extfieldsofstudy: Chemistry Medicine

text:
Isolation and Characterization of Phenolic Compounds and Anthocyanins from Murta (Ugni molinae Turcz.) Fruits. Assessment of Antioxidant and Antibacterial Activity

Berry fruit consumption has become important in the promotion of human health, mainly due to their phenolic compounds, which have been associated with protection against different pathologies, as well as antimicrobial and other biological activities. Consequently, there has been a growing interest in identifying natural antioxidants and antimicrobials from these plants. This study aimed to characterize the phenolic chemical composition and anthocyanin profile of murta (Ugni molinae Turcz.) fruit, and to evaluate the antioxidant and antimicrobial activity of its extracts (ethanolic and methanolic). LC/MS of the ethanolic extracts showed the presence of three major compounds: caffeic acid 3-glu, quercetin-3-glu and quercetin, while in the methanolic acid extract they were cyanidin-3-glucoside, pelargonidin-3-arabinose and delphinidin-3-glucoside. The antioxidant activity of ethanolic extracts (DPPH• and ORAC assays) was higher than that of methanol acid extracts or purified anthocynins. Furthermore, the methanol acid extract showed an inhibitory activity against the bacteria E. coli and S. typhi similar to that of standard antibiotics. The results suggest that the antioxidant activity of the ethanolic extract is regulated by the high content of phenolic compounds and the fruit’s characteristic color is due to the content of pelargonidin-3-arabinose and delphinidin-3-glucoside. The obtained results demonstrated the appreciable antioxidant and antibacterial activities, providing opportunities to explore murta extracts as biopreservatives.

Introduction
Berry fruit consumption has become important in the promoting of human health, mainly due to their phenolic compounds, which have been associated with protection against different pathologies (e.g., several types of human cancer), and because of their anti-inflammatory, gastroprotective, antimicrobial and other biological activities [1]. Consequently, there has been a growing interest to identify natural antioxidants and antimicrobials from these plants. Several edible Myrtaceae fruits including the Chilean berry "murta" (U. molinae Turcz.) have been shown to be good sources of polyphenolic antioxidants [2], comparable to those of blueberry [3].
Nowadays, it is essential to isolate, identify and characterize the phytochemicals of locally grown fruits as well as their antimicrobial activity in order to develop the fruits and plants as potential sources of antioxidant and therapeutic agents [4,5].
Phenolic compounds (PCs), including flavonoids and non-flavonoid types (phenylpropanoids, phenolic acids and cathechins), are of interest in food science because they affect the color quality, astringency and shelf stability of fresh and processed fruit products [6]. The bioactive properties of individual plant PCs are influenced by their structure, for example, antioxidant activity is greater in flavonoids that possess neighboring hydroxyl groups on their C phenyl ring [7]. Isolation and characterization studies of PCs, in order to understand the multiple mechanisms of antioxidant action, make their diverse group an interesting target in the search for phytochemicals that are of benefit to health and offer a possibility of extending the shelf life of lipid-rich foods [8].
Anthocyanins are a group of flavonoid compounds that are responsible for the colors of many flowers, vegetables, fruits and berries, providing attractive colors, such as orange, red and blue, thus being considered a valuable source of natural food colorants [9]. As a consequence of the social trend towards the consumption of natural products instead of synthetic ones, anthocyanins have received in the recent years increasing attention as natural colorants in food systems [10]. In addition, anthocyanins have attracted considerable interest due to their health benefits, including anti-cancer, anti-inflammatory and vasoprotective effects [11,12]. Among all common fruits and vegetables in the diet, berries, especially those with dark blue or red colors, have the highest content of anthocyanins and antioxidant capacities [13].
Murta (U. molinae Turcz.), a berry also known as murtilla, is a wild native plant occurring in the lowlands of the southern mountains of Chile (between the VII and X Region). This shrub produces a small globular fruit with a diameter of 0.7 to 1.3 cm used in homemade jams, syrups, jams and dessert liqueurs [14]. This is an aromatic species of the Myrtaceae family widely used in folk medicine as an analgesic for different types of pain and as an anti-inflammatory [15]. This plant has been historically appreciated because of the pleasant flavor of its edible fruits, and has been introduced into the UK and Australia for ornamental purposes. Due to its perennial shrub properties and applications in food manufacturing, it has been gaining attention from Chilean farmers, who have already domesticated this plant on a large scale [16]. Murta fruit has created increasing commercial and scientific interest during recent years, since its chemical and organoleptic properties make it a product with clear exportation potentials [17]. Additionally, murta fruit is considered a valuable source of high quality pectin that has a chemical composition similar to that of commercial citrus pectin [18]. Several reports show an association between the antioxidant activity of murta fruits and leaves and the levels of polyphenols [3,19,[20][21][22]. However, until now, the characterization of the phenolic compounds, including anthocyanin profile from this fruit and its antibacterial activity has not been reported.
In this context, the main objectives of the present study are: (a) characterization of phenolic compounds; (b) identification and quantification of anthocyanins; (c) determination of the in vitro antioxidant potential; (d) evaluation of the antibacterial activity of the methanol and ethanol extracts of murta fruits.

Identification of Phenolic Compounds of Murta Extracts
The content of phenolic compounds obtained by LC/MS is shown in Figure 1. Twelve compounds were identified under the analytical conditions used, of which three constitute the major proportion: caffeic acid-3-glucoside, quercetin-3-glucoside and quercetin ( Figure 1). The other compounds were rutin, gallic acid, quercitrin, luteolin, kaempferol, kaempferol-3-glucoside, luteolin-3-glucoside, p-coumaric acid and myricetin ( Figure 1). Figure 2 shows the structures of these compounds, confirmed by comparison with authentic standards and MS fragmentation patterns reported in the literature. Previous studies have reported that fruits of murta contain flavonols, flavan-3-ols and anthocyanins [3].
Caffeic acid, one of the most common phenolic acids, frequently occurs in fruits, grains and dietary supplements for human consumption as simple esters with quinic acid or saccharides. Glycosylated derivatives of caffeic acid are responsible for different biological activities such as suppression of humoral and cellular immunity [23].
Quercetin is the main flavonol present in our diet [24]. Because of its interesting chemical and biological properties [25], quercetin has been one of the most studied flavonoids. In recent decades, many claims on the beneficial health effects of quercetin have been stated, including protection against various forms of cancer, cardiovascular diseases and neurodegenerative diseases [26]. Furthermore, anti-inflammatory, antibacterial and muscle-relaxant activities have been ascribed to quercetin [27]. Indeed, quercetin is known as a potent competitive inhibitor of certain cytochromes P450 [28] and sulfotransferases [29]. It has been suggested that quercetin is a potent anticancer agent in man [30]. Quercetin is also a strong antioxidant that can contribute to the prevention of atherosclerosis [31]. Quercetin has been shown to reduce the carcinogenic activity of several cooked food mutagens, enhance the anti-proliferative activity of anticancer agents, and inhibit the growth of transformed tumorigenic cells [32]. Currently, kaempferol is of interest because of its antioxidant [33], anti-tumor, anti-inflammatory, and anti-ulcer activity [34].
Quercitrin is a compound with important antioxidant and antibacterial effects [35,36]. Along with rutin, it may protect the cells of the colon [37]. An important effect of quercitrin is in decreasing the phototoxicity of hypericin [35], a substance from Hypericum perforatum L. that is similar to the buckwheat phototoxic substance fagopyrin.

Identification and Quantification of Anthocyanins by HPLC/ESI-MS
The anthocyanin composition of murta berries was determined by HPLC/ESI-MS. Ten compounds were identified, based on HPLC retention time, elution order and ESI mass spectrometric data supported by comparison with our anthocyanin library and literature data [38]. Some general chromatographic trends were observed from this data. First, the general order of elution of anthocyanins under our analytical conditions was delphinidin, cyanidin, petunidin, malvidin and finally, peonidin. More specifically, the order of elution for a specific aglycone group was dependent on the attached glycoside; the order being galactoside < glucoside < arabinoside < rutinoside. By comparing the m/z values of each anthocyanin molecule and its fragmentation to the values in available published works, 10 peaks were tentatively identified in both kinds of extracts ( Figure 3, Table 1). Data acquisition in the MRM mode was used for anthocyanin quantification (relative content) in the murta extracts.  In crude acid ethanolic extract of murta, the main compounds found were pelargonidin-3-arabinose and delphinidin-3-glucoside with proportions of 49% and 40%, respectively. In the purified sample, the main compound detected was delphinidin-3-glucoside (73%) ( Table 1). The individual chromatograms for anthocyanins in both extracts (MRM mode) were used to identify and quantify the anthocyanins listed in Table 1. Figure 4 shows the structure of the main anthocyanins identified in murta fruits; the predominant anthocyanin family contained in murta fruits was that formed by the glucose or arabinose derivatives of delphinidin, petunidin and cyanidin. The total concentration of anthocyanins was not correlated with antioxidant activity, which could be attributed to the fact that glycosides (anthocyanins) are generally less active than aglycones (anthocyanidins). It is important to note that when the extract was purified in order to obtain a fraction rich in anthocyanins, only one compound strongly predominated, with the remainder of those present in the crude extract markedly decreased (Table 1).
Anthocyanins are bioactive food compounds with a double interest, one technological, due to their impact on the sensorial characteristics of food products, and the other their health related properties through different biological activities, for example their effect on cardiovascular disease risk protection [39]. Previous works had shown that murta leaf extracts contain hydroxybenzoic acids, flavan-3-ols, glycosylated flavonols [20] and triterpene acids [40]. The analgesic properties of murta infusion are associated with both flavonoid glucosides and triterpenoids [41]. Recently, Rubilar et al. [22] have reported an analysis of leaf extracts of murta, describing as the main components hydrobenzoic acid derivatives, quercetin dirhamnoside and myricetin rhamnoside, and several others not identified. The difference in term of compounds, between our work and that reported by Rubilar et al. [22] can be explained by the method of obtaining the extracts. Rubilar et al. [22] used a 50% ethanol solution, whereas in this work we used an 85% ethanol solution. The compounds extracted from plant material vary depending on the polarity of the solvent. In addition, Rubilar et al. [22] used dried plant material (fruit, leaves and stems). It has been reported that drying affects the quality of the extracts and consequently its biological activity [42].
Given the importance of murta in the economy of rural communities in Chile, the description of the components present in fruits should help to add value to products produced from this species. In addition, the LC/MS methodology optimized in this work may be useful in quality control of standardized murta extracts.

Antioxidant Capacity
Antioxidant capacity, total anthocyanins and phenolic compounds of murta extracts are shown in Table 2. DPPH • (antioxidant capacity) discoloration percentages of the crude extracts (ethanolic) were higher than for the methanol acid or purified extract. The antioxidant capacity of extracts was not significantly correlated with total anthocyanin content ( Table 2). The content of total anthocyanins was higher in the purified extract than in the crude extract. The antioxidant capacity of the crude extract correlated with the content of phenolic compounds (Table 2). Several authors have shown that phenolic compounds are often involved in the antioxidant capacity of plant extracts [43][44][45]. In addition, the ORAC value measured for murta extracts was higher in the ethanolic extract than in methanol acid or purified extracts. Furthermore, the ORAC values for murta extract is higher than those reported for blueberries [46].

Antibacterial Activity
The antibacterial activity of the extracts was assessed at different doses against E. coli and S. typhi (both Gram negative and common food contaminants [47]), and the results compared with the activity of some positive control antibiotics (Table 3). In the case of S. typhi the activity of 100 µL of methanolic extract was equivalent to the activity of all antibiotics tested (zone of inhibition = 22.5 mm), while for E. coli, the activity of 100 µL of extract was equivalent to the activity of tetracycline, amikacin, cefuroxim, cefotaxim ampicillin and ciprofloxacin (Table 3; Figure 4; p < 0.05). The contradictory effectiveness of leaf extracts of U. molinae has previously been reported [48,49].
There is no report about the antibacterial effect of murta fruit extracts. According to the demonstrated activity against E. coli and S. typhi the results support the use of this plant as a food additive due to its antimicrobial activity.
Resistance to antimicrobial agents has become an increasingly important and pressing global problem. Various plant extracts possess bacteriostatic and bactericidal effects due to the secondary metabolites they contain, namely alkaloids, tannins, flavonoids and phenolic compounds. Most of these secondary metabolites, other than possessing antimicrobial potential, can also act as potent antioxidants [50].

Plant Material
The ripe berries of murta (Ugni molinae Turcz.), genotype 14-4, were manually harvested and obtained from INIA Carillanca, Murta Gene Bank (Project FONDEF D05I1086), located in Puerto Saavedra (latitude, 38°45'S; longitude, 73°21'W), Chile, during summer 2009. Because the experimental field is located in a coastal area near to the Pacific Ocean, the weather is characterized as being a moderate oceanic climate with marine influence. Fruit from 5.5-year-old murta plants drip irrigated periodically during spring and summer, were used in the experiment. The fruits were placed in polyethylene bags and stored at −20 °C until use.

Extraction of Phenolic Compounds and Anthocyanins
Ten g of fresh fruit was mixed with 85% ethanol (100 mL, 85 v of absolute ethanol and 15 v of pure water) and then sonicated for 2 h. The extract was subsequently filtered through Whatman No. 2 filter paper. The final resulting solution was used as a stock solution for analyzing total phenolic content and profile, and the antioxidant and antibacterial activity.
Anthocyanins were extracted according to the methodology described by Fuleki and Francis [51]. One gram of fruit was macerated with ethanol (5 mL, 70% v/v acidified with 0.1% v/v of HCl), and the juice was stirred in the dark for 4 h and stored at 5 °C for 24 h. The samples were filtered through gauze and the solid residues were submitted to the same procedure, adding 10 mL of the extracting solvent. The resulting extract was completed with the extracting solvent to a volume of 30 mL.

Purification of Anthocyanins
Five milliliters of the anthocyanin aqueous solutions obtained from the extraction procedure were passed through an open column (20 mL) filled with ion exchange resin (Dowex Monosphere 88) in a sodium form. Anthocyanins were absorbed onto the column while sugars, acids and other water-soluble compounds were removed by washing the columns with 5 volumes of distilled water and 2.5 volumes of methanol. Anthocyanins were then eluted with methanol acidified with HCl in the following sequence: 7.5 volumes of methanol acidified with 0.1% HCl (v/v), 7.5 volumes of methanol acidified with 0.2% HCl (v/v), 7.5 volumes of methanol acidified with 0.3% HCl (v/v), 12.5 volumes of methanol acidified with 0.5% HCl (v/v), 12.5 volumes of methanol acidified with 1.0% HCl (v/v). To determine the volumes of solvent used and to control the releasing of anthocyanins from the column, spectrophotometric readings were carried out at 470 and 535 nm for each 10 mL eluted. The acidified methanolic fractions containing anthocyanins were combined and concentrated to 5 mL using a rotary evaporator at 38 ± 1 °C, then read in the spectrophotometer and frozen at −20 °C for further analysis of identification.

Analysis of Phenolic Content
Phenolic compounds in the ethanol extracts of murta were analyzed according to the methodology of Bravo et al. [52]. LC/MS analyses were performed using an Agilent 1100 series liquid chromatograph/mass selective detector equipped with a quadrupole (G6400) mass spectrometer (Agilent Technologies, Palo Alto, CA, USA). The liquid chromatographic system consisted of a quaternary pump, on-line vacuum degasser, and thermostatic column compartment, connected in line to a mass spectrometer. Data acquisition and analysis was carried out in an Agilent Mass Hunter. Sample (10 µL) was injected into the HPLC system, and separation was performed on a Nucleosil 120 C18 reversed phase column (250 × 4.6 mm i.d., 5 µm particle size) protected with an ODS RP18 guard column. A binary gradient of 1% formic acid in deionized water (solvent A) and acetonitrile (solvent B) was as follows: from 10% to 20% solvent B over 20 min, 20% to 25% solvent B over 10 min, 25% to 30% solvent B over 10 min. and then isocratically for 10 min. The flow rate was 1 mL/min. The mass spectrometer was fitted to an atmospheric pressure electrospray ionization (ESI) source, operated in negative ion mode. The electrospray capillary voltage was set to 3000 V, with a nebulizing gas flow rate of 9 L/h and a drying gas temperature of 300 °C. Mass spectrometry data were acquired in the Scan mode (mass range m/z 100-1000) and MRM mode.

Chemical Identification of Anthocyanins
Anthocyanins in the purified and crude extracts were separated by reverse phase HPLC on a Zorbax Eclipse XDB-C18 column (5 μm, 4.6 × 250 mm, Agilent Technologies, Santa Clara, CA, USA). The column was eluted with a mobile phase consisting of H2O:methanol:formic acid (75:20:5, v/v/v) with flow rate of 0.5 mL/min. The separated anthocyanins were further identified by LC-MS (Agilent) employing gel electrospray ionization (ESI) and operating in a triple quadruple mode. The instrument was scanned over the m/z range of 100 to 1000 in the ESI positive ion mode. The LC-MS was eluted with acetonitrile (ACN) and 0.5% NH4OH (90:10, v/v) with a flow rate of 0.4 mL/min. A binary solvent system was employed, following the work of Qin et al. [53]. Solvent A was 5% aqueous formic acid (v/v) and solvent B was 100% methanol (HPLC grade). The flow rate was maintained at 0.8 mL/min. The gradient elution profile was as follows: 0 min, 20% B; 1-10 min, 20%-27% B; 10-15 min, 27%-33% B; 16-20 min, 33%-20% B.

Analysis of Antioxidant Capacity (DPPH • and ORAC Assays)
DPPH • was dissolved in 85% ethanol [54] and the experiments were performed on freshly prepared solution. The assay conditions were as follows: 20 µL of each extract and 980 µL of DPPH • solution (64 µM). Absorbance was recorded in a UV-visible spectrophotometer (Agilent model 5420, Palo Alto, CA, USA) at 517 nm during an interval from 10 s to 4 min of reaction. The results were expressed as % of consumed DPPH • .
The ORAC value was estimated according to Alarcon et al. [55]. Briefly, a solution containing PGR (5 µM), AAPH (10 mM), with and without the extract sample (ethanolic or methanolic), was incubated at 37 °C in phosphate buffer 75 mM, pH 7.4. PGR consumption was evaluated from its absorption intensity (A) decrease at 540 nm. UV-visible experiments were carried out in a Hewlett Packard 8453 spectrophotometer (Palo Alto, CA, USA). Control solutions (in the absence of extracts, A0) showed a fast consumption of the probes (less than 20 min). Values of (A/A0) were plotted as a function of time.
Integration of the area under the curve (AUC) was performed up to a time such that (F/F0) reached a value of 0.2. These areas were employed to obtain ORAC values, according to Equation (1). All experiments were carried out in triplicate. (1) where: AUC = Area under curve in the presence of the tested extract sample, integrated between time zero and that corresponding to 80% of the probe consumption; AUC° = Area under curve of control (PGR or FL plus AAPH solution); AUCtrolox = Area under curve in the presence of Trolox; f = Dilution factor, equal to the ratio between the total volume of the working solution (target molecule plus AAPH, plus extract) and the added extract sample volume; [Trolox] = Trolox milimolar concentration.

Antibacterial Activity of Extracts
The antibacterial activity of the extracts was measured on strains of Escherichia coli (ATCC 25922) and Salmonella enteric serovar typhi (S. typhi) (ATCC 14028). Both strains were grown in Mueller-Hinton broth at 37 °C to saturation (OD600 = 0.6). For antibiotic analysis, 100 µL of saturated broth were poured onto plates with 15 mL of Mueller-Hinton agar medium and dispersed using a microbiological rake. Different aliquots of ethanolic and acidic methanolic extracts were evaluated (100, 75, 50, 25, 12, 10, 5 and 2 µL). The results were expressed as an inhibition diameter using the vehicle as a negative control. All samples were analyzed in triplicate.

Statistical Analysis
Determinations were based on two replicates in triplicate. Data statistical analyses were carried out by one-way ANOVA using the SPSS software (version 11.0, IBM Inc., Chicago, IL, USA). Significantly different means were compared using the multiple comparisons test of Tukey (p < 0.05).

Conclusions
LC/MS of ethanolic extracts of murta fruits showed the presence of three major compounds: caffeic acid-3-glu, quercetin-3-glu and quercetin, while the acidic methanol extract showed the presence of pelargonidine 3-arabinose, delphinidin-3-glucoside and petunidin-3-glucoside as major compounds. Our data suggested that the antioxidant and antibacterial activity shown by the ethanolic extract is regulated by the high content of phenolic compounds, and the characteristic color of fruits is mainly determined by the content of pelargonidine 3-arabinose and delphinidin-3-glucoside. The antioxidant activity of ethanolic extracts (DPPH • and ORAC assays) from murta fruit was higher than the activity of crude or purified anthocynins. Ethanolic and methanolic extracts of murta showed a strong antibacterial activity. The results provide opportunities to explore this fruit as biopreservatives due to its antimicrobial and antioxidant activity.


extfieldsofstudy: Biology Medicine

provenance: AgriculturalAndFoodSciences,1970-2015-Agricultural And Food Sciences-2015.jsonl.gz.gz

text:
Characterization and Functional Analysis of Pyrabactin Resistance-Like Abscisic Acid Receptor Family in Rice

Background Abscisic acid (ABA) plays crucial roles in regulating plant growth and development, especially in responding to abiotic stress. The pyrabactin resistance-like (PYL) abscisic acid receptor family has been identified and widely characterized in Arabidopsis. However, PYL families in rice were largely unknown. In the present study, 10 out of 13 PYL orthologs in rice (OsPYL) were isolated and investigated. Results Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that expression of OsPYL genes is tissue-specific and display differential response to ABA treatment, implying their functional diversity. The interaction between 10 OsPYL members and 5 protein phosphatase 2C in rice (OsPP2C) members was investigated in yeast two-hybrid and tobacco transient expression assays, and an overall interaction map was generated, which was suggestive of the diversity and complexity of ABA-sensing signaling in rice. To study the biological function of OsPYLs, two OsPYL genes (OsPYL3 and OsPYL9) were overexpressed in rice. Phenotypic analysis of OsPYL3 and OsPYL9 transgenic rice showed that OsPYLs positively regulated the ABA response during the seed germination. More importantly, the overexpression of OsPYL3 and OsPYL9 substantially improved drought and cold stress tolerance in rice. Conclusion Taken together, we comprehensively uncovered the properties of OsPYLs, which may be good candidates for the improvement of abiotic stress tolerance in rice. Electronic supplementary material The online version of this article (doi:10.1186/s12284-015-0061-6) contains supplementary material, which is available to authorized users.

Background
Abscisic acid (ABA) plays pivotal roles in regulating plant growth and development, including seed dormancy, germination, and seedling growth. More importantly, ABA is the key phytohormone that functions in a plant's response to abiotic stressors such as drought, high salinity, and extreme temperature (Cutler et al. 2010). During abiotic stress, ABA biosynthesis is activated, resulting in an increase in ABA levels in the plant. ABA binds to the pyrabactin resistant-like/regulatory components of ABA receptors, PYL/RCAR (hereafter referred to as PYLs for simplicity), an ABA receptor family that promotes the interaction between PYL with protein phosphatase 2C (PP2C), which then results in the release of SNF1-related protein kinase (SnRK)) from the repression of PP2C. Finally, the active SnRK phosphorylates and activates downstream transcriptional factors that promote the expression of ABA-regulated genes. An ABA signal is then generated, which in turn results in the acquisition of abiotic stress resistance in plants (Ma et al. 2009;Park et al. 2009;Cutler et al. 2010;Klingler et al. 2010). ABA receptor PYL proteins, which contain a conserved steroidogenic acute regulatory-related lipid transfer (START) protein domain, are the core components of this ABA sensing signaling pathway (Ma et al. 2009;Park et al. 2009). To date, PYL proteins have been identified from distinct plant species, including Arabidopsis, rice, tomato, and soybean, which present highly conserved PYL-mediated ABA-sensing signaling pathways (Ma et al. 2009;Park et al. 2009;Kim et al. 2012;Bai et al. 2013;Gonzalez-Guzman et al. 2014;He et al. 2014). The biochemical property and structure of PYLs have been extensively studied in the dicot plant model Arabidopsis (Ma et al. 2009;Melcher et al. 2009;Klingler et al. 2010;Joshi-Saha et al. 2011). Some AtPYLs are monomers that facilitate interactions with PP2C in the absence of ABA; some AtPYLs are in a dimeric state and require ABA to form a complex with PP2C (Hao et al. 2011). Recent studies have investigated the biological functions of various AtPYLs. The overexpression of AtPYL5 leads to ABA hypersensitivity during early seedling development, as well as enhanced drought stress tolerance (Klingler et al. 2010). AtPYL8 is involved in root growth and development, which is in line with its root-specific expression pattern ). Although AtPYL13 is not an ABA receptor, it can positively regulate the ABA signaling pathway by interacting with and inhibiting both the PYL receptors and the PP2C co-receptors (Li et al. 2013;Zhao et al. 2013). AtPY-L4 A194T forms stable complexes with PP2CA in the absence of ABA, and the overexpression of AtPYL4 A194T increases a plant's sensitivity to ABA-mediated inhibition of germination and seedling establishment, as well as enhances drought resistance ). In addition, AtPYLs are largely functionally conserved, and the analysis of higher order mutants have indicated that AtPYLs regulate stomatal conductance (Gonzalez-Guzman et al. 2012).
Unlike AtPYLs, information on PYL homologs in rice is limited. Studies have predicted that rice has 13 OsPYL members that share high sequence similarity with AtPYLs (Kim et al. 2012;He et al. 2014). Core components in ABA signaling, including OsPYLs, OsPP2C, OsSAPK2, and OsOREB1, have been identified in rice, and the ABA signaling transduction pathway has also been reconstituted in a protoplast system (Kim et al. 2012). The biochemical properties and structure of OsPYLs have also been recently reported (He et al. 2014). However, to date, only OsPYL/RCAR5 has been identified as a positive regulator in seed germination, early seedling growth, and drought and salt stress tolerance (Kim et al. 2012;Kim et al. 2014). Other features of OsPYLs such as expression pattern, subcellular localization, interaction specificity with OsPP2C, and biological function, have not been examined.
The present study examined the tissue-specific expression pattern and distinct response of OsPYL members to ABA treatment, which was suggestive of its differential biological function. An overall interaction map between 10 OsPYLs and 5 OsPP2C members indicated that OsPYLs were selective of their interaction partner, thus indicating the complexity and specificity of the rice ABA-sensing signaling pathway. Furthermore, using OsPYL3 and OsPYL9, we determined that OsPYLs play pivotal roles in ABA-mediated inhibition of seed germination, as well as drought and cold stress tolerance, which thereby could serve as potential targets for the improvement of abiotic stress tolerance in rice.

Expression Pattern of OsPYL Members
Using the AtPYLs sequence as queries and search in the rice genome database, a total of 13 OsPYL orthologs were identified in rice. Among these, three (OsPYL11-13) are thought to be nonfunctional that were caused by a large fragment deletion in the N or C terminal of the gene (Kim et al. 2012). To obtain an overview of the expression pattern of OsPYL members in different tissues, quantitative RT-PCR was performed. Because of the high level of sequence similarity among OsPYL members, it was difficult to design primers that could discriminate between OsPYL2 and OsPYL9, and OsPYL7 and OsPYL8. Most of the OsPYLs were detected in all tissues, although differentially expressed (Fig. 1a). OsPYL7/8 was upregulated in embryos, OsPYL3 and OsPYL5 were upregulated in leaves, OsPYL1 in roots, and OsPYL2/9 in all tissues (Fig. 1a). These diverse tissue-specific patterns of OsPYLs were indicative of its diverse biological functions.

OsPYLs are Localized in the Cytosol and Nucleus
To determine the subcellular localization of OsPYLs, green fluorescent protein (GFP)-OsPYL fusion proteins driven by a 35S promoter were transiently expressed in Nicotiana benthamiana leaves. All tested OsPYLs were localized in both the cytosol and nucleus (Fig. 2a), which were consistent with the results involving AtPYL9 and soybean GmPYL members (Ma et al. 2009;Bai et al. 2013). These results indicated that PYLs of different plant species are localized in the same cellular regions, which might partly explain the functional conservation of PYL proteins as ABA receptors.

OsPYL Members Selectively Interact with OsPP2C Members
PYL family proteins, as functional ABA receptors, interact with clade A PP2Cs to form PYL-ABA-PP2C triple complexes that facilitate in the transmission of ABA signals (Cutler et al. 2010;Joshi-Saha et al. 2011). A total of 10 predicted clade A OsPP2Cs have been identified in rice (Xue et al. 2008). In the present study, five clade A OsPP2Cs were isolated and investigated (Fig. 2b). To determine whether OsPYLs interacted with these OsPP2Cs, yeast two-hybrid assays were conducted in the absence or presence of ABA. The results showed that the interaction between OsPYLs and OsPP2Cs was selective and specific ( Fig. 3, Additional file 1: Figure S1). Some OsPYLs interacted with all OsPP2C members, except for OsPYL2 and OsPYL10. Most interactions were ABA-dependent or ABA-enhanced. A few interactions were ABA-independent or constitutive such as that observed with OsPYL1 and OsPP2C53 (Fig. 3). Most OsPYLs strongly interacted with OsPP2C30 and OsPP2C53 in an ABA-dependent or ABAindependent manner, respectively (Fig. 3). These results demonstrated that OsPYLs bind to OsPP2Cs in diverse fashions and with different intensities. To confirm these interactions in plant cells, BiFC experiments in N. benthamiana leaves were performed. The in vivo interaction results indicated that these could reproduce, as well as validated the results of the yeast two-hybrid assay (Fig. 4). Based on the results of the yeast two-hybrid and BiFC assays, an overall interaction map was generated between the 10 Fig. 1 Tissue-specific and ABA-regulated expression of OsPYLs genes. a Tissue-specific expression of OsPYLs was analyzed by quantitative RT-PCR in roots, leaves, stems, embryo, endosperm and panicles. The expression level of OsPYLs in roots was set as 1 and the fold change was analyzed via the 2 -△△CT method using the rice ubiquitin gene as an internal control. Values represent the mean ± SD of three biological replicates. b ABA-regulated expression of OsPYLs was analyzed by quantitative RT-PCR. Two-week-old seedlings were incubated in liquid MS medium containing 200 μM ABA for 0, 1, 3 and 6 h. The expression level of OsPYLs at 0 h was set as 1 and the fold change was analyzed via the 2 -△△CT method using the rice ubiquitin gene as an internal control. Values represent the mean ± SD of three biological replicates OsPYLs and 5 OsPP2C members (Fig. 3). These results provide evidence that the tested OsPYLs were capable of functioning as ABA receptors, and that the ABA-sensing mechanism was conserved among different plants.

OsPP2C Determines the Subcellular Localization of the OsPYLs-OsPP2C Complex in an ABA-independent Manner
The results of the BiFC assay indicated that the subcellular localization of the fused green fluorescent proteins varied among different OsPYLs and OsPP2C members, and most of the OsPYL-OsPP2C complexes were localized in the nucleus (Fig. 4), which is not consistent with the localization of OsPYLs (Fig. 2a). To explore the underlying mechanism of this activity, the subcellular localization of five OsPP2C members was investigated. OsPP2C53 was localized to both the nucleus and cytosol, whereas other OsPP2C members were detected only in the nucleus (Fig. 2b). Comparative analysis showed a similarity between the subcellular localization of OsPP2C members and that of OsPYLs-OsPP2C complexes (Figs. 2b and 4). In the BiFC assay, ABA was also injected to test whether ABA affected the interaction between OsPYLs and OsPP2Cs. The results showed that ABA treatment only enhanced interaction strength, and did not affect the subcellular localization of the OsPYL-OsPP2C complexes (Fig. 4). Consequently, the findings indicated that OsPP2C members can determine the subcellular localization of OsPYL-OsPP2C complexes in an ABA-independent manner.
Overexpression of OsPYL3 and OsPYL9 Confers ABA Hypersensitivity during Seed Germination To determine whether OsPYLs are functional ABA receptors in rice, OsPYL3 and OsPYL9 were selected for further investigation. Constructs of OsPYL3 and OsPYL9 driven by the 35S promoter were transformed into rice, and more than 20 independent transgenic lines were produced for each gene. Three independent transgenic lines of each gene were chosen for further analysis. The expression levels of OsPYL3 and OsPYL9 in the transgenic lines were measured by quantitative RT-PCR and RT-PCR, respectively ( Fig. 5a and b). OsPYL3 and OsPYL9 were overexpressed in the transgenic lines compared to the control lines ( Fig. 5a and b). A seed germination assay was conducted to examine the ABA-related phenotype of OsPYL3 and OsPYL9 overexpression lines. In the absence of ABA, OsPYL3 and OsPYL9 overexpression lines showed a slight delay in germination rate, and the OsPYL9 overexpression line germinated at a slower rate compared to that of the OsPYL3 overexpression line (Fig. 5c, d, and g). Application of different concentrations of ABA showed that the seed germination rate of the OsPYL3 and OsPYL9 overexpression lines was markedly slower than that of the controls (Fig. 5c, e, f, h, and i). OsPYL9 overexpression lines hardly germinated in a medium containing 3 μM of ABA until 5 days later (Fig. 5i). This finding indicated that the overexpression of OsPYL3 and OsPYL9 conferred ABA-hypersensitivity during seed germination and functioned as an active ABA receptor. In addition, a higher expression level of OsPYL9 than that observed with OsPYL3 in embryos may explain its enhanced phenotype during ABA-regulated seed germination (Figs. 1a and 5).

Overexpression of OsPYL3 and OsPYL9 Enhances Drought Stress Tolerance
To further investigate the biological function of OsPYLs, OsPYL3 and OsPYL9 overexpression lines were subjected to a drought tolerance assay. Three-week-old seedlings of (See figure on previous page.) Fig. 2 Subcellular localization of OsPYLs and OsPP2Cs. Confocal images were taken from Nicotiana benthamiana leaves epidermal cells. Constructs of (GFP)-OsPYLs (a) and (GFP)-OsPP2Cs (b) driven by the 35S promoter were infiltrated and observed at 3 days later. From left panel to right panel are GFP image, DAPI dye image, merged image and bright-field image. Empty GFP vector was used as control. The positions of nuclei were shown by DAPI staining Fig. 3 Interaction map between OsPYLs and OsPP2Cs. Interaction between OsPYLs and OsPP2Cs was first examined in yeast two-hybrid, "-" and "+" indicated without and with ABA supplement. Number of "*" indicate the interaction strength from weak to strong, one (*) to four star (****) represent that the yeast colony can grow on SD/-His /-Leu/-Trp plate containing different concentration of 3-AT (0, 3.0, 5.0 and 10 mM). "x" means no interaction. These interactions were further confirmed by BiFC in tobacco transient expression system (See figure on previous page.) Fig. 4 OsPYL3 and OsPYL9 interact with OsPP2Cs in planta. In planta interaction and subcellular localization analysis using agroninfiltrated Nicotiana benthamiana leaves. The interaction was detected by fluorescence in BiFC analysis. 100 μM ABA was injected at 24 h before observation. From upper panel to bottom panel are YFP image, DAPI dye image, merged image and bright-field image. The positions of nuclei were shown by DAPI staining Fig. 5 Overexpression of OsPYL3 and OsPYL9 confer ABA hypersensitivity during seed germination. a Quantitative RT-PCR analysis of OsPYL3 in overexpression transgenic lines. The expression level of OsPYL3 in control line was set as 1 and the fold change was analyzed via the 2 -△△CT method using the rice ubiquitin gene as an internal control. Values represent the mean ± SD of three biological replicates. b RT-PCR analysis of OsPYL9 in overexpression transgenic lines. Full length OsPYL9 primer was used and rice ubiquitin transcripts were used as control. c Representative photographs of seed germination of OsPYL3 and OsPYL9 overexpression transgenic lines. Seeds of control and transgenic lines were grown on half-strength MS medium containing indicated concentration of ABA for 5 days. Photographs were taken on day 5. (D to I) Germination time course of OsPYL3 (d-f) and OsPYL9 (g-i) overexpression transgenic lines and control in medium without ABA (d and g), 1 μM ABA (E and H) and 3 μM ABA (f and i). Data show the mean ± SD of three replicates. At least 50 seeds per genotype were measured in each replicate similar sizes showed the capability of withholding water for 10 days, control seedling leaves were curved and wilted and its seedling had fallen down, whereas OsPYL3 and OsPYL9 overexpression lines remained upright (Fig. 6a). After 7 days of re-watering, over 80 % of the OsPYL3 and OsPYL9 overexpression lines were turgid and survived; in contrast, only less than 10 % of the control lines remained alive (Fig. 6a-c). These results indicated that the ectopic expression of OsPYL3 and OsPYL9 enhanced drought stress tolerance in rice (Fig. 6a-c). Fig. 6 Overexpression of OsPYL3 and OsPYL9 exhibit enhanced drought stress tolerance. a Three-week-old OsPYL3 and OsPYL9 overexpression seedlings were subjected to drought conditions by withholding water and then rewatered. Photographs were taken before drought (upper panel) and 10 dayays after drought (middle panel), and 7 days after rewatering (bottom panel). b and c Survival rates of the drought treated OsPYL3 (B) and OsPYL9 (C) overexpression seedlings after 7 days of rewatering. Values are mean ± SD (n = 30 for each replicate) of three independent experiments. d and e Water loss rate of OsPYL3 d and OsPYL9 (e) overexpression lines. Leaves of the same developmental stages were excised and weighed at various time points after detachment. Values are means ± SD of three individual plants per genotype. Experiments were repeated at least three times with similar results. (f and g) Relative water content of OsPYL3 (f) and OsPYL9 (g) overexpression lines. Seedling of similar stage was withhold water for indicated days and used for measure relative water content. Values are means ± SD of three individual plants per genotype. Experiments were repeated three times Plants lose water mainly through stomata transpiration. To determine whether stomatal closure is involved in the enhanced drought tolerance of OsPYL3 and OsPYL9 overexpression lines, a water-loss assay was performed using detached leaves. The water loss rate of the OsPYL3 and OsPYL9 overexpression lines was significantly slower than that observed in the controls (Fig. 6d and e). Relative water content (RWC) more accurately reflects the physiological consequence of cellular water deficit. Fig. 6e and g shows that with drought stress, the OsPYL overexpression lines lost water at a slower rate and have a lower minimum relative water content that helped the plants survive during severe drought conditions. These results indicated that the OsPYL3 and OsPYL9 overexpression lines have a faster rate of stomatal closure and were hypersensitive to drought treatment compared to the controls, thereby contributing to a high level of drought stress tolerance.

Overexpression of OsPYL3 and OsPYL9 Increases Cold Stress Tolerance
The rice seedlings were also subjected to cold stress. To examine whether OsPYLs played a role during cold stress response, a cold tolerance assay was performed. Two-week-old rice seedlings of similar size were incubated in a 10°C growth chamber for 4 days. After 4 days, the leaves of the transgenic lines remained green and flats, and only the leaf tips were rolled up. In contrast, the leaves of the control lines were wilted, rolled up, and dry (Fig. 7a). After 7 days of recovery at the normal temperature, the survival rate of the OsPYL3 and OsPYL9 transgenic plant was >50 %, compared to the >95 % death rate of the control lines ( Fig. 7a-c). To evaluate the effect of cold stress on cell membranes, two-week-old rice seedlings were exposed to a 10°C environment, and relative ion leakage was measured. Without cold treatment, the relative ion leakage rate of the transgenic lines and control lines was similar. After 3 days cold treatment, the relative ion leakage rate of the control lines was 70 %, whereas that of the OsPYL3 and OsPYL9 overexpression lines was <10 % (Fig. 7d and e). These findings indicated that the increased cell membrane stability of OsPYL3 and OsPYL9 overexpression lines during cold stress partially, if not totally, contributed to the observed enhanced tolerance of cold stress. These results indicated that overexpression of OsPYL3 and OsPYL9 substantially improved cold stress tolerance in rice.

Expression of ABA-Regulated Genes is Enhanced in
OsPYL3 and OsPYL9 Overexpression lines Because OsPYL3 and OsPYL9 overexpression lines showed significant drought and cold stress tolerance at the seedling stage, we were prompted to determine whether the expression of ABA-regulated genes was also enhanced in these transgenic lines. Several ABAregulated genes (LEA3, RAB16A, and OsABA45) were selected, and its expressions levels between the control and OsPYL3 and OsPYL9 overexpression lines in response to ABA treatment were compared. Quantitative RT-PCR analysis indicated that ABA induced the expression of these genes, which was significantly higher than that observed in the controls (Fig. 8). Notably, in the absence of ABA treatment, the expression of these ABAregulated genes was still higher in the OsPYL3 and OsPYL9 overexpression lines than that in the control, indicating that the overexpression of OsPYL3 and OsPYL9 promoted the constitutive expression of ABA-regulated genes (Fig. 8), which possibly explains the delayed seed germination of OsPYL3 and OsPYL9 overexpression lines in the absence of ABA (Fig. 5c, d, and g). Taken together, the upregulation of ABA-regulated genes might also have contributed to the observed ABA hypersensitivity and the enhanced stress tolerance of the OsPYL3 and OsPYL9 overexpression lines.

Discussion
Members of the PYL protein family such as the ABA receptors and the core component in ABA-sensing signaling have been investigated in various plant species, including Arabidopsis, soybean, rice, and cucumber (Ma et al. 2009;Wang et al. 2012;Bai et al. 2013;He et al. 2014;Kim et al. 2014). Compared to the extensive studies on AtPYLs, the property, function, and mechanism of OsPYLs are largely unknown. In the present study, based on amino acid sequence analysis of AtPYLs and predicted OsPYLs in the rice genome annotation project, 10 out of the 13 OsPYLs were cloned and studied. OsPYLs were differentially expressed in the leaves, stems, roots, panicles, embryo, and endosperm, and some OsPYL members showed pronounced tissue-specific expression patterns (Fig. 1a). OsPYLs also displayed a differential response to ABA treatment (Fig. 1b). The diverse expression patterns of OsPYLs were indicative of their functional diversity. All OsPYLs were localized to the cytoplasm and nucleus (Fig. 2a), which was consistent with the results of previous reports on the subcellular localization of AtPYLs and GmPYLs (Ma et al. 2009;Bai et al. 2013).
The interaction between the OsPYLs and OsPP2Cs was investigated in both yeast and tobacco, and an overall interaction map was generated between 10 OsPYLs and 5 OsPP2C members in the absence or presence of ABA treatment (Figs. 3 and 4). OsPYLs selectively interacted with OsPP2Cs in ABA-dependent or ABA-independent manner. Combined with a recent report that OsPYLs inhibit OsPP2C activity (He et al. 2014), these data suggest that OsPYLs are functional rice ABA receptors.
In addition to the discovery of PYLs as ABA receptors, the present study has shown that these proteins also regulate ABA signaling and improve tolerance to abiotic stress. Overexpression of AtPYL4 and AtPYL5 leads to enhanced ABA hypersensitivity during seed germination and improved drought tolerance Pizzio et al. 2013). In addition, the rootspecific expression of AtPYL8 regulates ABA-mediated inhibition of root growth . However, information on the biological function of OsPYLs in rice, which is considered a monocot model, is limited. OsPYL5 has been shown to positively regulate seed germination and drought stress response (Kim et al. 2012;Kim et al. 2014). In the present study, transgenic rice overexpressing OsPYL3 and OsPYL9 were produced and analyzed in detail ( Fig. 5a and b). First, overexpression of OsPYL3 and OsPYL9 conferred ABA hypersensitivity during seed germination (Figs. 5c-i). In addition, overexpression of OsPYL3 and OsPYL9 in the absence of ABA significantly delayed seed germination, and OsPYL9 played more important roles, which was consistent with the upregulation of OsPYL9 in the embryo Fig. 7 Overexpression of OsPYL3 and OsPYL9 enhanced cold stress tolerance. a Two-week-old OsPYL3 and OsPYL9 overexpression seedlings were subjected to 10°C treatment and then recovered at room temperature. Photographs were taken at before cold (upper panel) and 4 days after cold treatment (middle panel), and 7 days after recovered (bottom panel). b and c Survival rates of the cold-treated plants after 7 days of recovery. Values are mean ± SD (n = 30 for each replicate) of three independent experiments. d and e Relative ion leakage in rice leaves after 10°C treatment for 0 d, 2 d and 3 d. Data represent means ± SD of three biological replicates compared to that of OsPYL9 (Fig. 5c-i). Second, overexpression of OsPYL3 and OsPYL9 enhanced drought stress tolerance, which can be partly explained by a slower water loss rate through the stomata in transgenic lines (Fig. 6). Third, OsPYL3 and OsPYL9 overexpression lines show increased membrane stability during cold stress and enhanced cold stress tolerance (Fig. 7). Last, expression of ABA-regulated genes in OsPYL3 and OsPYL9 overexpression lines was significantly higher than that in the controls (Fig. 8), which accounted for the increased stress tolerance of transgenic plants. Interestingly, compared to the controls, OsPYL3 and OsPYL9 overexpression lines did not show any negative effect on growth, grain yield, and other observable phenotypes, whereas these were observed in OsPYL5 (Kim et al. 2014). One possible explanation for this finding is that OsPYL5 was directed by the maize ubiquitin promoter, which is a very strong promoter in monocots, and OsPYL3 and OsPYL9 was directed by the 35S promoter, whose efficiency was less than that of ubiquitin in rice. Taken together, these results indicate that OsPYL3 and OsPYL9 can be used as a target gene for the improvement of abiotic stress tolerance in rice. Fig. 8 Overexpression of OsPYL3 and OsPYL9 increased the expression of ABA responsive genes. Fourteen-day-old control, OsPYL3 and OsPYL9 overexpression seedlings were treated with ABA solution (100 μM) for indicated time. Shoots were analyzed by quantitative RT-PCR. LEA3 (LOC_Os05g46480), RAB16A (LOC_Os11g26790), and OsABA45 (LOC_Os12g29400) were monitored. The expression level of ABA regulated gene in control at 0 h was set as 1 and the fold change was analyzed via the 2 -△△CT method using the rice ubiquitin gene as an internal control. Values represent the mean ± SD of three biological replicates

Conclusions
In the present study, we study the expression pattern of PYL orthologs in rice (OsPYL), generate an overall interaction map between 10 OsPYL members and 5 protein phosphatase 2C in rice (OsPP2C) members and show the biological function ofOsPYL3 and OsPYL9. Taken together, we comprehensively uncovered the properties of OsPYLs, which may be good candidates for the improvement of abiotic stress tolerance in rice.

Plant materials, and rice transformation
For cloning of OsPYLs and OsPP2Cs genes, Oryza sativa L. ssp. Japonica cv. Nipponbare was used. The coding sequence of OsPYLs and OsPP2Cs were cloned from the cDNA using standard PCR-based protocol. The primers used are listed at Additional file 1: Table S1. Full-length sequences of OsPYLs and OsPP2Cs were cloned into pENTR/D-Topo (Invitrogen), resultant constructs were confirmed by sequencing and saved for later use. To over express the OsPYLs in rice, OsPYLs was transferred to pH7WG2 vector via LR recombination reaction of the Gateway system. The resultant 35S:OsPYLs constructs, in which OsPYLs was drived by the cauliflower mosaic virus (CaMV) 35S promoter, were transferred into Agrobacterium tumefaciens EHA105. Finally, OsPYLs were transformed into Oryza sativa L. ssp. Japonica cv. Longjing 11 by the Agrobacterium-mediated co-cultivation method.

Yeast two-hybrid assay
Full-length sequences of OsPYLs and OsPP2Cs were cloned into pDEST32 or pDEST22 vector via an LR recombination reaction and used as baits or preys respectively. The resultant constructs were transformed into the yeast strain Y2H gold. Presence of the transgenes was confirmed by growth on a SD/-Leu/-Trp plate. To assess protein interactions, the transformed yeast cells were suspended in liquid SD/-Leu/-Trp to OD 600 = 1.0. The suspended cells were spread on plates containing SD/-His/-Leu/-Trp medium supplied with indicated concentration of 3-AT (3-amino-1, 2, 4-triazole) and ABA. The interactions were observed after 4 days of incubation at 30°C. The experiments were repeated three times with similar results.

Subcellular localization and Bimolecular fluorescence complementation in tobacco
For subcellular localization of OsPYLs and OsPP2Cs, fulllength sequences of OsPYLs and OsPP2Cs were cloned into the pH7WGF vector via an LR recombination reaction, in which the OsPYLs and OsPP2Cs fused with green fluorescent protein (GFP) was drived by 35S promoter. For Bimolecular fluorescence complementation (BiFC) assays, OsPYLs were fused with the C-terminal portion of the yellow fluorescent protein (YFP) via an LR recombination reaction; OsPP2Cs were fused with the N-terminal portion of the yellow fluorescent protein (YFP) via an LR recombination reaction. For transient expression, the fusion constructs were transferred into Agrobacterium tumefaciens GV3101. Agrobacterium tumefaciens strain harboring each construct along with the p19 strain were infiltrated into 4-week-old N. benthamiana leaves. For staining of the nuclei, 10 mg/ml 4, 6-diamidino-2-phenylindole (DAPI) was infiltrated into N. benthamiana leaves 3 h before observation. For microscopic analyses, leaf discs were cut 3 d after infiltration. The fluorescence signal was observed using confocal microscopy.

Analysis of genes expression
Total RNA was extracted using TRIzol (Invitrogen) and treated with DNaseI. cDNA was synthesized from 2 µg of total RNA using SuperscriptII Reverse Transcriptase. Real-time PCR was performed with SYBR Green PCR master mix (TransStart). Data were collected using Bio-Rad chromo 4 real-time PCR detector. All expressions were normalized against the Ubiquitin gene. The primers used are listed at Additional file 1: Table S1.

Germination and abiotic stress tolerance assay
Dehulled seeds were surface-sterilized and planted on half-strength MS medium supplemented with indicated concentration of ABA (A1049, Sigma-Aldrich). Seed germination was defined as the coleoptiles emerged from the seed and scored every 12 h for 5 days. Three independent T3 homologous transgenic lines and the control Longjing 11 were used for stress tolerance experiments. For the dehydration treatment, rice plants grown for 3 weeks were withhold water for 10 days and then rehydrated and grown under normal conditions for 7 days. For cold treatment, 14-day-old seedlings were transferred to 10°C for 4 days and then returned to normal growth conditions for 7 days. The survival rates were recorded. Approximately 50 seedlings of each line were used for each experiment, and three replicates of each experiment were performed. Tests for statistical analysis between transgenic lines and the controls were performed using Microsoft excel 2007.

Water loss assay and measurement of relative electrolyte leakage
For water loss assay, leaves of control and OsPYLs transgenic plants grown under normal conditions were detached from 3-week-old seedlings and weighed immediately on a piece of weighing paper, and then placed on a laboratory table and weighed at indicated time intervals. Three replicates were performed for each line.


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A REVIEW ON GONGRONEMA LATIFOLIUM, AN EXTREMELY USEFUL PLANT WITH GREAT PROSPECTS

Gongronema latifolium is a plant that has a wide range of nutritional and ethnomedical uses in different tropical African communities. Scientific reports on the chemical composition and bioactivity (anti-inflammatory, antimicrobial, antidiabetic, antioxidant, anticancer and allelopathic properties) of the plant material by different authors were discussed in this review. Future prospects of the plant extracts in the areas of herbal formulations, food preservation, alcoholic fermentation and beer production, drug discovery and allelopathy were also highlighted.

Figure 2: Basic sapogenin structure
Flavonoids are widely distributed and form major colouring components of plants. They large group of phenolic compounds and are responsible for a variety of pharmacological activities [21][22][23]. They exist as aglycones, glycosides and methylated deriva further divided into different groups like the flavones, flavon plant flavonoids have been shown to exhibit protective effects against infectious, cardiovascular, carcinogenic and age  [22,23].

phenyl-1,4-benzopyrone)
are large polyphenolic compounds that contain many hydroxyl and other groups, such as carboxyls, which usually form strong complexes with various macromolecules. Tannins medicinal properties [24]. The structure of tannic acid, a Flavonoids are widely distributed and form major colouring components of plants. They are a large group of phenolic compounds and are responsible for a variety of pharmacological tives and can be ols and flavanones [21]. Some plant flavonoids have been shown to exhibit protective effects against infectious, are large polyphenolic compounds that contain many hydroxyl and other groups, such as carboxyls, which usually form strong complexes with various macromolecules. Tannins e of tannic acid, a tannin, is It is hoped that in future the actual saponins, flavonoids and tannins present in leaves would be isolated, purified, characterized and subjected to bioactivity evaluations.
Eleyinmi [26] did nutritional analysis of the protein content was 27.2 %, dry weight, while the lipid extract, ash, crude fibre and nitrogen free extractives were 6.07%, 11.6%, 10.8%, and 443.3% dry matter, respectively. that leucine, valine, phenylalanine, the plant material, with aspartic acid, glutamic acid and glycine having 10.3%, respectively of the total amino acid composition. Likewise, saturated and unsaturated 6 Sitosterol is a plant sterol (phytosterol). Phytosterols are known to have beneficial effects in helping to reduce cholesterol absorption in the intestines [25].

Figure 5. B-Sitosterol
It is hoped that in future the actual saponins, flavonoids and tannins present in ified, characterized and subjected to bioactivity evaluations. did nutritional analysis of the G. latifolium leaves and reported that the crude protein content was 27.2 %, dry weight, while the lipid extract, ash, crude fibre and nitrogen ee extractives were 6.07%, 11.6%, 10.8%, and 443.3% dry matter, respectively. leucine, valine, phenylalanine, aspartic acid, glutamic acid and glycine were the plant material, with aspartic acid, glutamic acid and glycine having 13.8%, 11.9%, and 10.3%, respectively of the total amino acid composition. Likewise, saturated and unsaturated are known to have beneficial effects in It is hoped that in future the actual saponins, flavonoids and tannins present in G. latifolium ified, characterized and subjected to bioactivity evaluations. leaves and reported that the crude protein content was 27.2 %, dry weight, while the lipid extract, ash, crude fibre and nitrogen ee extractives were 6.07%, 11.6%, 10.8%, and 443.3% dry matter, respectively. He reported aspartic acid, glutamic acid and glycine were abundant in 13.8%, 11.9%, and 10.3%, respectively of the total amino acid composition. Likewise, saturated and unsaturated fatty acids were 50.2% and 39.4% of the oil, respectively. He also reported occurrence of minerals in the plant material [26].
latifolium. Both aqueous and methanolic extracts of the leaf significantly (P=0.05) inhibited the carrageenan-induced rat paw oedema in a dose-related manner [27]. The extracts also significantly inhibited the acetic acid-induced vascular permeability and the leukocyte migration assay conducted on experimental mice. The carrageenan-induced rat paw oedema test is an established protocol to test for acute inflammation [30, 31 while the leucocyte migration assay and acetic acid-induced vascular permeability experiments are additional established antiinflammatory tests [32,33]. Morebise et al [28] also reported that the methanolic extract of the plant leaf material significantly inhibited the nystatin-induced rat paw oedema and also significantly stabilized erythrocyte membrane subjected to heat-and hypotonic solutioninduced lyses.
To test for possible anti-inflammatory property of the plant in chronic inflammatory conditions, arthritis was induced in experimental rats and G. latifolium extracts were found to significantly and dose-dependently inhibit the arthritis formation and to lower the serum levels of Gammaglutamyl transferase, alanine aminotransferase and aspartate aminotransferase [29]. The total serum protein and the globulin fraction were significantly (P=0.05) raised while the albumin fraction and the serum glucose were significantly reduced [29].

Antidiabetic activity.
There have been several reports on the antidiabetic activity of the leave extracts of G.
latifolium. In the study conducted by Sylvester et al. [36], experimental rats were subjected to Streptozotocin-induced diabetes mellitus and then treated with G. latifolium leaf extracts. They iv.

Antioxidant activity.
Numerous reports have shown that G. latifolium leaf extracts exhibit antioxidant property.
Ugochukwu and Babady [38] reported that oral administration of aqueous and ethanolic extracts of G. latifolium leaf to Streptozotocin-induced diabetic rats significantly raised the activity of superoxide dismutase, glutathione reductase, glutathione peroxidase and glucose-6phosphate dehydrogenase (G6PD). The level of reduced glutathione was increased while lipid peroxidation was decreased. They concluded that G. latifolium leaves could exert their antidiabetic activity through their antioxidant properties [38].
Nwanjo et al. [39] reported the anti-lipid peroxidative property of the aqueous extract of G.
latifolium leaves in experimental rats subjected to Streptozotocin-induced diabetes. They found out that the extract significantly increased the activity of superoxide dismutase and lowered the level of the plasma lipid peroxidation product, malondialdehyde.
Eze and Nwanguma [12] reported the antioxidant activity of the tannin extract from G. latifolium leaves on partially purified lipoxygenase from Cucumeropsis manii seeds. The inhibitory effects of the tannin extract and of two known antioxidants: ascorbic acid and propyl gallate, on the partially purified lipoxigenase was investigated. The extract significantly inhibited the lipoxygenase and this inhibitory activity compared well with those of ascorbic acid and propyl gallate. They suggested that the extract could be included in food processing to hinder the deteriorative effects of lipoxygenase [12].

v. Anticancer activity.
G. latifolium leaf extracts were reported by Iweala et al.
[40] to exhibit strong inhibitory activity against human lung carcinoma and human breast adenocarcinoma in vitro. The extracts also exhibited free scavenger scavenging activity against 1,1-Diphenyl-2-picrylhydrazyl (DPPH) in vitro. Previous reports have indicated that phytochemicals could prevent cancer and other chronic diseases by their antioxidant activity as free radical scavengers [41][42][43].
vi. Allelopathic activity. Morebise and Fafunso [4] reported the phytotoxic activity of saponin extracts from G. latifolium leaves. The saponin extract showed strong inhibitory activity against the germination of bean and maize seeds, and against the growth of their seedlings. Inhibition of root and shoot lengths was concentration-dependent and the roots were more sensitive than the shoots to the effect of the extract [4].

vii. Beer production
Adenuga et al. [44] conducted an experiment in which sorghum beer was brewed with the extracts of G. latifolium, Vernonia amygdalina and Garcinia kola to impart bitter taste and flavor as substitutes for hops used for beer production. Sensory evaluation of the beer samples was done by trained panelists. The panelists adjudged the G. latifolium flavoured beer as better than the other samples and that it compared favourably with hopped beer in terms of flavor and taste.

viii. Toxicological study
Interestingly, Sylvester et al. [36] reported that acute toxicity of the ethanolic extract of the plant on mice showed 0% lethality when administered intraperitoneally at 1000mg/kg body weight, but 100% mortality when administered at 2000mg/kg. This clearly suggests that the plant material is not toxic at the safe doses being consumed. More research is needed in this area.

GENERAL DISCUSSION
The Catharanthus roseus [49,50]. A large percentage of Orthodox drugs in clinical management of 13 cancer have their roots in plants and other natural products [51] while more than 60% of cancer patients still resort to use of herbs to manage the disease [52].

ii. Herbal Supplements/ Formulations
Researchers should look into the possibility of using G. latifolium leaf extracts as approved medicinal formulations or supplements, especially for diseases like diabetes mellitus, sickle cell disease, malaria and other disorders for which the plant is being used ethnomedically. During the past decades, the World Health Organization (WHO) and several Governments in the developing countries have campaigned for the promotion and integration of herbal remedies in healthcare delivery as supplementary contributions to modern medical facilities [53].

iii. Food Production and Preservation
Based on the research findings highlighted in this review, commercial application of G.
latifolium extracts as alternative to hops in beer production should be looked into. Also, commercial use of the extracts as food supplements or for food preservation is an interesting area which researchers can focus on.

iv. Allelopathy
Allelopathy is an emerging approach to agricultural issues like inter-cropping, pest management, crop rotation and growth enhancement [54]. A lot of research has been done to explore the inhibitory potential of different allelopathic crops and trees for weed management [55][56][57][58]. The inhibitory property of allelochemicals has been attributed to the blockage or cessation of important physiological and metabolic processes of plants [54]. Since allelochemicals do not have residual toxic effects, they are better alternatives to synthetic herbicides [59].


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Evaluation of Salmonella Growth at Low Concentrations of NaNO2 and NaCl in Processed Meat Products Using Probabilistic Model

This study developed probabilistic models to predict Salmonella growth in processed meat products formulated with varying concentrations of NaCl and NaNO2. A five-strain mixture of Salmonella was inoculated in nutrient broth supplemented with NaCl (0%, 0.25%, 0.5%, 0.75%, 0.5%, 1.0%, 1.25%, and 1.75%) and NaNO2 (0, 15, 30, 45, 60, 75, 90, 105, and 120 ppm). The inoculated samples were then incubated under aerobic and anaerobic conditions at 4°C, 7°C, 10°C, 12°C, and 15°C for up to 60 days. Growth (assigned the value of 1) or no growth (assigned the value of 0) for each combination was evaluated by turbidity. These growth response data were analyzed with a logistic regression to evaluate the effect of NaCl and NaNO2 on Salmonella growth. The results from the developed model were compared to the observed data obtained from the frankfurters to evaluate the performance of the model. Results from the developed model showed that a single application of NaNO2 at low concentrations did not inhibit Salmonella growth, whereas NaCl significantly (p<0.05) inhibited Salmonella growth at 10°C, 12°C, and 15°C, regardless of the presence of oxygen. At 4°C and 7°C, Salmonella growth was not observed in either aerobic or anaerobic conditions. When NaNO2 was combined with NaCl, the probability of Salmonella growth decreased. The validation value confirmed that the performance of the developed model was appropriate. This study indicates that the developed probabilistic models should be useful for describing the combinational effect of NaNO2 and NaCl on inhibiting Salmonella growth in processed meat products.

INTRODUCTION
Salmonella are facultative and intracellular pathogens, and they cause salmonellosis with the consumption of contaminated food (Jantsch et al., 2011). In the EU, salmonellosis was the second highest zoonotic disease in 2011, following campylobacteriosis (EFSA and ECDC, 2013). Nontyphoidal Salmonella infections are estimated to affect 1.4 million people annually in the United States, resulting in 15,000 hospitalizations and 400 deaths (Voetsch et al., 2004). Salmonellosis is mostly caused byanimal foods such as poultry, eggs, pork, beef, and processed meat products (Meyer et al., 2010;EFSA and ECDC, 2013). Salmonella contamination of meat products occurs frequently at processing plants through cross contamination (Beery et al., 1988;Izat et al., 1989). According to a study by between the growth and no growth of bacteria or pathogens as a function of various factors (Ratkowsky and Ross, 1995;Schaffner and Labuza, 1997;Lee et al., 2013). For instance, Yoon et al. (2009) suggested a combination of minimum level of lactic acid, dipping time, and storage temperatures to inhibit Listeria monocytogenes growth in frankfurters using probabilistic model, and the model could be used to select conditions in meat products. Therefore, the objective of this study was to evaluate the combinational effect of low NaCl and NaNO 2 concentration on inhibiting Salmonella growth in processed meat products with probabilistic models.

Inoculum preparation
Salmonella Typhimurium NCCP10812, Salmonella Agona NCCP12231, Salmonella Enteritidis NCCP12243, Salmonella Enterica KACC11595, Salmonella Montevideo NCCP10141 were cultured in 10 mL nutrient broth (NB; Becton, Dickinson and Company, Sparks, MD, USA) at 35°C for 24 h. Subsequently, 0.1-mL aliquots of the cultures were subcultured in 10 mL NB at 35°C for 24 h. The subcultures of five strains were mixed, and the mixed subcultures were centrifuged (1,912×g, 15 min, 4°C). The pellets were then washed twice with phosphate buffered saline (PBS, pH 7.4; 0.2 g of KH 2 PO 4 , 1.5 g of Na 2 HPO 4 ·7H 2 O, 8.0 g of NaCl, and 0.2 g of KCl in 1 L distilled water). The suspension was serially diluted with PBS to obtain 4 log CFU/mL of inoculum.

Media preparation and inoculation
Twenty five microliter aliquot of the inoculum was inoculated into 225 µL NB supplemented with combinations of NaCl (0%, 0.25%, 0.5%, 0.75%, 1.0%, 1.25%, 1.5%, and 1.75%) and NaNO 2 (0,15,30,45,60,75,90,105, and 120 ppm) in a 96-well microtiter plate. Two hundred fifty microliter aliquot of NB was used as a negative control, and 225 µL of NB inoculated with 25 µL of the cell suspension was used as a positive control. The microtiter plates were then incubated at 4°C, 7°C, 10°C, 12°C, and 15°C for up to 1,440 h, 1,440 h, 1,152 h, 384 h, and 168 h, respectively. For the aerobic conditions, the microtiter plates were sealed with plastic paraffin film (Parafilm M; Bemis Company Inc., Neenah, WI, USA). For the anaerobic conditions, the microtiter plates sealed with paraffin film were placed into tightly sealed plastic containers with Anaerogen (Oxoid Ltd., Basingstoke, Hampshire, UK). The Anaerogens were replaced every 24 h.

Model development
In this study, the combinations of NaCl (0%, 0.25%, 0.5%, 0.75%, 1.0%, 1.25%, 1.5%, and 1.75%), NaNO 2 (0, 15,30,45,60,75,90,105, and 120 ppm), storage temperature (10°C, 12°C, and 15°C), and storage time (up to 1,440 h) were prepared. Each combination had eight observations (n = 8), and growth response was evaluated every 24 h during storage. If a well became turbid, it was assigned 1 (growth), otherwise it was assigned 0 (no growth). The growth response data were then analyzed by logistic regression using SAS (Version 9.3; SAS Institute Inc., Cary, NC, USA). The following equation was derived by the logistic regression and a stepwise selection method was used to select significant parameters (p<0.05). The selected parameters in the equation were used to describe the combinational effect of NaCl and NaNO 2 on Salmonella growth by producing growth/no growth interfaces for Salmonella at probabilities of 0.1, 0.5, and 0.9.

Validation
To evaluatethe performance of the developed model, the predicted growth probabilities were compared to the results obtained from the frankfurters used as model processed meat products. To prepare the frankfurters, the formulation described by Kim et al. (2010) was used as follows: fat (20%) and lean meat (60%) were well-mixed with ice (20%), isolated soy protein (1.0%), phosphate (0.3%), spice (0.5%), potassium sorbate (0.2%), sugar (0.5%), NaNO 2 (0 or 10 ppm), and NaCl (1.0%, 1.25%, or 1.5%). The mixtures were stored at 4°C for 1 h, followed by stuffing them into collagen casings (ca., 30 g/each) using an automatic sausage can filler (Konti A50; Frey, Herbrechtingen, Germany). The frankfurters were subsequently cooked at 75°C for 40 min in a smokehouse (MAXI 3501; Kerres, Backnang, Germany). After cooling, the sausages were vacuum-packaged, and then heated for a second time at 80°C for 15 min, followed by dipping into ice water for 10 min and storage at 4°C overnight. The frankfurters were cut into 25-g samples. Thirty frankfurters were immersed in 500 mL inoculum (3 log CFU/mL) in a sterilized plastic container for 2 min. The samples were air-dried under a laminar flow cabinet for 15 min to allow Salmonella attachment then transferred into vacuum bags (Gwak et al., 2015). The bags were sealed for aerobic storage or vacuum packaged for anaerobic storage using a vacuum packager (HFV-500; Fujee Inc., Hwaseong, Korea). The packaged samples were incubated at 4°C, 10°C , and 15°C for up to for 1,440 h. During incubation, Salmonella were enumerated on xylose lysine deoxycholate agar (XLD; Beckton, Dickinson, and Company) at the appropriate time interval (6 to 8 times), depending on the incubation temperature. If the Salmonella cell counts increased above 1 log CFU/g compared to the cell counts on day 0, it was considered as "growth" and if the cell counts decreased or increased less than 1 log CFU/g, it was considered "no growth" (Koutsoumanis et al., 2004;Yoon et al., 2009).

RESULTS AND DISCUSSION
The estimates of significant parameters are listed in Table  1. These estimates were used to predict the growth and no growth interfaces at probabilities of 0.1, 0.5, and 0.9. For both aerobic and anaerobic conditions, Salmonella growth was not observed at 4°C or 7°C (data not shown). Salmonella growth was initiated earlier in aerobic conditions than in anaerobic conditions at 10°C to 15°C (Figures 1 to 6). Møller (2012) reported a T min (theoretical minimum temperature for growth) for Salmonella in pork at 2.33°C and the T min for Salmonella suggested by Pin et al. (2011) was 4.27°C in broth media, which are both lower than the approximate lowest temperature (7°C) for Salmonella growth evaluated in this study. Fares (2007) and University of Nebraska Cooperative Extension (2005) also suggested 6°C to 7°C as the minimum temperature for Salmonella. Because T min is a theoretical lower temperature limit of growth below which the calculated growth rate is close to zero (Ratkowsky et al., 1991), T min and the minimum observed temperature in food are different. Even though low NaNO 2 and NaCl concentrations in processed meat products could be microbiologically unsafe, storage of the samples below 7°C can make the product safe. However, since NaNO 2 is very closely related to the inhibition of Clostridium botulinum germination (Christiansen et al., 1973), use of low NaNO 2 concentration for processed meat products should be considered very carefully.
At 10°C under aerobic conditions, no antimicrobial effects from NaNO 2 were observed at all concentrations with 0% NaCl, but 120 ppm NaNO 2 showed obvious antimicrobial effects with more than 1% NaCl compared to other NaNO 2 concentrations. The antimicrobial effect of NaNO 2 became more obvious with 1.75% NaCl (Figure 1). However, at 10°C under anaerobic conditions, the antimicrobial activity of NaNO 2 increased as the NaCl concentration increased and became more obvious with 1.75% NaCl (Figure 2). At 12°C, the antimicrobial activity of NaNO 2 on Salmonella growth was not obvious with 0% NaCl, but NaCl with 0 ppm NaNO 2 inhibited Salmonella growth (p<0.05) under aerobic conditions in a concentrationdependent manner. In addition, the combinational effect of NaNO 2 and NaCl was not obvious under aerobic conditions, but the antimicrobial effect of NaNO 2 increased as NaCl concentration increased under anaerobic conditions ( Figures  3 and 4). At 15°C, the antimicrobial activity of NaNO 2 was not observed with 0% NaCl under both aerobic and anaerobic conditions, and the combinational effect of NaNO 2 and NaCl was not obvious under aerobic conditions at 12°C, but the antimicrobial effect of NaNO 2 increased as NaCl concentration increased under anaerobic conditions ( Figures  5 to 6). Jo et al. (2014) also developed probabilistic models to describe Pseudomonas growth responses in processed meat products formulated with NaNO 2 and NaCl, and found that the antimicrobial effect of NaNO 2 on Pseudomonas increased as NaCl concentration increased. In addition, Pelroy et al. (1994) reported increased NaNO 2 antimicrobial activity against Listeria monocytogenes in salmon with high NaCl concentrations. These results suggest that NaNO 2 should be combined with NaCl in processed meat products to inhibit Salmonella growth, especially in products with low NaNO 2 concentrations.       To evaluate the performance of the developed probabilistic model, Salmonella growth response data were collected from frankfurters formulated with various concentrations of NaCl and NaNO 2 , and the growth response data were compared to the results predicted from the probabilistic model under aerobic and anaerobic conditions; growth probability above 0.5 was considered growth (Koutsoumanis et al., 2004). The percentage of agreement between the observed and predicted growth response was approximately 81% (Table 2), indicating that the developed probabilistic model in this study was acceptable for evaluating the effect of low NaCl and NaNO 2 concentrations on Salmonella growth.
In conclusion, processed meat products containing low concentrations of NaNO 2 and NaCl should be stored below 7°C to inhibit Salmonella growth, regardless of the atmospheric conditions. However, if the products must be stored above 7°C, low NaNO 2 concentrations should be combined with NaCl to inhibit Salmonella growth under both aerobic and anaerobic conditions. In addition, the developed probabilistic model is appropriate for predicting the growth probability of Salmonella in processed meat products formulated with NaNO 2 and NaCl, which should be useful for describing the effect of NaNO 2 and NaCl on Salmonella growth in processed meat products.


s2fieldsofstudy:
Agricultural And Food Sciences
Medicine

text:
Evaluation of biochemical alterations produced by acetaminophen overdose in Bubalus bubalis

Aim: Evaluation of the effect of acetaminophen (APAP) overdose on biochemical parameters in buffalo calves. Materials and Methods: The experiment was conducted on six healthy male buffalo calves of 6-12 months age. The APAP was administered intramuscularly at the dose rate of 250 mg/kg body weight (B.W.) on day 0, followed by two subsequent doses at the dose rate of 50 mg/kg B.W. on day 2 and 4, respectively. Biochemical parameters including alanine aminotransferase (ALT), alkaline phosphatase (ALP), amylase, blood urea nitrogen (BUN), creatinine, and total acid phosphate were estimated in the plasma samples collected on 0, 1, 2, 3, 4, 5, and 6th day from the start of treatment. Results: Significant increase in the plasma levels of ALT (446.0%), ALP (137%), BUN (216.8%) and creatinine (149.2%) was recorded on day 3, 4, 3, and 4, respectively, after the start of APAP dosing. However, a significant decrease was observed in amylase activity with a maximum decline of 48.3% on 6th day after the start of treatment. No significant alteration was observed in ACP activity after APAP overdose. Conclusion: Administration of APAP in overdose produced hepatic dysfunction as evidenced by a significant increase in the activities of ALT and ALP, whereas reduced amylase may indicate acute pancreatitis in buffalo calves. In addition, repeated dosing also resulted in renal impairment in these animals as seen by a significant elevation in BUN and creatinine levels, whereas negligible effect on prostatic function.

Introduction
Acetaminophen (APAP) is a regularly used non-narcotic analgesic as well as antipyretic agent in veterinary practice [1]. It has been reported that these effects of APAP are due to selective inhibition of cyclooxygenase (COX)-III, a variant of COX-I in the central nervous system and consequential inhibition of prostaglandin synthesis [2]. It is a safe drug when administered at therapeutic doses, but its overdose is quite common as it has narrow therapeutic index. Its overdose can lead to hepatic and renal damage in both humans and experimental animals [3].
The liver is a vital organ that regulate metabolism of xenobiotics including antimicrobials and also responsible for maintenance of body homeostasis. A number of chemical agents and drugs which are used routinely for the treatment including APAP produce cellular and metabolic liver damage [4,5].
APAP gets metabolized by conjugation with sulfate and glucuronidate and are excreted in the urine as an inert conjugate. Depending on the dose, a fraction of APAP is converted into N-acetyl-p-benzoquinone imine (NAPQI) -a highly reactive toxic intermediate by several P450 cytochromes. Significant amounts of NAPQI are efficiently eliminated by conjugation with glutathione (GSH). However, after a large dose of APAP, there is the saturation of sulfonation reaction and subsequently accumulated NAPQI depletes GSH in the liver. Unconjugated NAPQI binds to proteins and cellular structures and produces rapid cell death and necrosis that can lead to liver failure [5].
Kidney, another crucial organ for metabolism and excretion of many xenobiotics including antibiotics is at risk of APAP overdosing [3,6,7]. It is the second most sensitive organ of APAP toxicity and renal dysfunction occurs among patients with marked hepatic injury; though, nephrotoxicity after an acute overdose may even occur in the absence of hepatotoxicity [3].
Enzymes are proteins that catalyze the biochemical reactions in a sequence-specific manner. Exposure to APAP at high doses can cause cellular damage releasing these enzymes to plasma [3,4]. Thus, plasma enzyme levels can be used as a reliable marker for early diagnosis and prognosis of organ damage.
The use of NSAIDs is quite common in the treatment of bovines and its extra-label use may pose the risk of overdose in these species. Buffalo is an important dairy animal and contributes about 70% to the total milk production in India. Although effects of APAP overdose were evaluated in laboratory animals especially in rats, there were no reports to our perusal regarding the effect on liver as well as kidney in buffalo calves. The present study was therefore carried with the primary objective to see the effect of APAP overdose on biochemical parameters in buffalo calves.

Ethical approval
The study had been approved by the Institutional Animal Ethics Committee and experimental plan followed the ethical guidelines on the proper care as well as use of animals.

Animals and treatment
The experiment was conducted on six healthy male buffalo calves of 6-12 months age and weighing between 100-130 kg. Animals were kept under normal ambient conditions in the experimental animal shed of the department and maintained on green fodder, concentrates, and wheat straw. Water was provided ad libitum. The dosage schedule of APAP (Paracetol-Vet, Cadila Health Care, India) was as per followed by Sharma and Ul Haque [8], i.e. 250 mg/kg body weight (B.W.) intramuscularly on day 0, followed by 2 subsequent doses at the dose rate of 50 mg/kg B.W. on day 2 and 4, respectively.

Estimation of biochemical parameters
To study the biochemical parameters, blood samples were collected in heparinized vials from the jugular vein of animals on 0, 1, 2, 3, 4, 5, and 6th day from the start of APAP dosing. Plasma was separated by centrifugation at 3000 rpm for 15 min. Biochemical parameters including alanine aminotransferase (ALT), alkaline phosphatase (ALP), amylase, blood urea nitrogen (BUN), creatinine were estimated using Bayer Autopack kits (Bayer Diagnostics India Ltd., Baroda, Gujarat), and total acid phosphate (TACP) was estimated using Accurex (Accurex Biomedical Pvt. Ltd. Mumbai).

Statistical analysis
All these results were subjected to one-way Analysis of Variance and the significance was tested using Dunnett's test. The significance was assayed at 5% (p<0.05) as well as 1% (p<0.01) levels [9]. These statistical calculations were carried out with SPSS 16.0 software.

Results and Discussion
Therapeutic dose of paracetamol administered in buffalo calves was reported to be 50 mg/kg B.W. [10]. The dose of the APAP administered to the animals in the present investigation was toxic dose as confirmed by previous hepatic dysfunction model used by the authors to conduct pharmacokinetic studies in buffalo calves [8].
Results on the effect produced by APAP overdosing on ALT, ALP, amylase, BUN, creatinine, and TACP are presented in Table-1 and also depicted in Figures-1 and-2, respectively. The values of these biochemical parameters on day 0 were taken as 100 and the increase/decrease calculated in % given in the parenthesis. Significant increase in the plasma ALT level was recorded with maximum of 446.0% on day 3, while amylase activity was significantly decreased from day 1 onward with maximum decline of 48.3% on day 6 after repeated APAP overdosing. The activity of ALP was elevated significantly from day 1 onwards with a maximum elevation (137%) on 4th day. In addition, significant elevation in BUN levels was observed from day 1 onwards with a maximum increase of 216.8% on day 3 following repeated administration of APAP. Similarly, creatinine levels also exhibited significant elevation from day 2 onwards with a maximum increase of 149.2% on day 4 after the start of treatment. However, no significant alteration was recorded in plasma TACP levels.
Liver plays an important role in the biotransformation of xenobiotics by which toxic metabolites transformed into less harmful products and get excreted so as to reduce toxicity. However, during   overdose these intermediate metabolites are produced in a large quantity resulting in liver cell damage and produce toxicity to this organ. Hepatotoxicity is the most noteworthy feature of APAP overdose and it resembles other types of acute inflammatory liver diseases with a significant increase in levels of ALT. [11]. Elevated levels of ALT in the plasma often indicate the existence of medical problems for instance hepatitis, liver damage, bile duct problems, congestive heart failure, infectious mononucleosis or myopathy and diabetes [8]. The serum ALT is one of the most sensitive biochemical markers for the diagnosis of hepatic dysfunction [12]. During liver damage the transport function of hepatocytes is disturbed, resulting in consequential leakage of hepatic enzymes through plasma membrane therefore an elevation of aminotransferase activity in the extracellular fluid or plasma is considered as a sensitive indicator for hepatocellular damage [13]. The significant increase in the plasma ALT activity of APAP treated buffalo calves from day 1 to day 6 in the present study indicates liver impairment following its repeated administration [8,14].
ALP is a zinc-containing enzyme present in liver, biliary tracts, small intestines, bones, lungs, and kidney. Elevated levels in the blood may indicate damage to the liver and other organs [13,15]. The enhanced activity may be due to increased synthesis in response to hepatic damage as seen by the ALT levels in these buffalo calves after repeated administration of APAP [8].
Serum amylase is an important marker for acute pancreatitis and the reduced activity of this enzyme was reported after diazinon-induced acute pancreatitis  in rats [16]. Similarly in the present study, the levels of amylase were reduced significantly in buffalo calves. The studies on pancreatectomized rats have already demonstrated the distribution of amylase in liver [17]. In addition, abnormal serum activity in patients with hepatic disease postulated that liver can be one of the sources of serum amylase [18]. Hepatic dysfunction as observed through increased ALT and ALP levels might be a secondary reason for decreased amylase activity in the present study. Thus, a significant decrease in the amylase activity in the present investigation might be because of reduced synthesis from damaged pancreatic and/or hepatic cells after repeated administration of APAP at high doses.
APAP gets eliminated mainly through the renal route. Thus, it is obligatory to examine the effects of its overdosing on kidney functions. Studies in the laboratory animals proposed that the overall incidence of acute renal failure after APAP poisoning is <2%, but can occur at the frequency of 10-40% in cases of severe hepatic necrosis. Renal prostaglandin production is mediated principally by cyclooxygenase and plays a pivotal role in compensatory renal hemodynamics. Severe adverse renal effects upon acetaminophen overdose might have occurred due to vasoconstriction subsequent to inhibition of renal prostaglandin-mediated vasodilatation, decreasing renal blood flow, and consequential reduction in glomerular filtration rate [19]. Hemodynamic depression following APAP administration occurs in a dose-dependent manner and it produces direct tubular toxicity through enhanced local production of the toxic quinoneimine intermediate metabolite and proximal convoluted tubular necrosis [6]. Some workers also proposed that APAP-induced nephrotoxicity due to covalent binding of reactive electrophiles [11].
BUN test is an estimate of the amount of nitrogen in the blood coming from urea, a waste product of the body. BUN levels increase in blood during renal insufficiency or high protein diet [13,20]. Blood urea nitrogen indicates the glomerular filtration but 75% of the kidney should be non-functional for BUN elevation [13]. Creatinine, a breakdown product of creatine phosphate in muscle, is an important indicator of renal health because it is an easily measured by product of muscle metabolism that is excreted unchanged by the kidneys [20]. Creatinine is considered to be an important biochemical parameter for the diagnosis of renal impairment [14]. Plasma creatinine levels help in evaluating renal functions particularly the glomeruli since creatinine is excreted in the urine after filtration. Increase in plasma BUN and creatinine levels in buffalo calves are suggestive of renal damage and may be attributed to urinary obstruction that potentiates decreased secretion of urea from the body after APAP overdose [6,7,13,14]. The BUN levels increased after the first dose with a maximum increase on day 3 followed by decreasing trend with non-significant increase on 6th day indicates kidneys compensatory mechanism to paracetamol overdose. This might be due to vasodilatation through cyclooxygenase mediated increased renal prostaglandin synthesis. Creatinine levels also followed the similar trend as that of BUN, although these were increased from day 2 onward up to day 4 and the percent increase was comparatively less than BUN.
It is well known truth that APAP overdose affect the hepatic functions but in present investigation authors evaluated its effect on second major organ of metabolism, i.e. kidney and observed renal impairment in addition to well-known hepatopathy in buffalo species as evident by alterations in the BUN and creatinine levels.
TAP, i.e. sum of both prostatic acid phosphatase (PAP) and non-PAP (NPAP) is a sensitive marker for the prostatic impairment especially prostatic adenocarcinoma in dogs [21]. The change in values of TACP in the plasma was non-significant as compared to day 0. This clearly indicates that APAP administration at high doses have a negligible effect on the prostatic function in buffalo calves.

Conclusions
Repeated administration of APAP produced hepatic dysfunction as observed by a significant increase in the activities of ALT and ALP in the plasma of buffalo calves. Significantly reduced plasma amylase activity indicates the hepatic damage in addition to acute pancreatitis. Repeated high doses of APAP also resulted in renal impairment in these animals as seen by a significant elevation in the levels of BUN and creatinine, whereas negligible effect on prostatic function.