Isolation and in-silico characterization of Peroxidase isoenzymes from Wheat (Triticum aestivum) against Karnal Bunt (Tilletia indica)

To investigate the role of Peroxidase and its physiological significance under Karnal Bunt (KB) were determined in resistant (HD-29) and susceptible genotype (WH-542) of wheat during different developmental stages. The enzymes were expressed constitutively in both the susceptible and resistant genotype. In gel assay and differential expression analysis of POD was significantly higher (p >0.05) in Sv and S2, than the S1 and S3 stages. in silico analysis of Peroxidase for eg. physico-chemical properties, secondary structural features and phylogenetic classification for comparative analysis. Motif and Domain analysis of Peroxidase by MEME, to be important for the biological functions, and studies of evolution. Our results clearly indicate that the enhanced expression of POD at the WS2 stage, which reinforces its role in stage dependent immunity against Karnal bunt and role of POD metabolism provides genotype and stage dependant structural barrier resistance in wheat against KB.

widespread in the plant kingdom and POD isoenzymes are known to occur in a variety of plant tissues.The pattern of expression of each isoform varies in the different tissues of healthy plants and is developmentally regulated and influenced by environmental factors [2]; however, the role of each isoform is not fully understood.Various experiments suggest the involvement of plant POD not only in biosynthetic processes related to wall development such as lignification [3], suberization [2], and polymerization of hydroxyproline-rich glycoproteins [4], but also in the regulation of cell wall elongation [5] and wound healing [1].Also, the POD are sometimes listed as the class of pathogenesis related proteins (PR proteins), increases upon viral, bacterial, or fungal infection and resistance against infection by pathogens [5-9].Such a wide spectrum of functions is consistent with the presence of several isoforms suggesting that different POD isoenzymes might be involved in distinct processes.Moreover, POD can be considered useful markers for environmental stresses since their activity is affected by low temperature, air pollution, ozone, heavy metals, wounding, salts, UV radiation, and pathogen attack [10].In view of the above, present investigation has been undertaken to characterize the genotype and organ, stage dependent immunity using biochemical and in silico analysis during different developmental stages of resistant and susceptible wheat spikes.

Methodology: Collection of Wheat Genotypes
In present study two genotypes (WH-542, HD-29) of bread wheat (Triticum aestivum) were used.The seeds of these genotypes-one highly susceptible and another resistance to Karnal bunt based on pathogenecity testing under field conditions were collected [11].Different stages of developing wheat spikes were selected on the basis of % severity of KB induced by inoculating at different growth stages the data [11].
In gel Assay 10% native gel was prepared and sample was loaded with loading dye, gel was run for 2 hour at 80 mv.POD analysed after electrophoresis in PAGE with the use of Guaicol as a hydrogen donor.Gel was incubated in 0.02M guaicol for 30 minute, washed and then immersed in 0.01M H2O2 for band development [12].A densitometry analysis was done with the help of Gene Profiler software, Alpha Innotech Corporation USA.Briefly, individual gels were scored by placing the curser over individual band and recording the relative densitometry values of gels used for expression analysis [11].

Results & Discussion:
Karnal bunt (KB) caused by Tilletia indica (Syn.Neovossia indica) is an economically important disease of wheat.POD, a class of pathogenesis related proteins (PR proteins), increases upon fungal infection and resistance against infection by pathogens.Therefore, it is important to differentiate the resistant and susceptible genotypes based on expression of POD at, biochemical and also in silico analysis of different plant POD was carried out by using various computational tools.

In gel analysis of POD
In order to ascertain the role of basal expression level of enzyme POD was analyzed using in gel assay at different physiological stages of wheat spikes of resistance and susceptible genotypes.POD isoenzymes were electrophoreses on native-polyacrylamide gels and stained with guaicol and hydrogen peroxide (Figure 1).The native gel analysis showed that there were three different bands present in both the genotypes.Differential expression analysis of POD was carried out by using Gel documentation system, POD activity was somewhat increased significantly dominant in resistant genotype than susceptible.Surprisingly, POD2 isoenzyme was significantly increased in resistant genotype, suggesting that this isoenzyme is most likely involved in defense mechanism.The expression of POD was significantly higher (p<.05) in Sv and S2 stage.There was decreasing trend up to S3 stages of both genotypes indicating the enhanced lignification of cell walls in developing spikes after fertilization and grain development.During earlier stages, higher level of expression of defense enzymes and accumulation of chemical at vegetative leaf stages (Sv) certainly prevent the fungal mycelial colonization in leaves.POD activity in plants can increase in response to a variety of stresses including biotic stress, indicating that POD activities have been suggested to be involved in, cell wall biosynthesis by the polymerization of cinnamyl alcohol into lignin, in defense against attack by pathogens [14], and in the response to wounding [15].In addition, POD catalyzes polymerization of naturally occurring phenolics to produce a variety of bioactive products.POD activity is frequently increased in plant infected by pathogen, and the level of its activity is clearly correlated with disease resistance [15].The POD expressed constitutively in both susceptible and resistant genotype.However, the activity was higher in resistant genotype indicating that resistant genotype has significant high basal level of POD as compared to susceptible line and could be used as marker to define KB resistance.Hence, the S2 stage of wheat spike of susceptible genotype is more prone to KB infection as compared to resistant genotype.

In silico Study of Peroxidase
Secondary structure properties, PDB Structure, Motif and Physicochemical characterization, for all the Peroxidase, were carried out by using various computational tools.SOPMA analysis was done for Peroxidase in all monocot selected plants and it showed a alpha helix occupied the largest part of the protein followed by, random coil, extended strand and beta turns, while in dicoty plant random coil occupied the largest part of the protein followed by, random coil, extended strand and beta turn Table 2 (see supplementary material), PDB structure also showed (Figure 2).High value for random coil bears important significance in the study of protein tertiary structure and related functions.Functional analysis of these proteins includes identification of important motifs Table 3

(see supplementary material).
There is two important motif found in both monocot and dicot plants first motif PS00436 and PS00435.PS00436 occur at starting sequence like 57-67 and PS00435 is found at middle sequence like 177 -187.These motifs were 10 to 20 amino acids in length arise because specific residues and regions proved to be important for the biological function of a group of proteins, which are conserved in both structure and sequence during evolution.The ProtParam tool (http://web.expasy.org/protparam/) of ExPASy was used to compute glycine and proline composition (%), in the selected monocot plants.It was observed that the average percentage of glycine was higher than the proline.When compared the glycine and proline contain with in Poaceae family, Setaria italic have highest glycine contain while in Triticum urartu a diploid variety of Triticum have highest contain of proline.The amino acid proline is known to occur widely in higher plants and normally accumulates in large quantities in response to environmental (biotic and abiotic) stresses (Figure 3).The results of transgenic modifications of biosynthetic and metabolic pathways indicate that higher stress tolerance and the accumulation of compatible solutes may also protect plants against biotic and abiotic stress.The osmoprotectant role of proline has been verified in some crops by overexpressing genes involved in proline synthesis.The total number of positively (Arg + Lys) and negatively (Asp + Glu) charged residues of Peroxidase members were observed Table 4 (see supplementary material).Peroxidase showed both positively and negatively charged nature, varies with their Isoelectric point.Some monocot plant Peroxidase showed higher positively charged like Triticum aestivum, Cenchrus ciria, Avena, and Asparagus.This possible variation might be due to their isoelectric point in acidic range.For the remaining members, the isoelectric point was within alkaline range.For the separation of the protein on a polyacrylamide gel the computed isoelectric point will be useful.Extinction coefficient for all Peroxidase was observed with in a same range except Avena and Medicago.High extinction coefficient means higher concentration of lysine, tryptophan and tyrosine.for the calculation of protein concentration in the solution the extinction coefficient can be useful.Stability of protein is described in terms of its stability index whether a protein is stable or not, can be described by its instability index.Instability index of root peroxidase of Triticum aestivum, and Cucumis is higher than 40 and thus describing these proteins unstable.It is noteworthy that high aliphatic index was observed for all plants Peroxidase.The higher aliphatic index indicates higher concentration of alanine, valine, isoleucine and leucine occupying the relative volume of a protein.Grand Average of Hydropathy (GRAVY) was computed for all the members.GRAVY index indicate the solubility of protein and its range was observed from 0.171 to -0.089 in selected plants.
A positive and negative GRAVY value for POD designates it to be hydrophobic and hydrophilic in nature of Peroxidase respectively.Most of selected plant a negative GRAVY value for POD designates it to be hydrophilic in nature but diversity occurs in nature (Table 4).

Domain and MEME Analysis
In silico studies revealed that peroxidase belong to the Domains (functional and/or structural units of a Protein) cl00196 (CDD accession).Heme-dependent peroxidases similar to plant peroxidases (Figue 4) these enzymes belong to a group of peroxidases containing a heme prosthetic group (ferriprotoporphyrin IX), which catalyzes a multistep oxidative reaction involving hydrogen peroxide as the electron acceptor.Peroxidases are found in the extracellular space or in the vacuole in plants where they have been implicated in hydrogen peroxide detoxification, auxin catabolism and lignin biosynthesis [16], and stress response [17].Multiple Expectation-Maximization for Motif Elicitation (MEME) [15] is a suite of tools for motif discovery and searching.Twenty five different motifs (subdomain) between 6 and 50 residues were detected and distributed by MEME software.One to ten motifs sharing by almost all the plant groups with the few exception (Figure 5).Motifs one are almost conserved and found in every group and subgroup.These conserved motifs could be the essential elements determining the POD family's common molecular function among different plant species.Motif 19 absent all the monocotyledon plants.When compared a most conserved motifs in a Triticum group, Motifs 9 absent only in T.

Phylogenyn analysis of Peroxidase of Triticum aestivum with other Plants
Homology analysis depicts two main clusters A and B (Figure 6).On the basis of phylogenetical analysis it can conclude that overall 51.95% homology is present in monocot plants.From the thirteen species, eleven species present in cluster A and is divided in two sub-clad (A1 and A2) on the basis of homology.

Conclusion:
The function of POD in plant defense range from pre-formed or inducible.POD activity was significantly dominant in resistant genotype than susceptible.The expression of POD was significantly higher (p<.05) in S2 stage and in subsequent stages.
There was increasing trend in Sv and S2 stages followed by decrease in S3 stage of both genotypes indicating the enhanced lignification of cell walls in developing spikes after fertilization and grain development.In this study, physico-chemical properties, secondary structural features and phylogenetic classification will provide an insight for the biologists working with POD in order to understand the functionality of enzymes.
The POD is expressed constitutively in both susceptible and resistant genotype.However, the activity was higher in resistant genotype indicating that resistant genotype has significant high basal level of these enzymes as compared to susceptible line and could be used as marker to define KB resistance.

Figure 1 :
Figure 1: In Gel analysis of wheat POD at different stages (Sv, S1, S2 and S3) of developing wheat spikes in Resistant and Susceptible genotypes.

Figure 2 :
Figure 2: PDB structure of POD in selected plants.

Figure 3 :
Figure 3: Glycine and Proline percentage of POD in selected plants aestivum, Motif 5 absent in root part of T. aestivum.While Motifs 3&6 and2 & 10 absent in T.monoccocum and T. ururtus.Motif 16, 19 occurs only found in Brassicaceae plants.Unique and Absence of Motifs are either, substitution, accumulation of mutation or subjected to rearrangements.It is not necessary that changes their activity, because that do not have a direct impact on the active site contain altered residues.

Figure 4 :
Figure 4: Functional domain analysis of POD in selected plants.

Figure 5 :
Figure 5: Schematic diagram of motif distribution of POD Proteins.MEME4.6.0 was applied to show that different sub groups were distinguished by the motif distribution.
Cluster A, first group include Cenchrus, Zea mays, Triticum, Oryza Avena, consist 64 % homology and other that include, Avena sativa, Triticum aestivum , root peroxidase Triticum aestivum, Hordeum and Hordeum vulgare subsp.vulgare consist 77% homology.When homology determine in poacease family with respect to Triticum aestivum it observe that 84% homology present in peroxidase taken from root and leaf.Cluster B also divided in to sub-clad (B1 and B2).Monocot four species like Triticum urartu, Setaria italica, Spirodela polyrhiza, and Asparagus present in Clustur B along with dicot plants.This result suggests that POD genes transfer from dicotyledonous plants to monocotyledonous or vice versa.

Figure 6 :
Figure 6: Homology tree of POD with selected plants.

Table 1 :
Selected Monocotyledonous and Dicotyledonous plant with Accession Number

Table 2 :
Secondary structure prediction of Peroxidase through SOPMA

Table 3 :
Motif prediction of Peroxidase in selected plants

Table 4 :
Various parameters computed using Expasy's ProtParam tool of Peroxidase in selected plants