Molecular Role of Nitric Oxide in Secondary Products Production in Ginkgo biloba Cell Suspension Culture

Effects of sodium nitroprusside (SNP; nitric oxide donor) treatment on the enhancement of secondary metabolites production, oxidative stress mediators (O2) accumulation and antioxidant defense enzymes of Ginkgo biloba callus culture was investigated. On one hand, the obtained data showed a highly metabolic modification of chemical constituents, PAL activity and various antioxidant defense enzymes (APX, SOD), which gradually increased in response to SNP treatments. On the other hands the high NO levels significantly increased the accumulation of various oxidative burst of O2. MS basal medium supplemented with casein hydrolase (500 mg/L), NAA and BA at equal concentration (0.5 mg/L) recorded the highest number of regenerated shoots (4.81 cm) and shoot height (4.96 cm) as well as root number (2.25 cm) and root length (4.5 cm). The highest survival (40 %) was shown in acclimatization on the mixture containing sand, peat moss and vermiculite (1: 1: 1, v/v/v), which significantly confirmed and reflected the variation in survival percentage. Meanwhile, higher treatment (500 μM) of NO positively enhanced secondary products accumulation of total tannins, saponins, phenols and total flavonoids in G. biloba callus culture.


Introduction
Ginkgo biloba has been known as a popular remedy in traditional Chinese medicine for over 4000 years, and it has been a common herbal medicine in Europe since the 1730's.The Ginkgo tree, the only existing tree in the family Ginkgoaceae, is the world's oldest living tree and is thus sometimes referred to as a "living fossil".Ginkgo trees are now widely planted in China, Japan, Korea, France, Germany and the United States for both ornamental and medicinal purposes.Kuo et al. (2004) demonstrated that G. biloba extract competitively inhibited rat hepatic microsomal CYP1A-mediated enzyme activity, as determined by experiments performed on extracts containing terpene trilactones and ginkgo flavonol glycosides.Despite the fact that the plant appears to possess antioxidant activity, the mechanism of action of G. biloba is not known.Plant cells and tissue cultures have been used as alternative sources to the whole plant for the production of valuable phytochemicals such as flavors, fragrances, pharmaceuticals and nutraceuticals.Numerous strategies have been developed to improve the productivity of plant cell culture such as medium optimization, cell line selection, cell immobilization, precursor addition, elicitation, genetic transformation, organ or hairy root cultures, metabolic and integrated bioreactor engineering (Abdullah et al., 2005;El-Beltagi et al., 2011).
Nitric oxide as a bioactive molecule shows prooxidant as well as antioxidant properties in plants (Beligni and Lamattina, 1999;Delledonne et al., 2001;Beligni and Lamattina, 2002;Delledonne et al., 2002).The biosynthetic origin of NO during plant pathogen interactions involves L-arginine conversion into L citrulline.Nitric oxide induces a complementary set of plant defense genes, including two key enzymes of the phenylpropanoid pathway, so-called Phenyl alanine ammonia lyase (PAL) and chalcone synthase.Furthermore, NO-treated tobacco (Nicotiana tabacum) cells were shown to induce the accumulation of cyclic acid, a key molecule for the expression of systemic acquired resistance (Durner et al., 1998;Crawford and Guo, 2005).The free radical NO has a half-life of just a few seconds and can also react with the free medium with four week intervals.Data of explants callus formation responses were recorded.

Growth dynamic
After several subcultures of callus (5 g F.W.) they were transferred into 250-ml flasks containing 50 ml of the same medium without agar and maintained on a rotary shaker at 100 rpm.Microscopic observations were carried out on cell suspension cultures during the growth cycle, which revealed the presence of single cells and small cell aggregates.The number of cell per unit can be determined using a hemocytometer.Cell growth was measured every two days for a total period of 12 days; starting, 2, 4, 6, 8, 10 and 12 days.

Cells viability
Cells viability was determined using Evan's blue staining test.A 2 mL sample from tank was incubated into 0.25% Evan's blue stain for 5 minutes and then 700 cells were counted.The viability was calculated taking into account the non-viable cells (Evans et al., 2003).
Experimental design and cell culture treatment G. biloba cell suspension cultures were treated with various concentrations of sodium nitroprusside (SNP, 50, 100, 250 and 500 µM) after sub-culturing and followed by cells harvest at time intervals.All parameters and enzymes activity were monitored after 0, 12, 48 and 72 h post NO donors treatments.

The production rate of superoxide anion (O 2 -. )
The production rate of superoxide anion (O2 -. ) was measured by the modified method described by Elstner and Heupel (1976).Fresh mass (200 mg) from culture was homogenized in 1 ml of 50 mM phosphate buffer (pH 7.8), and the homogenate was centrifuged at 10,000 g for 10 min.Then 0.5 ml of the supernatant was added to 0.5 ml 50 mM phosphate buffer (pH 7.8) and 0.1 ml of 10 mM hydroxylamine hydrochloride.After 1 h reaction at 25 1C, 1 ml of 17 mM sulfanilamide and 1 ml 7 mM a-naphthylamine were added to the mixture at 25 1C, and after 20 min, the specific absorbance at 530 nm was determined.Sodium nitrite was used as standard solution to calculate the production rate of O2 -. .

Assay of total soluble protein
Soluble proteins were measured by the Bio-Rad micro assay modification of the Bradford (1976) procedure using crystalline bovine serum albumin as a reference.

Preparation of enzyme extracts and assay of protein content
The callus samples (1.0 g) were crushed into fine powder using liquid nitrogen.Soluble protein was extracted by homogenizing the powder in 5 mL 50 mM phosphate buffer (pH 7.8) containing..ethylenediamine-N,N,N,N-tetraacetic.acid(EDTA, 1 mM) and polyvinylpyrrilidone (PVP, 1%) in addition to ASC radical superoxide to form the reactive molecule peroxynitrite (ONOO), which can lead to the formation of NO2 and the potent oxidant hydroxyl radical, which is a very strong oxidizing specie that can rapidly attack biological membranes and all types of bio-molecules such as DNA and proteins leading to irreparable damage, metabolic dysfunction and cell death (Del-Rio et al., 2003).ONOO is responsible for tyrosine nitration and oxidation of thiol (Stamler et al., 1992;Tamir et al., 1993;Lamattina et al., 2003).Sodium nitroprusside (SNP) was used as the donor of nitric oxide (NO).SNP at high concentrations stimulated catharanthine formation of Catharantuse roseus cells, but inhibited the growth of the cells (Xu et al., 2009).It stimulated production of hypericin by Hypericum perforatum.The cells were significantly increased after elicitor treatment by NO, achieving 4.2-fold higher over the control (Xu et al., 2005).
Antioxidant defense enzymes such as superoxide distumase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and monodehydroascorbate reductase (MDAR) are the systems designed to minimize the concentrations of superoxide and hydrogen peroxide.A demonstrated strong inhibitory effect of NO on the catalase (CAT) peroxidase (POD) activity of cytc/CL complex combined with its increased amount in mitochondria suggest that NO may regulate lipid peroxidation and apoptotic activity of CL membrane bound cytc during apoptosis (Vlasova et al., 2006).
Recently, a function of nitric oxide (NO) in the protection of plants against oxidative stress under various adverse conditions was reported (Beligni and Lamattina, 2002;Shi et al., 2005).Many previous studies had reported the presence of NO in the plant kingdom and its involvement in growth, development and defense responses (Beligni and Lamattina 1999).In addition, Tu et al. (2003) found that 0.1 mM SNP delayed the senescence of wheat leaves by inhibition of the degradation of chlorophyll and soluble proteins, especially Rubisco enzyme.Therefore, this study was conducted to investigate the molecular role of nitric oxide in secondary products production in G. biloba cell suspension culture.

Plant materials and surface sterilization
All excised tissues of young stems of G. biloba were collected from Orman botanical garden, Giza governorate and stored temporarily in an antioxidant solution (150 mg/l citric acid and 100 mg/l ascorbic acid), prior to surface-sterilization in 1% chlorox for 20 min, 2 drops/ 100 ml solution of Tween 20 as wetting agent then rinsed three times with sterile distilled water.

Callus initiation and maintenance
Callus was initiated from the sterilized young stems of G. biloba.The callus was initiated in a MS-Medium (Murashige and Skoog, 1962) supplemented casein hydrolisate (500 mg/L) and different combinations of Naphthaleneacetic acid (NAA) and kinetin (kin) each with concentrations of 1, 2, 3 and 4 mg/L and sucrose (30 g/L) solidified with agar (6 g/L).Three jars (replicates) were used for each treatment.Cultures were incubated at 25±1 °C, and re-cultured 3 times in the same on MS medium supplemented with different combinations of NAA (1, 2, 3 and 4 mg/l) and kinetin with concentrations of 1, 2, 3 and 4 mg/l.Data in Table 1 represents the effect of plant growth regulators (NAA and kin) combinations on callus formation responses.The highest response has been recorded with NAA and Kin with equal concentrations (3 mg/L) each followed by (2 mg/L) and (1 mg/L).On the other hand the application of NAA (1 mg/L) and kinetin (3 mg/L) did not represent any callus formation response of Ginkgo explants in vitro.Suspension culture of the cell line was initiated from G. biloba callus on a liquid MS medium supplemented with NAA (2mg/L), kinetin (2 mg/L) and sucrose 30 g/L.These data were in accordance with Wang et al. (2006), who found that hypocotyls and cotyledons of Arctium lappa were induced to form callus by culturing on MS medium supplemented with 2, 4-D (2.0 mg/L) and BA (0.5-2.0 mg /L).
Data in Table 2 showed that, the growth rate and biomass were gradually enhanced and reached their optimum on the 8 th day of cultivation.These data are in agreement with Bulgakov et al. (2002), Chong et al. (2005) and Tanveer et al. (2012); they found a significant decrease in the culture growth.

Chemical analysis of different types of G. biloba explants
While data in Table 3 show that the percentage of tannins and saponins compounds were 2.02%, 0.59% and 1.15%, 0.79% based on the dry weight, respectively for the G. biloba leaves and cell suspension cultures.While the percentage of flavonoids and phenolic compounds were 1.75%, 1.09% and 2.95%, 1.68%.The highest concentration of tannins, saponins, flavonoids and phenolic compounds were found in the G. biloba leaves compared to its derived cell suspension culture.
As shown in Table 3, there were a high accumulation of total soluble phenols under various NO treatments.The (1mM) in the case of APX assay.The samples homogenate was centrifuged at 15,000 x g for 20 minutes and the supernatant was used for further enzyme activity assays.

Assay of superoxide dismutase (SOD) and ascorbate peroxidase (APX) activity
The activity of SOD was assayed by measuring its ability to inhibit the photochemical reduction of NBT using the method of Beauchamp and Fridovich (1971).APX activity was determined (13.7 mM -1 cm -1 ) using the method of Nakano and Asada (1981).

Assay of phenylalanine ammonia lyase (PAL)
PAL activity was determined as the rate of conversion of L phenylalanine to trans-cinnamic acid at 290 nm as described by Dickerson et al. (1984).

Callus formation responses
It was determined according to the rate of scaling by Pottino (1981), which included: -No response, + Poor response, ++ Average response, +++ High response and ++++ very high response.

Callus regeneration and development
Callus was cultured on MS basic medium supplemented with casein hydrolisate (500 mg/L) and auxin (NAA) Naphthalene acetic acid in combination with cytokinins (BA) 6-Benzyl amino purine was used (mg/L); with different concentrations, as follows: 0.2, 0.5, 1.0 and 2.0 (mg/L) each.Incubation was carried out in a growth room at 25±1 °C with 16 h photoperiod (1500 lux).After 6 weeks, data including the number of regenerated shoots, shoot height (cm) as well as no. of roots and root length (cm) were recorded.

Acclimatization
In order to realize a proper acclimatization, plantlets were removed from rooting medium, rinsed under tap water and then, planted in plastic pots (5 cm) filled with a soil mixture as follows: sand, peat moss and vermiculite (1:1:1, v/v/v).Plants were covered with transparent polyethylene sheet and irrigated if needed.Potted plants were incubated for 30 days in phytotron at 25 ± 1 ºC, relative humidity (80-90 %) and 16 h photoperiod with 3000 lux light intensity.Acclimatization was realized through gradual remove of the plastic sheet, every day, until it became removed after 30 days.Survival percent, the number of leaves/plant and leaf length (cm) were recorded.

Statistical analysis
All statistical analyses were carried out using SPSS 10 software.Mean and standard error were descriptive measures of quantitative data using the analysis of variance test (ANOVA) for independent samples.All determinations have been done in triplicate, p-Values < 0.05 were considered significant.

Establishment of Ginkgo callus culture
Callus formation occurred on young leaves excised from G. biloba tree.On one hand, explants were individually cultured highest accumulation has been observed with SNP (500 µM).All NO treatments significantly increased the total phenolic compounds, tannins, flavonoids and total saponins contents.These data were in accordance with Goodman et al. (1967), who found a multifold increase of phenols in inoculated plant cells after challenging with SNP.In the present study the increase of phenols it can be due to the excess production of H2O2 in elicited plant cells through increased respiration (Farkas and Kiraly, 1962) or due to the activation of hexosemonophosphate pathway, acetate pathway and release of bound phenols by hydrolytic enzymes.Moreover, callus cultures can be employed and exploited for secondary metabolites production (Jedinák et al., 2004;Radfar et al., 2012).In tissue culture procedures, this used frequently for production of flavonoids.The results also demonstrate that NO signaling is involved in ROS induced secondary products production of G. biloba cell suspension culture.In addition to NO, many other signal molecules such as hydrogen peroxide (H2O2), jasmonicacid (JA), and salicylic acid (SA) have also been reported to play important roles in secondary metabolite production (Hahlbrock et al., 2003).Whether these signal molecules are involved in NO-induced secondary metabolites production and their relationship and/or interaction with ROS in mediating the NO-stimulated secondary metabolite synthesis still needs further investigations.The role of nitric oxide in the biosynthesis of secondary metabolites in plant cells is not well established yet.Nitric oxide released from its donor sodium nitroprusside (SNP) enhanced the production of terpenoid indole alkaloids of Catharanthus roseus cells.Furthermore, one or more protein kinases were demonstrated to act downstream of nitric oxide and transduce the signal to indole alkaloid biosynthesis (Delledonne et al., 2002).
Data in Table 4 show that, all NO treatments increased the O2 -. content in a positive correlation with time duration.NO treatments increased the accumulation of defense related to enzymes such as PAL, APX, catalase and then increased the accumulation of phenols, secondary metabolites and reactive oxygen species (ROS).Data are in agreement with Lu et al. (2011), who found that Nitric oxide (NO) has recently emerged as an important signaling molecule in plants.It has been well documented that NO is widely involved in plant adaptive responses to biotic and abiotic stresses.Accumulating evidences indicate that this free radical, which was first identified as a second messenger in animals, plays an important role in stress-induced secondary metabolite accumulation in plants.
Data in Table 5 reveal that, the increase in phenylalanine ammonia lyase (PAL) activity has been triggered under all NO treatments.Furthermore, there was a positive correlation between administrated NO and affected PAL activity.In accordance to Goodman et al. (1967), Kobeasy et al. (2011), the administration of NO increase the PAL activity which might enhance the phenol content in challenge plant cells.In addition, NO treatments also increased the PAL activity which enhanced the phenolic compound and flavonoid accumulation (Durner and Klessig, 1999).These data indicate that many studies have implicated nitric oxide (NO) as a key regulator for many different physiological processes in plants.

Evaluation of elicitation induces antioxidant defense enzymes activity
Data in Table 5 show that, SOD and APX activities increased after elicitation of different SNP doses.Similar results were obtained by Qiao and Fan (2008) which confirms that, NO can protect oxidative stresses by decreasing the carbonyl group and H2O2 contents.The decrease of H2O2 concentration was probably produced by the direct NO and H2O2 interactions.Also, it is highly possible that the protective effect of NO may result from the increased expression of genes encoding active oxygen scavenging enzymes.Pretreatment of plants with NO also enhanced the antioxidant enzyme activities in concentrations (Mackernessa et al. 2001) compared with its corresponded control.In addition, the NO elicitation processes trigger a specific activation of peroxidase activity that interferes with the process of hypersensitive reactions against infection example: lignifications and antibacterial toxic compounds formation during and after the elicitation process.The application with 500 µM of SNP reduced the SOD and APX activities compared with its corresponded control.These data were in agreement with Zhu et al. (2009) and Cui et al. (2010) confirming that, NO treatment has led to inhibition in POD and SOD activity after fresh-cut of peach and copper toxicity of tomato plants.It has also been reported that NO inhibited the activity of haemcontaining enzymes, such as catalase and peroxidases, the important H2O2-scavenging enzymes during pathogen attack (Clark et al., 2000).

Conclusion
This study shows that the NO treatments in G. biloba cell suspension cultures induced significant increase in phenolic compounds and glycosides accompanied with a significant increase in oxidative burst of O2 -, which enhance specific enzymes activities of PAL and antioxidant defense enzymes SOD and APX.In addition, the optimal media for induced G. biloba embryo callus and subculture of callus were MS basaic medium supplemented with casein hydrolase (500 mg/L), NAA and BA with equal concentrations (0.5 mg/L).
(4.81 cm) and shoot height (4.96 cm) as well as the number of roots (2.25 cm) and root length (4.5 cm) as compared to the other studied concentration treatments tested.In this concern, Ginkgo embryos culture initiated on MS medium with 2, 4-D plus NAA for 5 weeks produced shoots and roots when were transferred to media with 4.5 µM 2, 4-D alone for an additional 5 weeks.Plants were transferred from the 2, 4-D media to pots and maintained in the greenhouse (Camper et al. 1997).
High growth regulators (NAA and BA) with equal concentrations and combination (1.0 and 2.0 mg/L) gradually decreased the number of regenerated shoots and shoot height as well as the completely inhibited root formation.

Acclimatization
Data presented in Table 7 show that soil mixture containing sand, peat moss and vermiculite (1: 1: 1, v/v/v) significantly confirmed and reflected survival rate variation which is mainly ascribed to the strong relationship between plantlets and their received in vitro auxin concentrations during growth and development on rooting phase before ex vitro transfer.In this concern, Al-Salih et al. (1986) suggested that, success or failure of transferred plantlets to greenhouse is dependent primarily on the quality and type of materials produced in the previous stages of in vitro propagation.Sand + Peat moss + Vermiculite (1: 1: 1, v/v/v) 0.00 c -Means within each column followed by the same letter are not significantly different at P< 0.05.

Table 1 .
Effect of plant growth regulators (NAA and kin) in combination with different concentrations on callus formation and growth of Ginkgo biloba explants grown in vitro

Table 2 .
Growth dynamic of suspension culture of Ginkgo biloba cultured for 12 days in vitro Table 6 demonstrate that MS medium supplemented with NAA and BA with equal concentration (0.5 mg/L) recorded the highest number of regenerated shoots

Table 3 .
Effect of various NO treatments on total tannins, saponins, flavonoids and Phenolic compounds contents (% Dry weight) in Ginkgo biloba leaves and cell suspension culture Means within the same column followed by different letters are significantly different at P< 0.05.Values are means of three replicates (±SE).

Table 4 .
Effect of administration of different SNP doses (0, 50, 100 and 250 µM) on superoxide anion (O2 -. ) contents (nmol/h g FW) in Ginkgo biloba elicited suspension culture Means within the same column followed by different letters are significantly different at P< 0.05.Values are means of three replicates (±SE).

Table 5 .
Effect of different SNP administrated doses (0, 50, 100, 250 and 500μM)on the activities (Unit /mg protein -1 /h) of phenylalanine ammonia lyase (PAL), ascorbate peroxidase (APX) and superoxide dismutase (SOD) in Ginkgo biloba suspension culture Means within the same column followed by different letters are significantly different at P< 0.05.Values are means of three replicates (±SE).

Table 6 .
Effect of plant growth regulators (NAA and BA) in combination with different concentrations on regeneration response of Gingko callus grown for 6 weeks in vitro -Means within each column followed by the same letter are not significantly different at P< 0.05.

Table 7 .
Effect of NAA and IBA on in vitro rooting and their subsequent relation to plantlets survival %, after transplanted ex vitro for 3 months for acclimatization