Introduction

Host plant resistance to herbivores can suppress herbivore population densities and offers a promising approach for managing insect pests in a sustainable, economical, and environmentally safe manner (Heng-Moss et al. 2004). Plant secondary metabolites play an important role in plant defense against insects and pathogens (Cai et al. 2004), the levels of which are often mediated by defense-related enzymes, such as phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), and peroxidase (POD; Carver et al. 1992; Rosenthal and Berenbaum 1992; Bi and Felton 1995; Bi et al. 1997a, b, c).

PAL is a key enzyme in the phenylpropanoid pathway that converts phenylalanine into trans-cinnamic acid. Trans-cinnimaic acid can be further hydroxylated and methylated to produce compounds (i.e., coumaric, caffeic, and ferulic acids) that are toxic to herbivores and pathogens (Cole 1984; Leszczynski et al. 1989; Verpoorte and Alfermann 2000; Morelló et al. 2005; Wang et al. 2006). PPO and POD are oxidases that catalyze the formation of quinones from various phenolic precursors (Hildebrand et al. 1986; Mayer 1987; Felton et al. 1989; Duffey and Stout 1996).

PAL, PPO, and POD are associated with plant defense against insects (Felton et al. 1992; Stout et al. 1999; Chaman et al. 2001, 2003; Ni et al. 2001). However, little is known about the dynamic changes of these enzymes during plant development and the influence of aphid infestation on their activity in resistant vs. susceptible cultivars, especially in wheat. The present research was initiated to investigate the constitutive and induced activity of these enzymes in Sitobion avenae-resistant and S. avenae-susceptible wheat cultivars at various developmental stages.

Methods and Materials

Plants and Insects

Wheat cultivars KOK1679, L1, Beijing 411, and Beijing 837 were grown in 2003 and 2004 at the Science Park of China Agricultural University, Beijing, China. Wheat seeds were sown 20 cm apart in a 2-m single-row plot on October 14, 2003. Each plot was used as one replicate and covered with white nylon mesh to protect plants from insects and mechanical damage during the growth.

KOK1679 and L1 cultivars are resistant and repellant to the cereal aphid species S. avenae, while Beijing 411 and Beijing 837 are susceptible and attractive to this species (Liu et al. 2001). In addition, both KOK1679 and L1 have antibiotic properties against S. avenae compared to Beijing 411 and Beijing 837 (Cai et al. 2004 and unpublished data).

S. avenae nymphs and adults were collected from field-grown wheat and maintained on the aphid-susceptible wheat cultivar, Beijing 411, under field conditions. Insects over one generation were used for the experiment. When the unfolded flag leaf and ear emerged in 49–56 and 58–60 of Zadoks code (Zadoks et al. 1974), 20 flag leaves or ears from each experimental plot were selected and evenly divided for experimental and control treatments. Ten nymphs (second to third instar) were released onto each flag leaf and ear in each treatment. The flag leaves and ears were covered with nylon mesh cages (15 × 4 cm) to prevent the aphids from escaping (Rafi et al. 1996; Argandona et al. 2001). The experiments were arranged in a randomized complete block design, and each treatment was replicated three times.

Sample Collection

A total of 60 tillers from each plot were sampled randomly at various developmental stages to determine the constitutive enzyme activity in wheat tissue. Whole plants, including stems and leaves at Zodaks stage 21–23 and the upper 3–5 young leaves and stem at Zadoks stage 31–34, were sampled (Zadoks et al. 1974). Flag leaves at stage 49–56 and ears at stage 58–60 also were sampled. Flag leaves and ears of all the treatments and controls were individually excised and collected 9 days after aphid release (Ni et al. 2001). Upon collection, samples were wrapped in aluminum foil and immediately dropped into liquid nitrogen for transport to a −80°C freezer before enzymatic analyses.

Plant Protein and Enzyme Assays

Plant tissue (100 mg) from each sample was ground in liquid nitrogen and homogenized in 20 mM HEPES buffer (500 μl; pH 7.2). The resulting mixture was centrifuged (Himac CR22E, Hitachi Koki Co. Ltd. Japan) at 10,000×g for 20 min at 4°C. The supernatant was used for analyses of total protein content and enzymatic activity.

The Bradford method (Bradford 1976) was used to determine total protein content in samples. Three aliquots (20 μl) of each sample were mixed with Bio-Rad reagent (180 μl, Bio-Rad, Richmond, CA, USA). Absorbance of the reaction mixture was determined at 595 nm, and protein content was determined from a standard curve established by using known quantities of bovine serum albumin (from Sigma Chemical Co.) and the above reagent.

PAL activity was determined following the method of Wang and Xue (1980) with enzyme extract (0.25 ml) in sodium borate buffer (1.5 ml, 30 mM; pH 8.8). The decrease of l-phenylalanine was monitored at 290 nm. PAL activity was estimated from dA 290. The assay was replicated three times.

POD activity was measured following a method previously described (Hildebrand et al. 1986; Hori et al. 1997). Enzyme extract (10 μl) was mixed with 1 ml of substrate containing hydrogen peroxide (10 μl; 30%, w/w), guaiacol (1 μl; 20 mM), and HEPES (100 μl; 200 mM, pH 7.0) in deionized water. POD activity was estimated from the increase in A 470. The measurement was repeated three times.

PPO activity was determined following the method of Hori et al. (1997). Enzyme extract (100 μl) was mixed with a solution containing catechol (500 μl; 1.6% w/w), HEPES buffer (100 μl; 200 mM, pH 6.0), and deionized water (800 μl). PPO activity was estimated from the increase in A 470. The analysis was repeated three times.

Statistical Analysis

Analysis of variance (ANOVA) in SPSS (SPSS 11.0) was used to analyze PAL, POD, and PPO activity in wheat cultivars at various developmental stages. Pairs of treatment means were compared and separated (α = 0.05) by the least significant difference (LSD) procedure. Before the ANOVA and LSD procedure, PAL, POD, and PPO activity was transformed using the logarithm transformation (ln) to normalize the data.

Results

Protein Content

Total protein content among wheat cultivars was significantly different at the different developmental stages (i.e., tillering, stem elongation, flag leaf, and ear stages), as well as at the flag leaf and ear stages in plants infested by aphids (df = 3, 11, F TS = 7294.6, F SES = 4887.1, F FL = 11040.8, F H = 9939.9, F AFL = 4883.9, F AH = 8267.3, P < 0.001). The constitutive protein content of KOK1679 and L1 was lower than that of Beijing 411 and Beijing 837 at all stages, with the exception that the protein content of KOK1679 was higher than that of Beijing 837 at the flag leaf stage (Table 1, Fig. 1). However, after flag leaf and ear stages were infested by aphids, total protein content increased in flag leaves of all cultivars and in ears of KOK1679 and L1 (P < 0.05; Fig. 1).

Fig. 1
figure 1

Effect of S. avenae infestation on total protein content in flag leaf and ear stages of resistant and susceptible wheat cultivars. Significant difference at *P < 0.05; NS no significant difference (P > 0.05)

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Table 1 Total constitutive protein contents (milligram per gram fw) of four wheat cultivars without aphid feeding at tillering and stem elongation stages
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Defense-Related Enzymes

KOK1679 and L1 exhibited greater constitutive PAL activity than Beijing 411 and Beijing 837 in the tillering and stem elongation stages (df = 3, 11; F TS = 28.13, P < 0.01; F SES = 6.53, P = 0.02; Table 2). The constitutive PAL activity was also higher in KOK1679 and L1 than in Beijing 411 and Beijing 837 in the flag leaf (df = 3, 11; F FL = 26.58, P < 0.001), but not in the ear stage (Fig. 2). Aphid infestation strongly enhanced the levels of PAL activity in the flag leaf and ear stages of all four cultivars (P < 0.05; Fig. 2).

Fig. 2
figure 2

Effect of S. avenae infestation on PAL activity in flag leaf and ear stages of resistant and susceptible wheat cultivars. Significant difference at *P < 0.05; NS no significant difference (P > 0.05)

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Table 2 Constitutive phenylalanine ammonia-lyase activity (dA 290 per minute per milligram protein) of four wheat cultivars at tillering and stem elongation stages
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Constitutive PPO activity was higher in KOK1679 and L1 than in Beijing 837 and Beijing 411 at all developmental stages (df = 3, 11; F TS = 9.81, P = 0.005; F SES = 7.34, P = 0.011; F FL = 18.72, P = 0.001; F E = 21.33, P < 0.001; Table 3, Fig. 3). Aphid infestation caused significant increases in PPO activity in the flag leaf and the ear stages of Beijing 837 and Beijing 411 and in the ear stage of KOK 1679 (Fig. 3).

Fig. 3
figure 3

Effect of S. avenae infestation on PPO activity in flag leaf and ear stages of resistant and susceptible wheat cultivars. Significant difference at *P < 0.05; NS no significant difference (P > 0.05)

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Table 3 Constitutive polyphenol oxidase activity (dA 470 per minute per milligram protein) of four wheat cultivars at tillering and stem elongation stages
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KOK1679 and L1 exhibited greater constitutive POD activity than Beijing 837 and Beijing 411 in the tillering and stem elongation stages (df = 3, 11; F TS = 21.78, P < 0.001; F SES = 15.28, P = 0.001; Table 4). The constitutive POD activity was also higher in the flag leaf stage of KOK1679 and L1 than in Beijing 837 and Beijing 411 (df = 3, 11; F FL = 15.79, P = 0.001; Fig. 4). Aphid infestation boosted POD activity in all cultivars, especially in Beijing 837 and Beijing 411 (P < 0.05; Fig. 4).

Fig. 4
figure 4

Effect of S. avenae infestation on POD in flag leaf and ear of resistant and susceptible wheat cultivars. Significant difference at *P < 0.05; NS no significant difference (P > 0.05)

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Table 4 Constitutive peroxidase activity (dA 470 per minute per milligram protein) of four wheat cultivars at tillering and stem elongation stages
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Discussion

Active defense in plants can be induced by both biotic and abiotic factors (Karban and Baldwin 1997; Kant et al. 2004). PAL, PPO, and POD are among the most important enzymes involved in the defensive responses of plants to insects and pathogens. PAL plays a key role in corn resistance to the maize sheath blight Rhizoctonia solani (Jin et al. 2003) as well as to Helminthosporium maydis and Helminthosporium turcicum (Wang and Xue 1980). Cole (1984) demonstrated that PAL activity is related to lettuce resistance to the lettuce root aphid Pemphigus bursarius (L). S. avenae infestation resulted in increased PAL activity in the flag leaf and ear stages of resistant and susceptible winter wheat cultivars (Ciepiela 1989; Havlickova et al. 1996). Increased PAL activity also was found in the leaves of Brassica oleracea seedlings infested by green peach aphid Myzus persicae (Sulzer; Zhang et al. 2005).

S. avenae-resistant wheat cultivars (i.e., KOK1679 and L1) had significantly higher levels of PAL activity than susceptible cultivars (Beijing 411 and Beijing 837) at the tillering and stem elongation developmental stages. PAL activity in the flag leaf and ear stages of aphid-resistant cultivars increased more dramatically than that in the aphid-susceptible ones after S. avenae infestation, suggesting that the resistant cultivars may have a stronger capability to synthesize secondary metabolites related to insect susceptible ones.

Increased PPO activity in susceptible wheat can be induced by both aphids and methyl jasmonate (Leszczynski 1985; Boughton et al. 2006). Our study showed that constitutive PPO activity was higher in nutritional organs of resistant wheat cultivars compared to susceptible ones (Table 3). PPO activity in the ear and flag leaf stages of both resistant and susceptible cultivars increased when plants were infested with aphids; the increase was greater in susceptible cultivars (Fig. 3). However, Chrzanowski et al. (2003) reported that PPO activity in the ear stages of both resistant and susceptible spring wheat cultivars was decreased by S. avenae infestation. This discrepancy may be due, at least in part, to genetic differences in the wheat cultivars. Ni et al. (2001) reported that Diuraphis noxia feeding did not elicit changes in PPO activity in either resistant or susceptible cultivars of wheat or barley. This difference may be associated with the specific feeding behaviors exhibited by different aphid species, e.g., D. noxia feeding elicits leaf-chlorosis while S. avenae feeding does not.

Previous studies have demonstrated that POD activity can be enhanced by insects and elicitors such as ethephon and methyl jasmonate (Bi and Felton 1995; Bi et al. 1997a, b; Takahama and Oniki 1997; Kielkiewicz 1998). Buffalograss that is resistant to Blissus occiduus had higher POD activity compared to susceptible plants (Heng-Moss et al. 2004). Andres et al. (2001) reported that wheat root resistance to the cereal cyst nematode was associated with increased POD activity. POD activity in B. oleracea leaves infested with M. persicae increased significantly compared with control leaves (Zhang et al. 2005). Macrosiphum euphorbiae (Thomas) feeding induced greater POD activity in tomato leaves (Stout et al. 1998). Our results revealed that at the tilling and stem elongation stages, resistant wheat cultivars had a greater constitutive POD activity compared with susceptible cultivars, whereas at the flag leaf stage, the constitutive POD activities were similar between the resistant and susceptible cultivars (Table 4, Fig. 4). Also, aphid infestation of susceptible wheat cultivars at the flag leaf and ear stages induced a higher POD activity (Fig. 4). The differences in POD activities among the growing stages may be attributed to normal wheat plant growth (Ni et al. 2001).

In summary, S. avenae-resistant wheat cultivars had greater constitutive PAL, PPO, and POD activity at the tillering, stem elongation, and flag leaf stages of development. When the flag leaf and ear stages were attacked by aphids, PAL activity increased more dramatically in the resistant cultivars than in the susceptible ones. Aphid infestation increased activities of PPO and POD more dramatically in the susceptible cultivars than in the resistant ones.