Oxidative enzyme changes in sorghum infested by shoot fly.

This research investigated the role of oxidative enzymes in the defense response of sorghum, Sorghum bicolor (L.) Moench (Poales: Poaceae), to the sorghum shoot fly, Atherigona soccata Rondani (Diptera: Muscidae). Changes in polyphenol oxidase and peroxidase activity and total protein content were observed in resistant and susceptible sorghum genotypes in response to A. soccata feeding. Resistant plants exhibited higher levels of peroxidase and polyphenol oxidase activities and total protein content compared with susceptible plants. Peroxidase and polyphenol oxidase activities and total protein content in the infested resistant and susceptible genotypes were higher when compared with their control plants, respectively. These findings suggest that resistant genotypes may be able to tolerate shoot fly feeding by increasing their peroxidase and polyphenol oxidase activities. Among the enzymes examined, differences in isozyme profiles for peroxidase and polyphenol oxidase were detected between control and infested IS 18551, M35-1, 296B, SSV 84, and DJ 6514 plants. Differences in protein profiles were observed between A. soccata infested and their respective uninfested controls of all the genotypes. In conclusion, this study revealed that these defense enzymes and proteins might attribute to the resistance mechanisms in sorghum plants against A. soccata infestation.

Sorghum bicolor (L.) Moench (Poales: Poaceae) is one of the most important crops in the world because of its adaptation to a wide range of ecological conditions, suitability for low input cultivation, and diverse uses ( Doggett 1988 ). Globally, sorghum is planted in 42 million hectares with an annual production of 58.5 million tons. India is a major sorghum-producing country in the world with an area of 8.5 million hectares and production of 7.2 million tons ( FAO 2009 ). Productivity of sorghum in India is very low (783 kg/ha) compared with global yield (1373 kg/ha). Most of yield loss is due to biotic stress ( Dhillon et al. 2005 ). The sorghum shoot fly, Atherigona soccata Rondani (Diptera: Muscidae), is one of the most destructive insect pests of sorghum in Asia, Africa, and Mediterranean Europe during the early stages of crop growth and establishment ( Sharma 1993 ). The typical dead heart symptom produced by A. soccata larvae is the result of death and drying of central whorl leaves that contain an active growing shoot apical meristem. The tillers produced as a result of loss of apical growth are also frequently attacked by A. soccata , resulting in a complete stunted growth leading to tremendous grain and forage yield losses. Worldwide, sorghum yield losses were estimated at US$274 million from A.soccata damage ( Sharma 2006 ). In India, the losses due to A. soccata damage have been estimated to reach as high as 90% of grain and 45% of fodder yield ( Sukhani and Jotwani 1980 , Jotwani 1982 ). The pest is especially serious in late-sown crops, but sometimes appears with early sowing also, when the preceding dry season is interrupted by frequent showers of rain ( Nimbalkar and Bapat 1987 ). The levels of infestation may go up to 90–100% under delayed sowing ( Hiremath and Renukarya 1966 ). Although these are old estimates, similar yield losses occur even today because of the lack of acceptable levels of genetic tolerance/resistance to this insect pest in parental lines, compounded further with unacceptability of chemical control measures by the farmers ( Padmaja et al. 2010a , b ).

Plant resistance offers a promising approach for managing A. soccata because it is sustainable, economical, and environmentally responsible. When developing insect-resistant crops, a thorough understanding of the underlying mechanisms of the resistance is critical for formulating optimal strategies for identifying and exploiting resistant sources. Although considerable progress has been made in identifying germplasm resistant to A. soccata , progress toward characterization of the physiological and biochemical mechanisms conferring the resistance remains limited. Plant chemistry is probably the most important source of information contributing to the final decision by an insect to oviposit or not and depends on the balance of opposing positive and negative cues evoked by phytochemicals that determine whether a plant is accepted or rejected by a herbivore ( Padmaja et al. 2010a , b ). Modifications in plant protein profiles and alterations in plant oxidative enzyme levels have been reported to be among a plant’s first response to insect herbivory ( Green and Ryan 1972 ; Hildebrand et al. 1986 ; Felton et al. 1994a , b ; Miller et al. 1994 ; Rafi et al. 1996 ; Stout et al. 1999 ; Chaman et al. 2001 ; Ni et al. 2001 ). These enzymes, because of their potential roles in plant signaling, synthesis of defense compounds, and/or oxidative stress tolerance, have been implicated in plant resistance to insect herbivores. Thus, this study was carried out with an objective of assaying defense enzymes and total proteins in sorghum plants and their role in host resistance to A. soccata infestation. More specifically, we opined that if the plant resistance is modified by herbivore feeding, it results in induced resistance. Thus, we measured the variations in induced defense responses in sorghum genotypes against herbivore feeding.

Material and Methods

Field Evaluation for A. soccata Resistance

A total of five sorghum genotypes, namely, IS 18551 (a stable A. soccata- resistant germplasm line), M35-1 (most popular rabi variety), DJ 6514 ( A. soccata -susceptible genotype), 296B (most popular seed parent of kharif hybrids), and SSV 84 (sweet sorghum variety), were evaluated at Directorate of Sorghum Research, Hyderabad, India (latitude 17° 19′28.5″ N and longitude 78° 24′13.4″ E) at an altitude of 524 above mean sea level during two rainy ( kharif ) and two postrainy ( rabi ) seasons for A. soccata resistance during 2011 and 2012. The fish meal technique was used for creating optimum levels of A. soccata infestations in the experimental field ( Soto 1974 ). The test material was planted in 4-m row plot and the rows were 60-cm apart. The experiment was carried out in a randomized complete block design with five replications. The plants were thinned at 7 d after seedling emergence (DAE) to maintain a spacing of 10 cm between plants. Overall resistance was recorded as the percentage of dead hearts (DH%) caused by A. soccata infestation. Plants with dead hearts were recorded in all the plots at 28 DAE. The DH% (ratio of the number of dead hearts to the total number of plants × 100) recorded at 28 DAE was used for evaluating resistance.

Sample Collection

Sorghum seedlings (leaves and stems) (1 g) of each of the five genotypes infested with A. soccata were collected from field for protein analysis at 21 DAE. Plants without dead heart symptom served as uninfested control.

Preparation of Samples

Samples were prepared for protein assay following the protocol by Hildebrand et al. (1986) . Soluble proteins were extracted by grinding plant tissues in a chilled mortar with 3.0 ml of 20 mM 4-2-hydroxy-methyl-1-piperazineethane sulfonic acid buffer (HEPES) buffer (pH 7.2) containing a protease inhibitor cocktail [0.3 g/1 g of tissue contains 4-(2-aminoethyl)benzenesulfonyl fluoride, bestatin, pepstatin A, E-64, leupeptin, and 1,10-phenanthroline] and 1% polyvinylpyrrolidone. The homogenate was centrifuged at 10,000 ×  g for 10 min at 4°C. The supernatant was collected and stored (<3 h) at 4°C until protein analyses.

Protein and Enzyme Assays

The effect of A. soccata feeding on plant protein content and enzyme (peroxidase [POX] and polyphenol oxidase) activities were examined using a spectrophotometer. Total protein content was measured with bovine serum albumin as a standard ( Lowry et al. 1951 ). POX activity was measured by monitoring the increase in absorbance at 470 nm for 1 min by using a protocol by Hildebrand et al. (1986) and Hori et al. (1997) . The enzymatic reaction was started by adding 10 µl of 30% hydrogen peroxide to a cuvet containing 300 µl of 18 mM guaiacol, 100 µl of 200 mM HEPES (pH 7.0), 585 µl of distilled water, and 5 µl of sorghum extract. The specific activity of POX was determined using the molar absorptivity of guaiacol at 470 nm (26.6 × 10 3 /M/cm). Polyphenol oxidase activity was measured following a protocol modified from Hori et al. (1997) . Polyphenol oxidase activity was monitored at 470 nm for 1 min after the start of the reaction. The reaction was initiated by adding 20 µl of sorghum extract to a cuvet containing 500 µl of 1.6% catechol in HEPES buffer, 380 µl of distilled water, and 100 µl of 200 mM HEPES buffer (pH 6.0). Polyphenol oxidase activity was calculated as the change in A470 per minute milligram of protein.

Native Gel Electrophoresis

Samples were analyzed for isozyme expression by native gel electrophoresis using precast 12-well 12.0% polyacrylamide gels. A continuous buffer system of Tris–glycine (3 g of Tris-base, 14.4 g of glycine, and 1,000 ml of distilled water) (pH 8.3) was used. Equal amounts of protein as determined by the BSA protein assay were loaded in each lane. Samples were diluted 1:1 with a gel loading buffer consisting of 62.5 mM Tris–HCl (pH 6.8), 40% glycerol, and 0.01% bromphenol blue before loading. Gels were electrophoresed at 120 V for 1.5 h at 4°C. Isozyme profiles for POX and polyphenol oxidase activity were visualized using histochemical methods. All gels were evaluated for the presence or absence of bands after incubation and staining. The incubation and staining procedures for the POX and polyphenol oxidase were adapted from Vallejos (1983) .

POX Staining

Gels were soaked at room temperature (25°C) for 10 min in 50 mM sodium acetate buffer (pH 5.0). After this initial incubation period, 10 mg of 4-chloronaphthol (dissolved in 0.5 ml of methanol) and 20 µl of 30% hydrogen peroxide were added to the buffer. Zones of POX activity occurred as blue bands after 15 min.

Polyphenol Oxidase Staining

Gels were soaked at room temperature for 30 min in 20 mM HEPES buffer (pH 7.2) containing 5 mM DL-ß-(3,4-dihydoxyphenyl) alanine. Polyphenol oxidase activity was observed as dark brown bands on a clear background after 30 min of incubation ( Cheung and Willetts 1973 ).

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

Samples were separated in 12% gels by using the procedures outlined by Laemmli (1970) . A continuous buffer system of Tris–glycine–sodium dodecyl sulfate (SDS) (3 g of Tris-base, 14.4 g of glycine, 1 g of SDS, and 1,000 ml of distilled water) (pH 8.3) was used. Samples were diluted 1:1 with a gel loading buffer consisting of 62.5 mM Tris–HCl (pH 6.8), 0.01% bromphenol blue, 25% glycerol, 5% β-mercaptoethanol, and 2% SDS before loading. The protein samples were then heated for 5 min at 95°C before loading on the gel. Equal amounts of protein were loaded in each lane. Electrophoresis was conducted at 120 V for 2 h at room temperature. Proteins were visualized by Coomassie brilliant blue staining according to standardized methods. The molecular weights of the different protein bands in each sample were determined from the standard curve drawn between log molecular weight and relative mobility.

Statistical Analysis

The data obtained from field experiments were subjected to analysis of variance using the statistical software Windostat ( Indostat services 2004 ). Genotypes were used as fixed effects and blocks and years as random effects. LSD was used to compare the treatment means. Enzyme activity values were analyzed using one-way Analysis Of Variance (ANOVA) using “PROC GLM.” The means were separated using Tukey’s Honest Significant Difference (HSD) test ( SAS Institute Inc. 2009 ).

Results

Evaluation of Resistance to A. soccata Under Field Conditions

There were significant differences among the genotypes for dead heart formation in both the seasons (rainy and post rainy) during both the years (2011 and 2012) ( Table 1 ). M35-1 was as good as the resistant genotype IS 18551 for A. soccata dead hearts, whereas the dead heart formation was greater on the genotypes 296B, DJ 6514, and SSV 84 ( Table 1 ).

Table 1.

Performance of genotypes resistant to shoot fly Atherigona soccata in sorghum

Genotypes	Dead hearts (%)
	Kharif 2011	Kharif 2012	Rabi 2011	Rabi 2012
296B	88.1 ± 2.1	88.5 ± 1.3	75.3 ± 2.4	76.7 ± 2.9
IS 18551	32.9 ± 1.2	33.0 ± 1.3	23.7 ± 1.4	24.5 ± 1.7
SSV 84	71.4 ± 3.2	73.6 ± 3.2	73.1 ± 3.1	68.6 ± 3.1
DJ 6514	82.6 ± 1.9	84.9 ± 1.6	79.7 ± 0.3	78.3 ± 1.8
M35-1	40.1 ± 0.8	36.6 ± 1.4	32.9 ± 0.9	32.9 ± 1.1
Mean	63.2	63.3	56.9	56.2
CV***	6.9	6.8	6.7	8.0
SEM	1.94	1.93	1.69	2.02
F ratio	167.4	190.5	243.6	160.2
P	0.0000	0.0000	0.0000	0.0000

Treatment means were compared using Least Significant Difference (LSD).

df = 4, 20.

Protein Assays

Infested plants showed an overall increase in total protein content compared with uninfested plants in all the five genotypes ( Fig. 1 ). A. soccata -resistant genotypes IS 18551 and M35-1 had a higher protein content compared with the susceptible genotypes 296B and DJ 6514 ( t  = −15.0; df = 48; P  ≤ 0.0001). However, SSV 84 recorded highest protein content (10 mg/g fresh weight).

Fig. 1.

Total protein content in sorghum genotypes infested by Atherigona soccata.

Enzyme Assays

A. soccata -infested IS 18551, M35-1, 296B, DJ 6514, and SSV 84 plants had higher levels of POX activity compared with their control plants ( t  = −11.98; df = 48; P ≤  0.0001) ( Fig. 2 ). A. soccata -resistant genotypes IS 18551 and M35-1 had a higher POX activity compared with the susceptible genotypes 296B, SSV 84, and DJ 6514. The greatest difference in POX activity between infested and control 296B and SSV 84 plants was observed. A. soccata -infested IS 18551 showed approximately twofold increase in POX activity.

Fig. 2.

POX-specific activity in sorghum genotypes after infestation by A. soccata.

Polyphenol Oxidase Activity

A. soccata -infested IS 18551, M35-1, 296B, DJ 6514, and SSV 84 plants had higher levels of polyphenol oxidase activity compared with their control plants ( t  = −3.66; df = 48; P  = 0.0006) ( Fig. 3 ). A. soccata -resistant genotype IS 18551 had a higher polyphenol oxidase activity compared with the susceptible genotypes 296B, SSV 84, and DJ 6514.

Fig. 3.

PPO-specific activity in sorghum genotypes after infestation by A. soccata .

Isozyme Profile Studies

Native gel electrophoretic separation of enzyme extract from different genotypes of sorghum plants showed different Peroxidase (PO) isoform patterns. Native gels stained for POX-specific activity showed four isozymes (PO1, PO2, PO3, and PO4) in 296B plants infested with shoot fly, whereas two isoforms (PO1 and PO2) in control plants ( Fig. 4 A). Two of them (PO3 and PO4) were induced after infestation with shoot fly compared with their controls. In contrast, resistant genotype IS 18551 expressed two isoforms (PO1 and PO4) in both infested and control plants. There were four POX isozymes in SSV 84, DJ 6514, and M35-1 plants infested with A. soccata with visible difference between infested and control plants ( Fig. 4 A).

Fig. 4.

(A) Native gel stained for peroxidase activity. Lane 1, 296B control; lane 2, 296B infested; lane 3, IS 18551 control; lane 4, IS 18551 infested; lane 5, SSV 84 control; lane 6, SSV 84 infested; lane 7, DJ 6514 control; lane 8, DJ 6514 infested; lane 9, M35-1 control; and lane 10, M35-1 infested. (B) Native gel stained for polyphenol activity. Lane 1, 296B control; lane 2, 296B infested; lane 3, IS 18551 control; lane 4, IS 18551 infested; lane 5, SSV 84 control; lane 6, SSV 84 infested; lane 7, DJ 6514 control; lane 8, DJ 6514 infested; lane 9, M35-1 control; and lane 10, M35-1 infested.

Native gels stained for polyphenol oxidase-specific activity showed four isozymes in 296B, SSV 84, DJ 6514, and M35-1 plants infested with A. soccata , whereas there were only two isoforms in respective control plants ( Fig. 4 B). Two of them were induced after infestation with A. soccata compared with their controls. In contrast, resistant genotype IS 18551 expressed two isoforms in both infested and control plants.

SDS Gel Electrophoresis

Electrophoretic separation of proteins from five genotypes of sorghum plants showed different protein profiles ( Table 2 ). Differences in protein profiles were observed between A. soccata -infested 296B, IS 18551, M35-1, DJ 6514, and SSV 84 plants and their respective uninfested controls ( Table 2 ). In resistant genotype IS 18551, no bands were stained in A. soccata -infested plants.

Table 2.

Protein profile of different genotypes of sorghum infested with shoot fly

Molecular weight (kDa)	Sorghum genotypes
	296B		IS 18551		SSV 84		DJ 6514		M35-1
	C	I	C	I	C	I	C	I	C	I
112.2					+				+	+
104.7						+	+
95.5	+	+	+
63.1	+					+	+		+	+
25.1										+
22.9					+	+	+	+

+ = stained bands; C = control plants; I = infested plants.

Discussion

An increase in the activities of phenolic-related enzymes and the accumulation of phenolic compounds has been correlated with resistance of cereals to biotic stresses ( Mohammadi and Kazemi 2002 ). Plant resistance to biotic and abiotic stresses is often regulated by the metabolism of phenolic compounds. Sorghum phenolic compounds, or allelochemicals, are involved in plant resistance to all kind of stresses ( Lo et al. 1999 , Weston et al. 1999 , Weir et al. 2004 ). POXs play an important role in stress-related resistance. One of the important physiological roles of POXs is the synthesis of cell-wall polymers (lignin and suberin), which constitute physical barriers for both biotic and abiotic stresses ( Cosgrove 1997 ). In sorghum, POXs are involved in thermal tolerance ( Choudhary et al. 1993 ) and resistance to fungal infection ( Luthra et al. 1988 ). Polyphenol oxidases play an important role in plant defense via the oxidation of endogenous phenolic compounds into o-quinones, which are toxic to invading pathogens and pests ( Mohammadi and Kazemi 2002 ).

In this study, we examined A. soccata feeding-induced damage on sorghum plants and its subsequent effects on the plant biochemical and enzymatic changes. Infestation by A. soccata increased the total extractable protein content of five sorghum cultivars. In most of the insect-wounded plants due to herbivory, there has been quantitative increase in the levels of biochemicals such as proteins, phenols, and carbohydrates and an increase in enzymes activities. Ni et al. (2001) reported an increase in total protein in wheat infested with the Russian wheat aphid, Diuraphis noxia (Mordvilko), and the corn leaf aphid, Rhopalosiphum padi (L.). We found that shoot fly-resistant cultivar IS 18851 had nearly twofold increase in POX-specific activity after infestation compared with uninfested plants. Plant oxidative enzymes (e.g., POX, polyphenol oxidase, and catalase) play an important role in the plant’s response to biotic and abiotic stresses ( Van Loon 1976 ; Castillo et al. 1984 ; Hildebrand et al. 1986 ; Felton et al. 1994a , b ; Zhang and Kirkham 1994 ; Stout et al. 1999 ; Chaman et al. 2001 ; Ni et al. 2001 ).

Our enzyme activity assays and protein profiles suggest that A. soccata feeding leads to a loss in POX activity in susceptible sorghum genotypes. Tolerant genotypes, however, may be able to tolerate A. soccata feeding by increasing their POX activity. Hydrogen peroxide is thought to be produced in response to plant stress such as insect feeding ( Dowd and Lagrimini 1997 ). The level of hydrogen peroxide is mediated by the presence of oxidative enzymes such as POX and catalase ( Levine et al. 1994 , Mehdy 1994 , Allen 1995 ). Hildebrand et al. (1986) suggested that increased POX activity in resistant plants may allow the plant to detoxify peroxides and therefore sustain less tissue damage than susceptible plants. POX is also involved in lignin synthesis in cell walls. Lignification can be beneficial to the plant because it serves to strengthen and reinforce cell walls ( Fincher and Stone 1986 ). The synthesis of lignin in response to insect feeding may provide cell wall reinforcement and thereby increase the plant’s tolerance to insect feeding. The increase of the Peroxidase (POD) activity in herbivore-damaged plants can be attributed to the fact that these are the key enzymes that participate in several plant cell wall building processes ( Chittoor et al. 1999 ). The final products of such enzymatic activities would be considered antinutritive because they cannot be effectively digested and assimilated by insects ( Constabel 1999 ). From the above evidences, it is assumed that the activity of PO might attribute to the reduced A. soccata damage and their preference to sorghum seedlings.

In our studies, all the five genotypes had higher levels of polyphenoloxidase (PPO)-specific activity as a result of A. soccata infestation. The association of PPO activity with host plant resistance to insects occurs in many plants including tomato, potato, coffee, and poplar ( Duffy and Felton 1991 , Constabel et al. 1996 , Chaman et al. 2001 , Wang and Constabel 2004 , Thipyapong et al. 2006 ). Polyphenol oxidase reduces the nutritional quality of infested plants by converting soluble phenolic compounds into quinones that eventually prevent the digestion of proteins in insects. Similarly, considerable evidence from the earlier work implicates that increased accumulation of PPO in plants against tomato fruit borer ( Helicoverpa armigera and Helicoverpazea ) has affected the growth and development of these insects ( Isman and Duffey 1982a , b ). Saravanakumar et al. (2007) demonstrated that increased activity of PPO reduced the growth and development of Cnaphalocrocismedinalis on rice plants. From the earlier reports, it is suggested that PPO might interrupt with the insect fecundity and digestion which ultimately leads to delay in developmental period. Further research is needed to determine the role of these enzymes in the defense response of sorghum genotypes to A. soccata feeding and to evaluate these differences as potential molecular markers for selecting A. soccata -resistant sorghums.