75%) and chromatography on DEAE-cellulose / CM cellulose, Sephadex G-200 and BioGel 0.5m / UltrogelAcA 34 (Table 2A and 2B). Comparative elution profiles are depicted in Figure 2 (a-f). The overall purification of cytosolic MIPS and MIPP was around 27and 39-folds respectively over homogenate with about 40% and 35% yields (Table: 2A and 2B). In case of this red alga, D-glucose-6-phosphate served as the principal substrate for MIPS. However, a very faint activity was recorded when D-glucose-6-phosphate was replaced by D-glactose-6phosphate (Table 3). In contrast, D-myo-inositol-1-phosphate served as the exclusive substrate for MIPP (Table 3). Other hexose phosphates (viz, D-glucose-6-phosphate, D-fructose-6-phospahte, etc.) under identical concentrations were completely ineffective as substrates (Table 3). It is noteworthy to mention here that although partially purified cytosolic MIPS from H. venusta exhibited coenzyme specificity primarily on NAD, a basal level of activity was observed in presence of few other coenzymes tested (viz, FAD, NADP and

Recovery (%) Fold purification
. This is indicative of the possible presence of a 'bound NAD' in this system. However, such 'built-in' NAD was in no way associated with thermo-tolerance, since no significant result could be obtained on removal of this endogenous NAD by means of charcoal treatment as conducted previously in animal systems [11].
The apparent molecular weights of cytosolic MIPS and MIPP as determined by gel filtration on a Sephadex G-200 column were 198 kDa and 193 kDa respectively. Native PAGE of partially purified cytosolic MIPS from H. venusta exhibited only one high intensity band along with few insignificant bands. By means of gel slice assay the major band was found to coincide with the enzymatic activity (Figure 3a). The PAGE profile of MIPP on the other hand exhibited two major high intensity bands along with two low intensity insignificant bands. Slice assay was effectively used to determine the actual band of interest (Figure 3b).
The stability of algal MIPS and MIPP varied at different stages of purification. The 11, 400 g supernatant retained activity for about 10-15 days in case of MIPS and 50-60 days for MIPP when stored at -20 0 C. However, the BioGel 0.5m or Ultrogel AcA 34 fractions of MIPS and MIPP retained their activity up to 20 days and 60 days respectively when stored at identical temperature. Repeated freezing and thawing resulted in considerable loss of activity particularly in case of MIPS but not in MIPP.
When assay was carried out using increasing concentration of MIPS protein (0-90 µg) and MIPP protein (0-70 µg) it was revealed that the activity of both the enzymes increased linearly with respect to protein concentration of about 75 and 80µg respectively  (Figure 3c,d).When 50 mMTrisacetate buffer was used in the pH range of 6.0-9.0, the algal MIPS exhibited optimum activity at pH 7.5, while optimum activity of MIPP was detected in between pH 7.0 -7.5 (Figure 3e,f).
In case of MIPS, among the monovalent cations tested, NH 4 + was highly stimulatory, K + exhibited an insignificant stimulatory role and in contrast, Li + was strongly inhibitory to this enzyme. Using similar concentrations of divalent cations it was observed that Mg 2+ played an ephemeral stimulatory role and Ca 2+ exhibited inhibitory effect (Figure 4a).
Among the monovalent cations tested, in case of MIPP, K + played a fleeting stimulatory role; Li + on the other hand was strongly inhibitory. Using identical concentrations of divalent cations it was observed that Mg 2+ played an appreciable stimulatory role while Ca 2+ exhibited moderate inhibitory effect. (Figure 4b).

DISCUSSION
The coast of Okha situated in the 'Gulf of Kutch' is one of the most important places of interest for natural algal growth in India. This area is characterized by the presence of flat rocky intertidal belt, clear waters, several tide pools, puddles and crevices which offer an ideal habitat for various Phaeophytes and Rhodophytes. Taking into account their diversity, antiquity, presence of variable morphology, growth forms, capacity of sustaining hyper saline environments, great abundance in the coast of Okha and also the paucity of information regarding their inositol synthesizing potential, members of Rhodophyceae were selected for the present investigation.  [24]. In contrast, the inshore and near shore marine environments are subjected to influence of tides, freshwater inputs, evaporation and various anthropogenic contaminants [24,25,26].These results in considerable variability in salinity level on daily and seasonal basis. The results obtained in the present communication thus confirm the fact that fluctuating salinity is a local parameter which strongly influences the distribution, diversity and physiological performance of seaweeds, at least in case of red sea weeds. Work done by previous workers indicate that myoinositol and other related cyclitols undergo isomerisation and methylation to produce O-methyl inositols, which play vital roles in salinity related responses and are known to accumulate upon salt stress in several halotolerant plant species including algae [4,13,26,27,28,29]. The results obtained in the present investigation clearly elucidate the fact that Halymenia venusta along with other Rhodophyceae members also support the saltinduced enhancement of MIPS and MIPP as reported earlier in higher plants by Ray Choudhury et al. [30]. However, it cannot be stated with certainty whether myo-inositol is the only cyclic polyol which is involved in stress protection, the possible involvement of other cyclic polyols (D-ononitol, D-pinitol etc.) along with myo-inositol cannot be ruled out.
Since the cytosolic enzyme MIPS exhibited a basal activity in absence of NAD + (Table 3) as an assay component, it can be presumed that this partially purified MIPS from Halymenia venusta did possess a 'built in' NAD + associated with thermal stability in its molecular architecture, like the MIPS obtained from mammalian adult brain [11] and from Euglena gracilis [31].
During the course of the present investigation we attempted to detect its actual role in the investigated sample following the method of activated charcoal treatment and non-treatment experiments, as reported earlier [11]. Incidentally, even after several repetitive trials, concurrence with the previous results could not be obtained. Hence, the rationale of the presence of such 'built-in' NAD + in Halymenia venusta is still obscure and its real metabolic significance is yet to be unravelled.
The MIPS and MIPP exhibited temperature optima of 35 o C, which is in complete congruence with majority of the published reports in various plant and animal systems [13]. The enzymes exhibited maximum activity within a pH range of 7.0 to 7.5 with pH optima of 7.5. This is also in complete agreement with previous reports [7,29,30,31].
In case of cytosolic MIPS obtained from Halymenia venusta, the K m value for G-6-P was recorded to be 3.599 mM. This is in slight disagreement with previous reports obtained in lower eukaryotes and in mammalian systems. In case of rat testes enzyme, the K m was found to be 3.89 mM [9], while in case of Euglena gracilis [31] and yeast [32], the recorded K m values were much lower (2.1 mM and 1.18 mM respectively) in comparison to the value obtained in the present study. With respect to the concentration of the coenzyme (NAD) of MIPS, the K m value for NAD + was found to be 0.2366 mM, which is nearly identical in comparison to the values recorded for the same enzyme from other sources [13]. The K m value for MIP was found to be 0.4070 mM which appeared much higher than vascular cryptogamic MIPPs viz., 0.068 mM (recorded in Lycopodium clavatum) and 0.076 mM (recorded in Selaginella monospora) [33].
Among the monovalent cations tested K + had trivial stimulatory effect, Na + played an impartial role and Li + was sturdily inhibitory to these enzymes. It has been mentioned earlier that both MIPS and MIPP have two isoforms located in cytosol and chloroplasts in various higher plant systems [8,30]. However, during the course of the present investigation the study of chloroplastidial MIPS and MIPP was not attempted.
The tropical Indian waters harbour a diversity of habitats which provide excellent support for a plethora of seaweeds in different proportions and densities, many of which are effectively utilized as important phycocolloid sources; few are consumed as food while the rest are insufficiently known. Members of Rhodophyceae are considered as unique and valuable from economic as well as from eco-physiological stand points. Although various aspects on red seaweed research have been carried out for decades, enzymological investigations in red algae have been a highly neglected area of research. The present study thus not only proves the existence of inositol biosynthesis in marine Rhodophyceae members for the first time but also establishes a connection between enhanced sugar-alcohol synthesis and salinity stress. However, it must be taken into account that our study has been conducted primarily with the cytolosic part of the red algal cells as the enzyme source. The possible contribution of chloroplasts in this context have to be essentially unveiled in future for understanding the correlation between variable ecological rhymes and stress related cyclitol synthesis in red algal group. The existence of diverse pigment system, complex carbohydrate components like agar and carrageenan, demand an extensive study of carbohydrate metabolism in this group using radio-labelled precursors. A molecular approach can also possibly resolve the unsolved questions if suitable DNA probes or sequence information is available.