Study of Germin Like Oxalate Oxidase Enzyme in Monocot Plants

Cell wall associated proteins and enzymes actively work to reshape the wall architecture during cell growth and wall fortification during pathogen attack. When a host plant cell detects the presence of a potential pathogen, enzymes such as NADPH oxidase, impels an oxidative burst that produces reactive oxygen species capable of damaging the invading organisms. Reactive oxygen molecules also help strengthen the cell wall by catalysing cross-linkage between cell wall polymers, thus serving as a signal to neighbouring cells. Plant cells respond to microbial attack by callose formation between the cell wall and cell membrane adjacent to the invading pathogen. (Freeman and Beattie. 2008). Plants have evolved various defence mechanisms to combat against infection by various pathogenic microorganisms (Jones and Dangl. 2006; Zhao et al., 2008). These defence mechanisms include pre-existing physical and chemical barriers, as well as inducible defence responses. The pre-existing biochemical defence mechanisms include, secretion of various defence molecules like phenolics, phenolic glycosides, unsaturated lactones, saponins, cyanogenic glycosides, glucosinolates, 5-alkylated resorcinols and dienes (Osbourn, 1996). The inducible defences include the production of reactive oxygen species (ROS), hypersensitive cell death, reinforcement of cell wall, synthesis of phytoalexins and production of pathogenesis-related (PR) proteins (Mellersh and Heath. 2004). The level of accumulation of products or the rapidity of the induction of these defence genes in a host plant is correlated with the degree of its disease resistance (Lawrence et al., 1996, Zhang et al., 2013).
 
ROS species such as O₂^- and H₂O₂ are commonly produced under stress conditions and are strong oxidizing species that can rapidly attack all types of biomolecules thereby damaging them. Plant cells contain both oxygen radical detoxifying enzymes such as catalase, peroxidase and superoxide dismutase and non-enzymatic antioxidants such as ascorbate peroxidase and glutathione-S-transferase for protecting themselves from oxidative stress. These enzymes protect the plant cells from oxidative damage at the site of ROS generation. The antioxidants, not only plays an important role in scavenging ROS, but also maintains the physiological redox status of organisms.
 
Oxalate oxidase (also known as germin), is expressed in cereal crops in response to a number of biotic and abiotic stresses including the powdery mildew infection of barley. This enzyme, characterized in some plant species including barley (Hordeum vulgare) can degrade oxalate to CO₂ and H₂O₂ (Sugiura et al., 1979; Pietta et al., 1982), but the specific biochemical interactions are yet to be established. It has been reported that wheat (Triticum aestivum) and barley oxalate oxidase, which belong to the germin family of proteins, play a role in plant defence mechanism (Lane et al., 1994). Germins which are glycoproteins by chemical nature are detected in monocotyledons such as wheat, oat, rye and barley (Grzelczak et al., 1985), are induced during germination of wheat embryos (Grzelczak and Lane, 1984). Millets also accumulate oxalate (as soluble and/or insoluble form) to potentially toxic concentrations (Dhillon et al.,1971). Oxalate is a common constituent of plants. Various studies prove that oxalate plays a pivotal role in calcium regulation, ion balance, plant protection, tissue support and heavy metal detoxification (Libert and Franceschi. 1987). The present study reports a comparative analysis of oxalate oxidase  enzyme activity from rice, wheat, maize, millets, barley. We have identified that the main objective is to compare the oxalate oxidase enzyme activity and to investigate which among them is more resistant to oxalate.

Plant material
 
The monocot plants between which a comparative study has been carried out are rice (Oryza sativa) cultivar khitish, maize (Zea mays), wheat-PBW 343 (Triticum aestivum) barley (Hordeum vulgare L.) and Foxtdl millet-4rc-484 (Setaria sp) seeds, procured from NIPGR, New Delhi, were surface sterilized and allowed to germinate for 48 hours (Fig 1), at about 30°C in dark and humid conditions till young seedlings appeared.
 

       
Then the seedlings were treated with 2mM oxalic acid (OA) on foliar tissue. The seedlings (both OA treated and control) were then harvested after 8 days for further experiments.
 
H₂O₂ content
 
The H₂O₂ content from the above mentioned monocot seedlings was measured as described by Velikova et al., (2000). 0.25g tissue was extracted with 1.25 ml of TCA (0.1%, w/v) at 48°C and homogenate was centrifuged at 12,000 rotations per minute (rpm) for 15 minutes. To 0.5 ml of the supernatant, 0.5 ml of 0.05 M sodium phosphate buffer (pH 7.0) and 1 ml of 1M potassium iodide solution were added and absorbance was measured at 390 nm.
 
Sod activity
 
Enzyme extraction procedures were carried out at 48°C. 0.5 g plant sample was homogenized in 2.5 ml of pre-chilled 0.1 M sodium phosphate buffer (pH-7.0). The homogenate was centrifuged at 12,000 rotation per minute (rpm) for 20 minutes and supernatant was used for assay. Superoxide dismutase (SOD; EC 1.15.1.1) activity was assayed by using the nitroblue tetrazolium method (Giannopolitis and Ries. 1977). The reaction mixtures contained 2.5 ml 80mM Tris-HCl buffer (pH 7.5) containing 0.12 mM EDTA and 10.8 mM TEMED, 0.1 ml 3.3×10^-3 % BSA, 6 mM NBT, 0.1 ml of 0.6 mM riboflavin and 0.1 ml enzyme extract. The reaction started by the addition of riboflavin and placing the reaction mixture under fluorescent lamp (60 mmol m^-2 s^-1) for 10 minutes. The absorbance was measured at 560 nm.
 
Catalase activity
 
Enzyme extraction procedures were carried out at 48°C. 0.5 g plant sample was homogenized in 2.5 ml of pre-chilled 0.1 M sodium phosphate buffer (pH 7.0). The homogenate was centrifuged at 12,000 rotation per minute (rpm) for 20 minutes and supernatant was used for assay. Catalase (CAT; EC 1.11.1.6) activity was determined as the amount of potassium permanganate (KMnO₄) consumed in terms of H₂O₂ (Casper and Laccoppe. 1968). The reaction mixture contained 0.5 ml of 0.1 M sodium phosphate buffer (pH 7.0), 1 ml of 3% H₂O₂ (v/v) and 1 ml of enzyme extract. After incubation, 3 ml of 10% H₂SO₄ (v/v) was added to stop the reaction. The residual H₂O₂ was titrated against 0.02(N) KMnO₄.
 
Ascorbic acid oxidase
 
Enzyme extraction procedures were carried out at 48oC. 0.5 g plant sample was homogenized in 2.5 ml of pre-chilled 0.1 M sodium phosphate buffer (pH 7.0). The homogenate was centrifuged at 12,000 rotation per minute (rpm) for 20 minutes and supernatant was used for assay. Ascorbic acid oxidase (AOX; EC 1.10.3.3) activity was measured according to (Olliver, 1967). Reaction mixture consisted of 3 ml of 0.1 M sodium phosphate buffer (pH 7.0), 1 ml of 0.025% ascorbic acid (w/v) and 1 ml enzyme extract. After 30 min of incubation the reaction was stopped by adding 5 ml of 10% TCA (w/v) and titrated with DCPIP ( 2,4-dichlorophenolindophenol) solution.
 
Estimation of phenolic content
 
Seedlings (1 g) were homogenized in 2.5 ml of 80% methanol and agitated for 15 minutes at 70°C. 1 ml of the methanolic extract was added to 5 ml of distilled water and 250 ml of Folin Ciocalteau reagent (1 N) and the solution was kept at 25°C. After 3 min, 1 ml of saturated solution of Na₂CO₃ and 1 ml of distilled water were added and the  reaction mixture was incubated for 1 hour at 25°C. The blue colour developed was measured using a spectrophotometer at 725 nm wavelength. (Zieslin and Ben-Zaken 1993).
 
Determination of PAL (Phenylalanine Ammonia Lyase)
 
Phenyl ammonia lyase (PAL) activity was studied by assaying the rate of conversion of L-phenylalanine to trans-cinnamic acid and the absorbance was measured at 290 nm, as described by Dickerson et al., (1984). Seedlings (0.25g) were homogenized in 1.25 ml of 0.1 M borate buffer, pH 7.0 containing 0.1 g polyvinylpolypyrrolidone (PVP). The homogenate was centrifuged at 10,000 rotation per minute (rpm) for 35 minutes at 48°C. The supernatant was collected and used in the enzyme assay. The reaction mixture contained 0.4 ml of enzyme extract, 0.5 ml of 0.1 M borate buffer (pH 8.8) and 0.5 ml of 12 mM L-phenylalanine in the same buffer. The reaction mixtures were incubated for 30 minutes at 30°C in a water bath and absorbance was read at 290 nm.
 
OxO Activity assay
 
In solution, detection of OxO activity was also performed by adding 80 mg of protein sample to 1 mL of OxO solution developer [40 mM succinic acid / NaOH, pH 3.8, 60% (v/v) ethanol, 2 mM OA, 5 units mL^-1 horseradish peroxidase, 20 mL/100 mL N, N dimethylaniline, 8 mg 100 mL^-1 4-antipyrine] and incubating for 2 hours at 37°C. The concentration was measured spectro- photometrically at an absorbance of 555 nm. The reaction was terminated by the addition of 20 µl of 1 M NaOH before measuring the absorbance. The spectrophotometer was normalized by protein mixed with control developer without OA.
 
Histochemical staining of leaves
 
Tissue localization of OxO activity was assayed by histochemical assay according to method described previously (Dumas et al., 1995). Sliced leaves from barley and millets (both treated and control) plants were incubated with OA (2.5 mM) in succinate buffer (25 mM succinic acid, 3.5 mM ethylene diamine tetraacetic acid, pH 4.0) containing 4-chloro-1-naphthol (0.6 mg mL^-1) as staining reagent at room temperature in the dark for overnight. As control, leaves from treated and control plants were also incubated in staining solution without OA. The leaves were then hand sectioned by a sharp blade and visualized under light microscope (Leica).

A comparative study was done among rice, maize, wheat, millet and barley to study the production of innate oxalate oxidase production on oxalic acid treatment. The enzyme was first isolated and characterized from barley (Hordeum vulgare) and wheat (Triticum aestivum) (Lane et al., 1994). Later oxalate oxidase (oxo) was isolated from many other plant species like maize (Zea mays), oat (Avena sativa), rice (Oryza sativa), rye (Secale cereale) and pine (Pinus sylvestris) (Lane, 2002).
       
The objective behind this study is to identify the monocot plant synthesizing maximum oxalate oxidase on oxalic acid treatment. Many fungal pathogens produce oxalic acid (OA) which plays important role in fungal pathogenicity. The germin oxos (g-oxos), or simply germins, are involved in cereal defense responses to invasion by fungal pathogens (Lane, 2002).
 
H₂O₂ content
 
In the graph, the plant materials under study has been kept on the x-axis and the H₂O₂ content per µg per gram fresh weight has been plotted on the y-axis. The maximum amount of H₂O₂content is found in maize as analysed from the difference between control and treated sets. The H₂O₂ content increases in treated plants compared to that of their control. H₂O₂ generated in the oxidative burst, perform multiple important functions in early defence responses of the plant. H₂O₂ has been shown to inhibit the growth and viability of diverse microbial pathogens (Peng and Kuc, 1992; Kiraly et al., 1993; Wu et al., 1977), which may directly suppress attempted invasion by the pathogens. The oxidative potential of H₂O₂ also contributes to plant cell wall  strengthening during plant-pathogen interactions through the peroxidase-mediated cross-linking of proline rich structural proteins (Bradley et al., 1992; Brisson et al., 1994). Moreover, H₂O₂ has been implicated to play a role not only in triggering hypersensitive cell death but also in limiting the spread of cell death by induction of cell protectant genes in surrounding cells (Levine et al., 1994; Tenhaken et al., 1995). H₂O₂ induces PR protein synthesis and systemic resistance (Chen et al., 1995). Fig 2 reveals that all the treated sets with oxalic acid produces more H₂O₂ compared to their control. This elevation in H₂O₂ production is due to defence response against oxalic acid.
 

 
Catalase activity
 
Active oxygen species (AOS), including hydrogen peroxide (H₂O₂), have been implicated in macrophage-mediated destruction of pathogens and in the activation of stress responses (Sohal and Weindruch, 1996). Catalase scavenges photorespiratory H₂O₂. H₂O₂ in the leaves is not strictly compartmentalized, being able to diffuse through membranes (Willekens et al., 1997). In the above graph, plant materials under study are represented on the x-axis and catalase activity (µg/g FW) is shown on the y-axis. The catalase activity in maize and millet (treated) is 8976 µg/g and 344µg/g respectively as dipicted in Fig 3. So it very evident that the catalase activity increases more for maize compared to millets. In treated sets increased catalase breaks the produced H₂O₂ (2H₂O₂ = O₂ + 2H₂O). This indicates that maize possess stronger defence mechanism in comparison to others.
 

 
Ascorbic acid oxidase activity
 
Activity of ascorbic acid oxidase is maximum in maize whereas least in wheat. Ascorbic acid oxidase (AOX) is a copper-containing enzyme available in cytoplasm and cell wall fractions, which oxidizes ascorbic acid in presence of oxygen producing dehydroascorbic acid and water. During oxidative stress, this enzyme becomes active to protect plant cells (Choudhury et al., 2011). In the above graph, the plant materials under study are shown on the x-axis whereas the ascorbic acid decomposed (µg/g FW) is shown on the y-axis. We estimated the activity of different monocot seedlings and found that there was an increase in activity of AOX after treatment in maize than in wheat, concluding that defensive response to oxidative stress is more in maize compared to other monocots.
 
SOD activity
 
Within the cell, the superoxide dismutase (SODs) constitute the first line of defence against ROS. O^2- is produced at any location where an electron transport chain is present and hence O₂ activation may occur in different compartments of cell (Elstner, 1991). Peroxisomes are the most important generators of ROS. Phospholipid membranes are impermeable to charged O^2- molecules (Takahashi and Asada, 1983). Therefore it is crucial that SODs are present for the removal of O^2- (Alscher, 2002). We estimated that the SOD activity increases in oxalic acid treated plants. In the above graph, the plant materials under study are represented on the x-axis whereas the H₂O₂ decomposed (µg/g FW) is shown on the y-axis. The activity of SOD can be analysed to be maximum in wheat considering the difference in control and treated sets. SOD activity increases in seedlings after treatment with oxalic acid. The SOD activity increased to scavenge the produced superoxide (O^2-) on oxalate treatment. The graph reveals that SOD activity increases more in barley suggesting that its defence capacity is more than others.
 
Phenolic content
 
In the above graph, plant materials under study are represented on the x-axis while the phenolic content (µg/g FW) is represented on the y-axis. Phenolics serve in plant defence mechanisms to counteract reactive oxygen species (ROS) in order to survive and prevent molecular damage and damage by microorganisms, insects and herbivores (Vaya et al., 1997). The phenolic content of the rice control, rice treated, millet control and millet treated is 864 μg, 1248 μg, 604 μg, 624 μg respectively. Fig 6 indicates that the increase in phenolic content is more in rice and least in millet. The phenolic content is comparatively more in rice than in millets conferring better resistance in rice.
 
PAL Activity (Phenylalanine ammonium lyase)
 
In the above graph, the plant materials are represented on the x-axis whereas the phenyl alanine activity (µg/g FW) is shown on the y-axis. PAL is the key enzyme of phenyl propanoid metabolism in higher plants which catalyzes the conversion of phenylalanine to transcinnamic acid which supplies the precursors for flavonoid pigments, lignins and phytoalexins (Hahlbrock and Scheel 1989). Inhibition of PAL affects subsequent biosynthetic pathways of phenolic compounds. (Carver et al., 1992). The PAL activity increases in OA treated sets. Fig 7 indicates that the increase in PAL activity after treatment is more in maize than in millets.
 

 
OxO Activity
 
In the graph, plant materials are represented on the x-axis whereas the OD value at 550 nm is shown on the y-axis. The OD 550 of maize control is 0.282 and in treated it 0.845., while the value in barley control is 0.227 and 0.351 in treated is lowest as shown in graph. The maximum activity is shown in rice and maize while it’s least in millets. The control samples developed a very faint purple colour while the treated samples developed dark purple colour. In OxO activity assay, OA treated plants showed the production of a dark blue stain due to production of H₂O₂ from oxalate and its subsequent interaction with 4-chloro-1-naphthol in the presence of horseradish peroxidase. Fig 8 reveals the leap in enzyme activity in maize for its control and experimental set is much more in comparison with others.
 
Histochemical analysis
 
The histochemical staining reveals that OA treated green leaves of both barley and millets are stained blue, while no such staining was observed in their respective controls. Interestingly, barley treated acquired a darker stain when compared to millets. In the histochemical staining (Fig 9) the blue stain was mainly found in the cut end and vein region (vascular region) of the leaves, which indicates the localization of OxO in this apoplastic region. A similar result was obtained in the case of rye grass OxO1 and wheat OxO, where most of the OxO activity was recovered in the cell wall and in the fluid of the apoplastic (Davoine et al., 2001; Walz et al., 2008). The stain in the cut end appears relatively darker. One reason for such a result may simply be due to the greater accessibility to the staining solution. Another reason may be the involvement of OxO in wound (cutting)-induced production of endogenous H₂O₂ (LeDeunff et al., 2004).
 

       
Oxalic acid is produced by several plant pathogenic fungi. These enzymes, that can catabolise oxalate, is oxalate oxidase. It has been isolated from a number of plant species including barley (Chiriboga.1966). Oxalate oxidase catalysed the degradation of oxalate to CO₂ and H₂O (Thompson et al., 1995). Reactive oxygen species (ROS) developed as a response to biotic and abiotic environmental stimuli and programmed cell death. ROS plays an important signalling role in plants. It results in the evolution of highly efficient scavenging mechanisms that overcome ROS toxicity. ROS signalling is controlled by regional production and scavenging (Mittler et al., 2006). During pathogenesis by some fungi oxalic acid (OA) is produced in advance of mycelia growth (Maxwell and Lumsden 1970; Mc-Carroll and Thor 1985). We studied the occurrence of oxalate oxidase in monocot seedlings. Several experiments were performed to determine the amount of oxalate oxidase produced in different plant species like Oryza sativa, Zea mays, Triticum aestivum, Hordeum vulgare and Secale sp. We had, instead of inoculating the plants with fungi, 2mM of oxalic acid was used, a pathogenesis factor, sprayed externally over the green leaves.
       
In H₂O₂ assay it was found that production of H₂O₂ was maximum in Zea.sp followed by Oryza sp and then millet. Experiments were also performed to determine the activity of catalase, ascorbic acid oxidase, SOD, phenylalanine and oxalic oxidase (Fig 4, 5 and 6) among the plant species considering both control and treated plant, similar result is found, that Zea sp produces the maximum amount of enzyme among all the plants. The principal H₂O₂ -scavenging enzyme in plants is catalase acting on substrate H₂O₂, thereby liberating oxygen and water (Willekens et al., 1997). The two fold increase in catalase activity in maize is the requirement for cleavage of high amount of substrate H₂O₂. The increase in catalase activity is more in maize with respect to others, which can be contributed to the fact that H₂O₂ content in OA treated maize is greater than other plant materials used in this study. The expression of ascorbic acid oxidase was studied in zucchini squash (Cucurbita pepo L.) by (Lin et al., 1990). It is one of the most abundant natural sources of the enzyme.
       

 

 

 
Ascorbate is a well-known scavenger for active oxygen species, such as superoxide, hydroxyl radical and hydrogen peroxide, especially in the chloroplast. (Lin et al.,1990). From our experiment we see that there is a significant rise in the activity of ascorbic acid oxidase in maize but a low rise in millets and wheat. So it can be concluded that maize is more resistant to oxidative stress than millets. The SOD activity is much more in maize in comparison with others considering rice that showed lowest activity. This result is probably due to the relation between this compound, i.e. the radical scavenging activity of SOD, is effective only when it is followed by increase in activity of catalase and other peroxidises. SOD generates H₂O₂ as a product which is in turn more toxic to the cells and requires catalase or peroxidases to scavenge. Thus a concomitant increase in catalase and or peroxidase enzyme is essential to counteract the superoxide dismutase activity. Zea mays exhibited significantly greater catalase activity, SOD, oxalate oxidase and ascorbic acid oxidase.
       
Plant phenolics are secondary metabolites, which are involved in the defense mechanisms of plants against fungal pathogens. Phenol content of a plant increases under biotic stress. Among the five plant species, Oryza sativa showed increased activity with respect to Triticum aestivum, Hordeum vulgare, millets and Zea mays. Phenolics are able to alter peroxidation kinetics by modifying lipid packing order, thereby stabilizing membrane fluidity, hindering the diffusion of free radicals and restricting peroxidation reaction. There was a significant increase in the concentration of the total phenolic content in the OA treated plants, compared with the control. This indicates that upon pathogen attack, the plant phenolic content increases as a result of defence mechanism.
 
A sharp increase was observed in enzymatic activity of phenylalanine ammonia lyase (PAL) in the plants species that were treated with oxalic acid when compared with the control plants. Phenylalanine ammonia-lyase (PAL) has a crucial role in secondary phenylpropanoid metabolism and responses to biotic and abiotic stress. PAL plays an important role in plant defence and is involved in the biosynthesis of salicylic acid (SA), which is an essential signal involved in plant systemic resistance. Therefore their level of expression reflects the occurrence of stress condition. Among the five monocot plants Zea mays showed the highest production.
 
A significant variation is also noticed in case of solution assay of OxO activity among the plant species considering both the control and treated sets. Oryza sativa and Zea mays with the highest production of H₂O₂ followed by Triticum aestivum and with lowest production is of millets (Fig 8).
 

 
An anatomical study was performed to study the activity of oxalate oxidase assay between the treated and control sets of three plant species. The oxalate oxidase, found in the leaf tissue plays a central role for the regulation of the hypersensitive response. During pathogen-plant interactions, the generation of AOS has been considered as an important phenomenon (Baker and Orlandi. 1995; Low and Merida. 1996). Among the five plant species it was found that a dark-blue stain was observed after incubation in the developer solution containing oxalic acid, that is, H₂O₂- requiring staining reaction is dependent on oxalic acid which strongly indicates specificity for oxalate oxidase. However, no such staining was observed in the control sets.
 
It was observed that the maximum experiments carried out gave the high abundance of oxalate oxidase enzymatic activity in Zea mays mostly signifying a strong defence mechanism to combat against biotic and abiotic stress. Triticum aestivum also proved to have a good defence mechanism with respect to Oryza sativa and Hordeum vulgare while millets showed poor response, exhibiting to have a weak defence mechanism with respect to others. The highest activity of oxalate oxidase in Zea mays is efficient in imparting resistance against pathogens secreting oxalic acid and it can be used for generating transgenic crops with better resistance.