Introduction
The grain aphid, Sitobion avenae, is a major Triticeae pest, causing serious losses in grain yields through direct damage by sucking plant sap and mediating the global transmission of plant viruses (Glinwood et al., Reference Glinwood, Liu, Hu, Keller, Zhao, Wu and Liu2014). The enhancement of host resistance (defence) to aphids has been considered as a safe, effective and environment-friendly method of aphid management through which the plant injury is largely alleviated (Cai et al., Reference Cai, Zhang and Cheo2004; Razmjou et al., Reference Razmjou, Ramazani, Naseri, Ganbalani and Dastjerdi2011). Some pre-existing phenolic compounds in Triticeae plants largely contribute to resistance to aphids (Hu et al., Reference Hu, Han, Zhao, Yang, Huang, Lou and Cai2008). The synthesis of these defence compounds in plants is often regulated through changing the defence enzymes involved in the phenylpropanoid pathway by some plant hormones (Dong et al., Reference Dong, Wan and Liang2010; Alvarez, Reference Alvarez and Alvarez2014), which suggest the application of that phytohormone in vitro may improve the resistance of plants to pests.
Phytohormones, such as jasmonic acid (JA) and salicylic acid (SA), play crucial roles in regulating plant growth and development processes (Pieterse et al., Reference Pieterse, Van der Does, Zamioudis, Leon-Reyes and Van Wees2012) and mediating plant defence system against herbivores and pathogens (Smith et al., Reference Smith, De Moraes and Mescher2009; Pyati et al., Reference Pyati, Bandani, Fitches and Gatehouse2011). The roles of phytohormones in inducing plant defence in signal-transduction pathways have been well documented (Smith et al., Reference Smith, De Moraes and Mescher2009; Pyati et al., Reference Pyati, Bandani, Fitches and Gatehouse2011; Zhang et al., Reference Zhang, Li, Huang, Zhang, Xu and Lu2013). In general, JA-mediated signalling pathways are implicated in the regulation of antiherbivore defence via the phenylpropanoid pathway which involves the biosynthesis of lignin, flavonoids and other aromatic metabolites (Kim and Jander, Reference Kim and Jander2007; Vogt Reference Vogt2010; Campos et al., Reference Campos, Kang and Howe2014). The phenylpropanoid pathway starts with phenylalanine which is transformed by phenylalanine-ammonia-lyase (PAL) into cinnamic acid and p-coumaroyl-CoA, the general precursor of phenolic acids and flavonoids (Valiñas et al., Reference Valiñas, Lanteri, ten Have and Andreu2017). Whereas SA pathway is associated with defence responses against pathogens (Smith et al., Reference Smith, De Moraes and Mescher2009). Nevertheless, recent reports showed that JA was also engaged in plant defence against necrotrophic pathogens (Ballaré, Reference Ballaré2011) and SA-mediated responses affected the phloem feeders (Urbanek Krajnc et al., Reference Urbanek Krajnc, Kristl and Ivancic2011). These phenomena probably result from mutual interferences among phytohormones (Engelberth et al., Reference Engelberth, Viswanathan and Engelberth2011).
The phenylpropanoid pathway is a well-known JA-mediated metabolic pathway, in which some metabolic enzymes are involved in secondary metabolism such as PAL and 4-coumarate:CoA ligase (4CL), PAL induction by SA has not been reported, 4CL is also not reported to be induced by both JA and SA (Reymond and Farmer, Reference Reymond and Farmer1998). Recent evidence indicated that SA could fine-tune plant defence responses by modulating JA signalling pathways (Smith et al., Reference Smith, De Moraes and Mescher2009; Thaler et al., Reference Thaler, Humphrey and Whiteman2012), and mediating plant-herbivore–pathogen interactions (Hatcher et al., Reference Hatcher, Moore, Taylor, Tinney and Paul2004). For example, SA induced an increase of PAL activity in Vitis vinifera (Wen et al., Reference Wen, Chen, Kong, Pan, Wan and Huang2005), and activated the typical insect-elicitor-induced JA in SA-treated corn seedlings as well (Engelberth et al., Reference Engelberth, Viswanathan and Engelberth2011). Polyphenol oxidase (PPO) is regarded as a marker of JA response (Thaler and Bostock, Reference Thaler and Bostock2004). Peroxidase (POD) and catalase (CAT) are also pivotal antioxidases in plants to function against pathogens and herbivores (Kim and Jander, Reference Kim and Jander2007). The activities of these enzymes mediate the biosynthesis of some defence compounds in plants (Smith and Boyko, Reference Smith and Boyko2007).
With regard to phytohormone-mediated resistance to phloem feeders, although JA-inducible resistance was considered to deter some chewing herbivores before (Thaler et al., Reference Thaler, Humphrey and Whiteman2012; Lazebnik et al., Reference Lazebnik, Frago, Dicke and van Loon2014), the current opinion is that SA-inducible resistance is involved in protection against some phloem-feeding insects via eliciting JA signalling pathway (Alba et al., Reference Alba, Schimmel, Glas, Ataide, Pappas, Villarroel, Schuurink, Sabelis and Kant2015; Jonathan Gogbeu et al., Reference Jonathan Gogbeu, Mathurin Okoma, Beranger N'Goran and Odette Dogbo2015). For example, SA was confirmed to be necessary for resistance to Macrosiphum euphorbiae in the tomato (Bhattarai et al., Reference Bhattarai, Xie, Pourshalimi, Younglove and Kaloshian2007). By contrast, SA activation increased whitefly infestation in Arabidopsis thaliana through downstream suppression of JA defences (Zhang et al., Reference Zhang, Li, Huang, Zhang, Xu and Lu2013). This evidence further suggests that SA-inducible defence against sap-sucking feeders depends on different plant species and may be controlled through crosstalk with the JA-mediated pathway.
To determine whether SA engages in the activation of defence system (resistance to aphids, phenolic acids and the defensive enzymes) for wheat aphids, we investigated the effects of exogenous SA application in aphid-susceptible wheat variety on important performances of S. avenae, the activities of some defence-related enzymes and contents of major phenolic acids. These findings provide crucial information for further understanding the effect of exogenous application of SA on the improvement of wheat resistance against S. avenae.
Materials and methods
Plant and aphid culture
Seeds of winter wheat, Triticum aestivum cv. Lovin 10, which is susceptible to S. avenae (Cai et al., Reference Cai, Zhang and Cheo2004), were germinated at room temperature, and sowed in plastic pots (20 cm diameter) with nutritional soil. The plastic pots with wheat seeds were kept in a climate chamber at 24 ± 1°C, 60%–70% relative humidity and 14 h illumination. The grain aphid, S. avenae, were collected from a winter wheat field at the Shangzhuang experiment station of China Agricultural University, and reared on Lovin 10 in a climate chamber over ten generations.
Salicylic acid treatment
The wheat seedlings were treated by a SA solution following a previously described method (Ananieva et al., Reference Ananieva, Christov and Popova2004) with some modifications. Briefly, SA (Sinopharm Chemical Reagent Co., Ltd., Beijing, China) was dissolved in distilled water to 1.0 mM with 1.0% Triton X-80 (V/V) following Radwan methods (Radwan, Reference Radwan2012), the control contained distilled water with 1.0% Triton X-80 (V/V). The wheat seedlings at the third- or fourth-leaf stage (three plastic pots, 15–20 seedlings per pot) were sprayed at 8:00 am with SA or the control solution using a 10 ml portable ultra-low-volume sprayer until runoff at daytime in mesh room. The experiment was carried out in triplicate. Each pot containing sprayed wheat seedlings was immediately covered with a sealed glass box (35 × 35 × 60 cm3) for 24 h in a mesh room. Subsequently, the sealed glass box was removed and the seedlings were maintained for growth in the mesh room. Another management procedure was as previously described. To assay defence-related enzyme activities and contents of phenolic acids, the wheat leaves of SA treatment and control without aphids were randomly harvested at 24, 48, 72 and 96 h post-SA treatment (p.s.t.), wrapped in aluminium foil, snap-frozen in liquid nitrogen and stored at −80°C until use in the assays.
Aphid performance assay
To evaluate the effect of SA application on the performance of wheat plants against aphid, referring to Radwan's methods (Radwan, Reference Radwan2012), wheat seedlings at the second- or third-leaf stage were treated using SA solutions of 1.0 mM with 1.0% Triton X-80 and distilled water with 1.0% Triton X-80 for 24 h as described above.
The life table of S. avenae was established on the wheat seedlings of SA treatment and control with the methods of Aziz et al. (Reference Aziz, Iftkhar and Hanif2013). One newborn nymph was inoculated on the leaves treated with SA treatment or control plants, respectively, and covered with a clip leaf cage (1 nymph per cage) to prevent the nymphs from escaping. Thirty-replicate assays were performed for the treatment and the control. All leaves with aphids were moved to the growth chamber and reared at 18–25°C and 50–70% RH under 16 h illumination. Ecdysis, stadium and the numbers of aphids (survived, died and newborn) were recorded every day until adult aphids started reproduction. Some biological parameters were calculated using the following formulae:
Here, x: age of the insects in days; lx: survival rate at the beginning of each interval; mx: the number of newborn nymphs produced per female from time x–1 to time x (days).
Aphid growth and development
Wheat seedlings were treated following the methods described above. Twenty weighed newborn nymphs were transported to SA-treated and control wheat seedlings by using a soft brush and reared in a growth chamber. Aphid-inoculated seedlings were covered with nylon mesh to prevent the cross escape of aphids among pots of treatments and controls. Subsequently, these aphids were weighed again on a Poton electronic balance (Mettler-Toledo, Germany) at 1, 3, 5 and 7 days post-inoculation, and the mean aphid weight was calculated to evaluate the effect of SA treatment on aphid growth and development. The experiments were repeated three times.
Assay of defence-related enzyme activity
Plant tissue (300 mg) from each sample was ground in liquid nitrogen and homogenized in 1.5 ml of potassium phosphate buffer (0.1 M; pH 7.0) containing 1% polyvinylpyrrolidone, 1.0 mM Na2-ethylene diamine tetraacetic acid and 7.0% glycerin. The resulting mixture was centrifuged using a Himac CR22E centrifuge (Hitachi Koki Co., Ltd., Japan) at 10,000 × g for 30 min at 4°C. The supernatant was used for the analyses of total protein content and enzymatic activity.
The Bradford method (Bradford, Reference Bradford1976) was used to determine total protein contents in samples. Three aliquots (20 μl) of each sample were mixed with 180 μl of the Bio-Rad reagent (Bio-Rad, Richmond, CA, USA). The absorbance of the reaction mixture was measured at 595 nm, and the protein content was determined using a standard curve, plotted using known quantities of bovine serum albumin (Sigma Chemical Co., USA).
Phenylalanine-ammonia-lyase
PAL activity was assayed using the method of Kováčik and Klejdus (Reference Kováčik and Klejdus2012) with slight modifications. In a cold mortar and pestle, 0.3 g of fresh leaves was homogenized in 2 ml of sodium borate buffer (pH 8.8) with quartz sand, and the resulting homogenates were immediately centrifuged (10,000 × g, 30 min, 4°C). The reaction mixtures, consisting of 800 μl of sodium borate buffer and 200 μl of homogenates, were preincubated at 37°C (3 min), and the reaction was started by adding 400 μl of L-phenylalanine (Sigma-Aldrich, Germany). After incubation at 37°C for 1 h, the reaction was stopped by adding 6 M HCl. Controls were prepared using the buffer instead of enzyme extract. The absorbance of the mixtures was monitored through spectrophotometry at 290 nm (extinction coefficient 9630 M−1 cm−1). Assays were conducted in three replicated samples. The activity of PAL was expressed as micromole per minute per milligram of protein.
4-Coumarate:CoA ligase
4CL activity was tested following a previously described method (Knobloch and Hahlbrock, Reference Knobloch and Hahlbrock1977; Saballos et al., Reference Saballos, Sattler, Sanchez, Foster, Xin, Kang, Pedersen and Vermerris2012). The reaction mixture contained 5.0 mM p-coumaric acid, 50 mM ATP, 1.0 mM CoA-SH, 15 mM MgSO4.7H2O and 0.4 ml of enzyme extract. The reaction was carried out at 40°C for 10 min and then stopped. Controls were prepared using the buffer instead of enzyme extract. Assays were conducted in three replicated samples. 4CL activity was estimated on the basis of the increase in absorbance at 333 nm (extinction coefficient 21,000 M−1 cm−1). 4CL activity was expressed as micromole per minute per milligram of protein.
Polyphenol oxidase
PPO activity was determined following a previously described method (Han et al., Reference Han, Wang, Bi, Yang, Huang, Zhao, Hu and Cai2009). 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). Controls were prepared using the buffer instead of enzyme extract. Assays were conducted in three replicated samples. PPO activity was estimated from the increase at 470 nm. PPO activity was expressed as U (activity unit) per minute per milligram protein, that is, one unit of PPO activity was defined as the amount that caused a change of 0.001 in absorbance per minute (Aksoy, Reference Aksoy2020).
Guaiacol peroxidase
Guaiacol POD activity was measured following a previously described method (Han et al., Reference Han, Wang, Bi, Yang, Huang, Zhao, Hu and Cai2009). 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. Controls were prepared using the buffer instead of enzyme extract. Assays were conducted in three replicated samples. POD activity was estimated from the increase in absorbance at 470 nm (extinction coefficient 26.6 mM−1 cm−1). POD activity was expressed as millimole per minute per milligram of protein.
CAT activity was determined using previously described methods (Horváth et al., Reference Horváth, Janda, Szalai and Páldi2002) with slight modifications. An aliquot (0.4 ml) of enzyme extract was added to 2 ml of a mixture containing 15 mM H2O2 in 0.15 mM potassium phosphate buffer (pH 7.0). The enzyme activity was measured as the decrease in absorbance at 240 nm (extinction coefficient of 36 mM−1 cm−1). Assays were conducted in three replicated samples. The activity was expressed as millimole per minute per milligram of protein.
Quantification of phenolic compounds
SA-treated wheat leaves were dried in a freeze dryer (Biocool Co., Beijing, China) for 12 h and powdered. Phenolic compounds were extracted using the methods of Ruelas et al. (Reference Ruelas, Tiznado-Hernandez, Sanchez-Estrada, Robles-Burgueno and Troncoso-Rojas2006). Dry powder (100 mg) was mixed with 3 ml of extracting solution (4% NaOH: absolute ethyl alcohol = 2:1, v/v), in brown tubes containing 2 g l−1 benzaldehyde in triplicate. The resulting mixture was homogeneously agitated, crushed in a JY92 ultrasonic cell crusher (Ningbo Scientz Biotechnology Co., Hangzhou, China) for 30 min, homogeneously mixed with 500 μl of HCl (6.0 M) and centrifuged at 6700 × g for 20 min. The supernatant was collected and dried using a rotary evaporator. The resulting phenolic compounds were redissolved in 1.0 ml of mixed solvent (methanol: sodium acetate buffer = 1:1, v/v; pH 5.4) and filtered using a 0.45 μm filter membrane. The final extract was stored in brown tubes for HPLC analysis. Treatment or control assay was conducted in three replicated samples.
The Agilent 1100 HPLC system was used throughout this study. It consisted of a binary pump (G1312A), an autosampler (G1313A), a vacuum degasser (G1322A), a thermostatted column compartment (G1316A) and a diode array detector (G1315B). A Phenomenex column (250 × 4.6 mm, 5.0 μm, Phenomenex, CA, USA) was used for analysis. The column temperature was maintained at 30°C. Chromatographic separations were performed by following the methods of Ruelas et al. (Reference Ruelas, Tiznado-Hernandez, Sanchez-Estrada, Robles-Burgueno and Troncoso-Rojas2006) and Zhang et al. (Reference Zhang, Meng, Yao and Zhou2010). Solvent A (methanol) and solvent B (0.05 M acetic acid, pH 5.4) under gradient conditions were used at a flow rate of 1.0 ml min−1, and the injection volume was 20 μl. A 22 min gradient program was used in this study. The initial percentages of solvents A and B in the solvent mixture were 12 and 88%, respectively, for 8 min, and these percentages were held for 12 min; subsequently, the percentage of solvent A was ramped up to 17% for 21 min and then ramped down to 12% for 22 min. The percentage of solvent B was adjusted accordingly to achieve a total of 100%. The detection wavelength was 316 nm.
Phenolic compounds were quantified using a ten-point regression curve constructed on the basis of p-coumaric acid, caffeic acid, ferulic acid and chlorogenic acid (Sigma, USA). The contents are expressed as milligrams per gram of tissue dry weight (mg g−1 dw).
Statistical analysis
The statistical analysis of all data was performed using SPSS software (version 11.5). Based on the aphid life table, aphid parameters such as r m, fecundity, wingless aphid rate, female reproduction time, time to onset of reproduction and life time were calculated as described above. The aphid biological parameter, aphid body mass, enzyme activities and phenolic contents of wheat plants between treatment and control were compared using paired sample t tests with a 95% confidence interval of the difference. Correlation of enzyme activities with phenolic acids was conducted using bivariate correlation analysis, significant difference is at 0.05 level.
Results
Aphid performance
Compared with control seedlings, intrinsic rate of natural increase (r m) and fecundity of the grain aphids as well as apterous rate were significantly lower in the SA-induced seedlings than that in the controls (df = 29, t rm = 2.408, P rm = 0.023; t fecundity = 3.664, P fecundity = 0.001; t apterous rate = 2.262, P apterous rate = 0.031; fig. 1a–c), whereas female reproduction time, time to onset of reproduction and lifetime of the aphids that fed on SA-treated seedlings were not significantly affected (P＞0.05) (fig. 1d). Aphid body mass was significantly reduced on day 7 post-feeding treated wheat seedlings with 1.0 mM SA (t = 7.748, P = 0.016), but on day 5 post-feeding, significant suppression of aphid body mass was not observed in SA-treated wheat seedlings (t = 1.015, P = 0.417) (fig. 2). Results indicate the SA-induced wheat seedlings can significantly inhibit aphid key indices that determine aphid growth and population development.
Figure 1. Effect of SA-treated wheat seedlings on key aphid performance. Data (mean ± SD) was displayed. *, ** and *** indicated significant difference at 0.05, 0.01 and 0.001 levels, respectively, ns is no significance P > 0.05 (n = 30). (a) innate rate of increase; (b) female fecundity; (c) apterous aphid rate; (d) lifetime, female reproduction time (FRT) and time to onset of female reproduction (TOR).
Figure 2. Effect of SA-treated wheat seedlings on aphid biomass. Data (mean ± SD) was displayed. Asterisk (*) indicated significant difference at 0.05 level, ns is no significance P > 0.05.
PAL and 4CL activities
In the wheat seedlings treated with SA, the PAL activity was significantly higher than that of controls at 48 and 72 h.p.t (t 48 = ‒8.100, P = 0.015; t 72 = ‒11.414, P = 0.008) (fig. 3a), whereas a significant suppression of 4CL activity was shown at all tested time points (t 24 = 17.427, P = 0.003; t 48 = 14.571, P = 0.005; t 72 = 13.994, P = 0.005) (fig. 3b). This indicates that SA-induced PAL activity in wheat seedlings is associated with the time of duration, but the application of exogenous SA can suppress 4CL activity in wheat seedlings.
Figure 3. Effect of exogenous SA on the activity of defence-related enzymes in wheat seedlings. The enzyme activity (mean ± SD) was plotted. *, ** and *** indicated significant difference at 0.05, 0.01 and 0.001 levels, respectively, ns is no significance P > 0.05. (a) PAL (phenylalanine-ammonia-lyase); (b) 4CL (4-coumarate:coenzyme A ligase); (c) PPO (polyphenol oxidase); (d) POD (peroxidase); (e) CAT (catalase).
PPO, POD and CAT activities
Our results revealed that exogenous SA significantly increased PPO activity in wheat seedlings at both 48 and 72 h.p.t. (t 48 = ‒8.176, P = 0.015; t 72 = ‒10.708, P = 0.009), but inhibited the activity at 24 h.p.t. (t 24 = 38.864, P = 0.001) (fig. 3c). POD activity in SA-induced wheat seedlings was significantly higher than that in control wheat seedlings only at 72 h.p.t. (t 72 = ‒7.567, P = 0.017) (fig. 3d). Notably, CAT activity in SA-treated seedlings was significantly reduced at all tested time points post-treatment (t 24 = 6.065, P = 0.026; t 48 = 14.850, P = 0.005; t 72 = 20.415, P = 0.002) (fig. 3e). The findings show that the inducing effect of SA on PPO and POD activities is enhanced with time p.s.t., CAT activity in wheat seedlings always is inhibited by exogenous SA at all sampling time.
Phenolic compound content
When wheat seedlings were treated with exogenous SA, four phenolic acid content dynamics in the seedlings can be regulated at sampling time points. Coumaric acid content was significantly reduced at 48 h, but increased at 96 h p.s.t. (t 48 = 6.289, P = 0.024; t 96 = ‒5.362, P = 0.033) (fig. 4a). Ferulic acid content in SA-treated wheat seedlings was significantly higher than that in control ones at 24 and 72 h and lower than that in control ones at 48 h (t 24 = ‒21.671, P = 0.002; t 48 = 73.635, P < 0.001; t 72 = ‒45.813, P < 0.001) (fig. 4b). A significantly low caffeic acid content was displayed in SA-treated wheat seedlings at 24 and 48 h p.s.t. (t 24 = 6.953, P = 0.020; t 48 = 24.371, P = 0.002) (fig. 4c). Regarding chlorogenic acid content, exogenous SA triggered a significant increase in SA-treated wheat seedlings at 24, 48 and 96 h, but significantly inhibited chlorogenic acid content at 72 h (t 24 = ‒136.858, P < 0.001; t 48 = ‒21.886, P = 0.002; t 72 = 55.932, P < 0.001; t 96 = ‒82.674, P < 0.001) (fig. 4d). In terms of increase or inhibition, there is a fast response (24 h or/and 48 h) to exogenous SA in ferulic acid, caffeic acid and chlorogenic acid contents, whereas in SA-treated wheat seedlings, the coumaric acid content presents a delayed response to exogenous SA (at 96 h).
Figure 4. Content dynamics of coumaric acid (a), ferulic acid (b), caffeic acid (c) and chlorogenic acid (d) in SA-treated and control wheat seedlings. Second- or third-leaf wheat seedlings were treated with a SA spray for 24 h. Then, the leaves were harvested at 24, 48, 72 and 96 h.p.t. for determining the contents of phenolic compounds in leaf tissues through HPLC. Data are represented as the mean ± SD from three replicates. *, ** and *** indicated significant difference at 0.05, 0.01 and 0.001 levels, respectively, ns is no significance P > 0.05.
Correlation of defence-related enzymes with phenolic compounds
To understand the association of defence-related enzyme activity with the synthesis of phenolic acid, in SA-treated wheat seedlings or control ones, the correlation between each enzyme activity and compound content was analysed with sampling time. In wheat seedlings of control, CAT activity was significantly negatively and positively correlated with ferulic acid content (P = 0.005) and chlorogenic acid content (P = 0.022), respectively, but not in SA-treated wheat ones (P > 0.05). On the contrary, induced PPO activity in wheat seedlings by exogenous SA was significantly associated with caffeic acid content (P = 0.031). No significant correlation was observed between other enzyme activities and phenolic acid contents in SA-treated and control wheat seedlings (P > 0.05) (table 1).
Table 1. The correlation of defence-related enzyme activity with phenolic compound content in SA-induced wheat seedlings.
PAL, phenylalanine-ammonia-lyase; 4CL, 4-coumarate:coenzyme A ligase; PPO, polyphenol oxidase; POD, peroxidase; CAT, catalase; CoA, coumaric acid; FA, ferulic acid; CA, caffeic acid; ChA, chlorogenic acid.
Asterisk (* or **) following P value indicated a significant correlation at 0.05 or 0.01 level.
Discussion
Chemical defence against herbivores and pathogens is prevalent in plants. The synthesis of defensive compounds in plants is always related to some secondary metabolic pathways, among which the phenylpropanoid pathway has been regarded to be associated with plant resistance to herbivorous insects (Alvarez, Reference Alvarez and Alvarez2014). Plant hormones such as JA and SA are increasingly recognized as crucial mediators in the interactions of plants with the associated organisms (Lazebnik et al., Reference Lazebnik, Frago, Dicke and van Loon2014), generally through the activation of defence-related enzymes and accumulation of some secondary compounds in the phenylpropanoid pathway to potentiate plant defence against pests (Chu et al., Reference Chu, Wegel and Osbourn2011). Considerable evidence has shown that these hormones can trigger the activities of defensive enzymes or the accumulation of plant secondary metabolites involved in the resistance to herbivores or/and pathogens (Robert-Seilaniantz et al., Reference Robert-Seilaniantz, Grant and Jones2011); however, few studies explored specific hormone-mediated association among defence-related enzyme activity, compound content and plant defence against pests. Our findings in the current study revealed that exogenous SA application triggered the activities of some defence-related enzymes and increased specific phenolic content in aphid-susceptible wheat plants at a specific period, and reduced biological indices of S. avenae.
Phytohormones, particularly JA and SA as well as their derivatives, have been considered to regulate plant defences against either pathogens or herbivores. The activation of the defence system by phytohormones is favourable for plant resistance to herbivores (Thaler et al., Reference Thaler, Agrawal and Halitschke2010). Application of exogenous SA reduces Lolium multiflorum resistance to Rhopalosiphum padi by reducing the concentration of loline alkaloids (Bastias et al., Reference Bastias, Martinez-Ghersa, Newman, Card, Mace and Gundel2018). Previous evidence also indicated no significant effect of SA application on the biological index of S. avenae in wheat seedlings (Cao et al., Reference Cao, Wang and Liu2014). However, contradictory to this notion, our results showed exogenous SA significantly repressed the intrinsic rate of natural increase of aphid (r m), female fecundity and apterous rate, and aphid body weight at day 7 post-feeding wheat seedlings as well (figs 1a‒c and 2). This indicated that the application of exogenous SA improved wheat resistance to the aphid. The difference may result from the following reasons, first, the wheat variety adopted in our study is susceptible to S. avenae, their seedlings may be more sensitive to exogenous stimuli such as insect feeding, pathogen infection (Han et al., Reference Han, Wang, Bi, Yang, Huang, Zhao, Hu and Cai2009; Thaler et al., Reference Thaler, Agrawal and Halitschke2010) and phytohormones. Second, to enhance SA activation, we used previous SA-treated methods (Ananieva et al., Reference Ananieva, Christov and Popova2004) that SA-sprayed wheat seedlings were covered with a sealed glass box for 24 h. Therefore, the inducible effect of exogenous phytohormone on plant defence system might be closely associated with a variety of characteristics (Han et al., Reference Han, Wang, Bi, Yang, Huang, Zhao, Hu and Cai2009) and SA treatment methods.
SA and its derivatives mediate plant systemic acquired resistance to pathogens (Park et al., Reference Park, Kaimoyo, Kumar, Mosher and Klessig2007), some evidence suggests that SA could fine-tune plant defence responses to specific pests by eliciting JA signalling pathway (Smith et al., Reference Smith, De Moraes and Mescher2009) in some dicotyledonous plants, and resulting in the accumulation of some products downstream in the pathway via crosstalk (Engelberth et al., Reference Engelberth, Viswanathan and Engelberth2011; Thaler et al., Reference Thaler, Humphrey and Whiteman2012). In our study, exogenous SA also activate PAL activity in wheat plants, a monocotyledon (fig. 3a), but inhibited 4CL activity with sampling time points in both SA-treated and control wheat plants (fig. 3b). Until recently, although no evidence indicates how phytohormones affect 4CL activity in plants, some 4CL isoforms were thought to participate in the biosynthetic pathway of flavonoids and lignin as well as in the production of additional phenolic compounds (Ehlting et al., Reference Ehlting, Buttner, Wang, Douglas, Somssich and Kombrink1999). Further studies will facilitate understanding the effect of phytohormones on 4CL activity and its biological function.
Antioxidases such as PPO, POD and CAT play a crucial role in the phenylpropanoid pathway, some of their products are often vital defence compounds against herbivores and pathogens (Dong et al., Reference Dong, Wan and Liang2010; Jonathan Gogbeu et al., Reference Jonathan Gogbeu, Mathurin Okoma, Beranger N'Goran and Odette Dogbo2015). SA can enhance the activities of these three oxidases in many plants (Valverde et al., Reference Valverde, Giménez, Guillén, Valero, Martínez-Romero and Serrano2015), but the activities of POD and CAT are suppressed by SA in other plants (Mohase and van der Westhuizen, Reference Mohase and van der Westhuizen2002). Our findings showed that potentiated activities of PPO and POD at 48 and 72 h were observed in the SA-treated wheat seedlings and dependent on sampling time (fig. 3c, d). These results further confirmed the conclusions made by others (Ananieva et al., Reference Ananieva, Christov and Popova2004; Tasgin et al., Reference Tasgin, Atici, Nalbantoglu and Popova2006). Interestingly, SA reduced CAT activity in the present study, this result was consistent with previous reports in wheat plants (Mohase and van der Westhuizen, Reference Mohase and van der Westhuizen2002) and the apoplast of wheat leaves (Tasgin et al., Reference Tasgin, Atici, Nalbantoglu and Popova2006), but are different from the results observed in barley seedlings (Ananieva et al., Reference Ananieva, Christov and Popova2004) and cherry fruits (Valverde et al., Reference Valverde, Giménez, Guillén, Valero, Martínez-Romero and Serrano2015). Therefore, CAT activity induced by SA appears to be dependent on plant species.
Plant phenolics have been considered to be vital defence components against biotic and abiotic stresses, plant hormones are indispensable regulators in the synthesis of these defence compounds (Dong et al., Reference Dong, Wan and Liang2010; Lajara et al., Reference Lajara, López-Orenes, Ferrer and Calderón2015). Exogenous application of plant hormones is an effective way to induce the defence reactions in plants and results in stronger plant defence against particular herbivores and pathogens because of the increased contents of some defence compounds (Mohase and van der Westhuizen, Reference Mohase and van der Westhuizen2002; Park et al., Reference Park, Kaimoyo, Kumar, Mosher and Klessig2007). SA-inducible phenolic metabolism has been reported in SA-treated plants (Kováčik et al., Reference Kováčik, Grúz, Bačkor, Strnad and Repčák2009). The levels of coumaric acid at 96 h.p.t., ferulic acid at 24 and 72 h.p.t. and chlorogenic acid at 24, 48 and 96 h.p.t. were significantly increased in SA-treated wheat seedlings, but caffeic acid contents at 24 and 48 h.p.t. were significantly reduced in SA-treated wheat seedlings (fig. 4c). The results indicate that exogenous SA affects the dynamics of phenolic metabolism in wheat plants, suggesting that the SA-induced role may be different in various phenolic acids.
The biosynthesis pathways of many aromatic compounds have the first three enzymatic steps in common with the phenylpropanoid pathway in plants. That is, PAL, cinnamate 4-hydroxylase and 4CL in the upstream portion of the pathway successively catalyse phenylalanine deamination, hydroxylation and synthesis of p-coumaroyl CoA ester (Li et al., Reference Li, Kim, Pysh and Chapple2015). Growing evidence showed that both endogenous and exogenous SA stimulated the accumulation of phenylpropanoids through the activation of PAL in plants (Kováčik et al., Reference Kováčik, Grúz, Bačkor, Strnad and Repčák2009; Dong et al., Reference Dong, Wan and Liang2010); however, little is known about the relationship between SA, PAL and the accumulation of specific phenolics in plants. Our results indicate that the accumulation of all tested phenolic acids was not statistically significantly correlated with PAL and 4CL activities in SA-treated wheat seedlings (table 1). This implies that the sensitivity of the metabolism of these compounds to PAL and 4CL could be associated with the location of their metabolic branches, in which they are synthesized (Li et al., Reference Li, Kim, Pysh and Chapple2015). Although the first and last shared step is catalysed by PAL and 4CL (Li et al., Reference Li, Kim, Pysh and Chapple2015), respectively, and PAL activity at 48 and 72 h.p.t. was induced by exogenous SA, 4CL activity was significantly reduced in SA-treated wheat seedlings (fig. 3b). This suggests that PAL and 4CL may not be directly involved in the metabolism of terminal compounds that could belong to metabolic branches in wheat tissue.
Plants can defend themselves against pathogens and herbivores by using phenolics in two ways: first, large quantities of phenolic compounds are accumulated in plant tissues to prevent infestation, and second, some compounds with immense antibiotic capability are synthesized by oxidizing phenols of low toxicity in plant tissues (Chrzanowski et al., Reference Chrzanowski, Ciepiela, Sprawka, Sempruch, Sytykiewicz and Czerniewicz2003). As mentioned, the exogenous application of SA has been reported to trigger the increases in PPO, POD and CAT activity and the content of total phenolics and individual phenolic acids in a variety of plant species (Ananieva et al., Reference Ananieva, Christov and Popova2004; Dong et al., Reference Dong, Wan and Liang2010). PPO can oxidize mono- or dihydroxy phenols to the corresponding quinones, which provide resistance to pathogens and herbivores (Wang and Constabel, Reference Wang and Constabel2004). POD and CAT oxidize phenolic compounds in the presence of H2O2 or reactive oxygen species in plants (Fry, Reference Fry2004). These enzymatic activities were found to be inhibited by some substances and oxidative products (Le Bourvellec et al., Reference Le Bourvellec, Le Quéré, Sanoner, Drilleau and Guyot2004). Our results showed that a significant positive correlation existed between PPO activity and caffeic acid content in SA-treated wheat seedlings but not in control seedlings (table 1). CAT activity in wheat seedlings of control was negatively and positively correlated with ferulic and chlorogenic acids, respectively, whereas the correlation was not observed in SA-treated wheat seedlings (table 1). The findings suggest that exogenous SA could change the constitutive content of phenolic acids in wheat plants by interfering with the activities of some defence-related enzymes in the phenylpropanoid pathway.
In summary, our findings demonstrated that the application of SA in aphid-susceptible wheat seedlings significantly reduced r m, female fecundity, apterous rate and aphid biomass of S. avenae, these reduced indices reflect the inadaptation of aphids to host plants. It is worth noting that high-quality wheat varieties such as strong-gluten wheat often are susceptible to aphids in China, and can provide sufficient nutrition for aphid growth and development (Wang et al., Reference Wang, Yuan, Chen, Lei and Wu2006). Therefore, the application of SA in wheat production may provide an environmental-friendly way of controlling the aphid population to reduce wheat loss.
On the other hand, exogenous SA also potentiated the activities of PAL, PPO and POD with time, but reduced 4CL and CAT activities, and the constitutive contents of some phenolic acids were also modified in SA-treated wheat seedlings. Nevertheless, a correlation between defence-related enzyme activity with phenolic acid content was not observed by a simple correlation analysis, suggesting metabolic complexity of phenolic acids in plants. Therefore, we yet have no enough information about the phytochemical metabolism regulated by SA. Further studies are needed to determine the precise mechanism of SA-mediated relationship between a defence-related enzyme and phytochemical biosynthesis.
Acknowledgements
This research was funded by the Nation Key Projects on Science and Technology Innovation of High Yield and Efficiency of Grain, grant numbers 2016YFD0300203.
Conflict of interest
None.
