Effect of salicylic acid on jasmonic acid-related defense response of pea seedlings to wounding

Scientia Horticulturae 128 (2011) 166–173

Contents lists available at ScienceDirect

Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti

Effect of salicylic acid on jasmonic acid-related defense response of pea seedlings to wounding Hao-Ru Yang a,1,2 , Ke Tang a,1 , Hong-Tao Liu b , Wei-Dong Huang a,∗ a b

College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences, Beijing 100101, China

a r t i c l e

i n f o

Article history: Received 26 July 2010 Received in revised form 14 January 2011 Accepted 19 January 2011 Keywords: Jasmonic acid Salicylic acid Defense response Pea (Pisum sativum L. cv. NingXia)

a b s t r a c t Interactions between jasmonic acid (JA) and salicylic acid (SA) signaling pathways play important roles in the regulation and fine-tuning of induced defenses which are activated by pathogen, insect attack, or wounding. In this study, endogenous SA and JA level in pea seedlings presented opposite trends in response to wounding. Results exhibited that SA impaired the resistance to wounding of pea seedlings by means of suppressing the action of JA. Based on the fact that the wound-induced PPO activity was suppressed by SA, it could be concluded further that SA had negative effect on JA-related defense response. However, synergy between JA and SA also existed in phenolic metabolism related to PAL. Moreover, endogenous SA and salicylic acid 2-O-ˇ-d-glucose (SAG) levels in wounded pea seedlings also appeared opposite variation tendency, and newly synthesised SAG was detected. In contrast to SA, SAG showed some synergistic action in JA-related defense response in terms of MDA, PPO activity and PAL activity, which was quite different from SA. In conclusion, these results revealed both the negative effect and some synergistic effect of SA on JA-related defense of pea in response to wounding, particularly the role of SAG. © 2011 Published by Elsevier B.V.

1. Introduction Plants encounter various types of inevitable environmental stresses through their lives, such as pathogen attacks, insect herbivores, and various abiotic stresses. Therefore, they have evolved particular self-protection systems regulated by hormones to survive in such adverse conditions. The plant hormones, such as salicylic acid (SA), jasmonic acid (JA), and ethylene play instrumental roles in the signaling networks against biotic and abiotic stresses (Grant and Lamb, 2006; Von Dahl and Baldwin, 2007). One of the best characterized examples of defense-related signal cross-talk is the interaction between the SA and JA response pathways (Bostock, 2005; Beckers and Spoel, 2006).

Abbreviations: DEPC, diethylpyrocarbamate; EDTA, ethylene diamine tetraacetic acid; JA, jasmonic acid; MDA, malondialdehyde; NDGA, nordihydroguaiaretic acid; PAC, paclobutrazol; PAL, phenylalanine ammonialyase; PPO, polyphenol oxidase; ROS, reactive oxygen species; SA, salicylic acid; SAG, salicylic acid 2-O-␤-d-glucose. ∗ Corresponding author at: 301#, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China. Tel.: +86 0 10 6273 7024; fax: +86 0 10 6273 7553. E-mail address: [email protected] (W.-D. Huang). 1 The authors contribute equally to this work. 2 School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China. 0304-4238/$ – see front matter © 2011 Published by Elsevier B.V. doi:10.1016/j.scienta.2011.01.015

Salicylic acid is a key compound in the pathway that regulates resistance to fungal, bacterial, and viral pathogens. It provides a signal for the expression of pathogenesis-related proteins and other potentially protective factors induced following pathogen challenge (Beckers and Spoel, 2006). Jasmonic acid, produced in the octadecanoid pathway via lipoxygenation of linolenic acid, serves as a signal expressing a number of proteins including polyphenol oxidase (PPO, o-diphenol: oxygen oxidoreductase, EC 1.10.3.1) and proteinase inhibitors, which appear to contribute to plant resistance against many insect attackers (Bostock, 2005). Generally, it can be stated that herbivore damage as well as artificial wounding causes rapid increases in JA, triggering systemic defenses against herbivores and necrotrophic pathogens, but some exceptions (Thaler et al., 2004). In contrast, infection by biotrophic pathogens causes rapid increases in SA (Beckers and Spoel, 2006) and systemic expression of defense genes against these pathogens. It has been implied that SA- and JA-signaling pathways are ˜ antagonistic to each other (Pena-Cortés et al., 1993; Bostock, 2005; Beckers and Spoel, 2006) and there is evidence that the SA signal can strongly inhibit JA-dependent defense signaling (Felton et al., 1999). As a result of negative cross talk between SA and JA, activation of the SA response should make a plant more susceptible to attackers that are resisted via JA-dependent defenses and vice versa (Bostock, 2005; Stout et al., 2006; Spoel et al., 2007). On the one hand, the JA signal can be a potent inhibitor of SA-dependent signaling (Cui et al., 2005). However, depending on their relative

H.-R. Yang et al. / Scientia Horticulturae 128 (2011) 166–173

167

Table 1 Application of treatments used in these experiments. Treatments

Descriptions

Meanings

N+W

Application of NDGA before wounding Application of SA before wounding

Investigating the role of JA in wound defense response Investigating the effect of SA on JA-induced wound defense response through changes in MDA, PPO and PAL Investigating whether the effect of SA and NDGA on JA-induced wound defense response is additive Investigating the effect of SGA on JA-induced wound defense response through changes in MDA, PPO and PAL Testifying the role of JA on wound-induced defense response in the opposite side in contrast to N + W treatment Investigating the role of SA on JA-induced wound defense response directly Investigating the effect of SA on JA-induced defense response through changes in MDA, PPO and PAL Investigating the effect of SGA on JA-induced wound defense response through changes in MDA, PPO and PAL

S+W

S+N+W

Application of SA and PAC before wounding

P+W

Application of PAC before wounding

J+W

Application of JA before wounding

S+J+W

Application of SA and JA before JA

S+J

Application of SA before JA

P+J

Application of PAC before JA

JA, jasmonic acid; MDA, malondialdehyde; NDGA, nordihydroguaiaretic acid; PAC, paclobutrazol; PAL, phenylalanine ammonialyase; PPO, polyphenol oxidase; SA, salicylic acid; SAG, 2-O-␤-d-glucose.

concentrations or on the duration of co-accumulation (Mur et al., 2006), a more complex perspective by which the two mediators affect the plant gene expression antagonistically or synergistically (Schenk et al., 2000; Van Wees et al., 2000) emerged. It is interesting that the SA pathway may be exploited by herbivores to reduce the expression of jasmonate-dependent responses (Bostock et al., 2001). Polyphenol oxidase, which catalyzes the oxidation of odiquinones and hydroxylation of monophenols, is the major anti-nutritive protein caused by wounding, systemin and the octadecanoid pathway (Constabel et al., 1995). Quinones resulting from the mixture of PPO and phenolic substrates during feeding by grazing herbivores alkylate essential amino acids of the dietary protein, making them nutritionally unavailable to herbivores (Felton et al., 1992). The effectiveness of PPO as a defense component has been confirmed by experiments in which purified PPO plus a substrate inhibited the growth of tomato pest (Thaler et al., 2002). Tomato over-expressing PPO gene processed enhanced resistance to common cutworm (Mahanil et al., 2008). Gradually, PPO has been found to be a reliable indicator of systemic-induced responses to herbivore in solanaceous plants including tobacco (Constabel and Ryan, 1998). Phenylalanine ammonialyase (PAL) catalyzes the first committed step in phenylpropanoid biosynthesis, which is involved in defense and wound responses of plants at the level of phenol synthesis and oxidation. NDGA is an inhibitor of JA biosynthesis, and paclobutrazol (PAC) is an effective inhibitor of SA biosynthesis-related benzoic acid hydroxylase enzyme (Leon et al., 1995). In this study, the relationship between JA and SA was investigated using the inhibitory effect of NDGA and PAC (Fig. 1). The converse behavior of wound-induced JA and SA levels in pea seedlings and the negative effect of SA on JA-related resistance to wounding were evaluated, including the inhibition of malondialdehyde (MDA) level and PPO activity. The synergistic correlation was also observed, which was represented by the enhanced PAL activity accumulation in pea seedlings treated with SA prior to JA and wounding.

Fig. 1. Sketch map of the relationship among compounds in this study. JA, jasmonic acid; MDA, malondialdehyde; NDGA, nordihydroguaiaretic acid; PAC, paclobutrazol; PAL, phenylalanine ammonialyase; PPO, polyphenol oxidase; SA, salicylic acid; SAG, 2-O-ˇ-d-glucose. Dashed lines indicate inhibitory action. Grey lines indicate unknown action.

2. Materials and methods 2.1. Plant materials Pea seeds (Pisum sativum L. cv. NingXia) were grown for 10 d in a growth chamber containing pre-fertilized soil. Light intensity was maintained at 200 ␮mol m−2 s−1 , with 15 h-day/9 h-night. Temperatures were controlled at 25/22 ◦ C at 50% relative humidity. 2.2. Material treatments 2.2.1. Wounding treatment One of the upper fully expanded leaves in each seedling was quickly wounded twice at the central zone perpendicular to the main vein with a pair of scissors. After the treatment was finished, the pea leaves were immediately harvested at the time points indicated in figures, frozen in liquid nitrogen (N) and stored at −80 ◦ C until used. Leaves from intact seedlings were used as control. 2.2.2. Chemical treatment Seedlings were immerged into (±) JA (10 ␮M, Sigma) and SA (10 ␮M, Sigma) under light at 25 ◦ C for a 1 h absorption through roots as JA and SA treatments. Salicylic acid (10 ␮M, Sigma) and PAC (150 ␮M, Sigma) were absorbed for 1 h through roots before wounding treatment, respectively. Nordihydroguaiaretic acid (200 ␮M, Sigma) was sprayed on the pea seedling leaves until drips formed, for a 40 min infiltration period, then wounding treatment was conducted as described above as the inhibitor pre-treatment. After treatments were completed, the pea leaves were harvested at the time points indicated in figures, immediately frozen in liquid N2 and stored at −80 ◦ C until further use. Omission of the corresponding compounds was used as controls of the treatments above (Table 1). 2.3. Extraction and determination of SA, salicylic acid 2-O-ˇ-d-glucose (SAG) and JA content Purification and quantification of free SA were carried out according to methods previously described by Rasmussen et al. (1991). A 2 g sample of pea leaf was ground in liquid N2 and extracted twice, once with 90% methanol followed by with 100% methanol. After 5 min centrifugation at 10,000 × g, the combined supernatants were condensed by rotary evaporation at 40 ◦ C and ultrapure water added to a total volume of 4 ml. The extract was then adjusted to pH 3.0 by metaphosphate and partitioned three times in succession with ethyl acetate. The organic phase containing free SA was evaporated under a stream of N2 . The residue obtained after N2 drying was dissolved in 200 ␮l of 95% methanol. For the purification of free SA, the methanol fraction containing the free SA was spotted onto silica gel 60 A chromatography plates (Whatman) and developed in toluene/dioxane/acetic acid

168

H.-R. Yang et al. / Scientia Horticulturae 128 (2011) 166–173

(90/25/4, v/v/v). Free SA was visualized on the plate under UV light (302 nm). The fluorescent band corresponding to SA was eluted from the silica gel with 4 ml of 95% methanol and the methanol removed under a stream of N2 . The residue was dissolved in 200 ␮l of the mixture (methanol, 45%; phosphoric acid, 0.025%). Quantitative analysis was performed by high-performance liquid chromatography (HPLC) linked to fluorescence detectors (excitation wavelength = 305 nm, emission wavelength = 407 nm). Salicylic acid 2-O-ˇ-d-glucose was indirectly quantified by acid hydrolysis of the compounds remaining in a sodium acetate buffer after organic extraction and analyzing the released SA by HPLC. Acid hydrolysis was performed by incubating the samples in a boiling water bath at pH 1–1.5 for 30 min and then extracting the free SA as above. ˇ-Glucosidase digestion was performed as described by Southerton and Deverall (1990). For each sample, the dried methanol extract was re-suspended in 5 ml of water at 80 ◦ C and the solution divided into two equal portions. One portion of equal volume of 0.2 M acetate buffer (pH 4.5) containing 0.1 mg ml−1 ˇglucosidase was added, while buffer alone was added to the other portion. Both portions were incubated at 37 ◦ C overnight. After digestion, samples were acidified to pH 1–1.5 with HCl. Salicylic acid was then extracted and back extracted from each half for quantification by HPLC. Jasmonic acid was analyzed by enzyme linked immunosorbent method using monoclonal antibodies of JA according to Deng et al. (2008).

modifications. Briefly, 4 g of pea leaves was homogenized with mortar and pestle in 12 ml of extraction buffer (50 mM Tris–HCl buffer, pH 8.9, 15 mM ˇ-mercaptoethanol, 5 mM EDTA, 5 mM ascorbic acid, 10 ␮M Leupeptin, 1 mM PMSF, 0.15%, w/v, PVP). The homogenate was filtrated through four layers of cheesecloth and centrifuged at 12,000 × g for 20 min at 4 ◦ C. The supernatant was used as a source of crude enzyme for assaying PAL activity. The reaction mixture (3 ml) contained 16 mM l-phenylalanine, 50 mM Tris–HCl buffer (pH 8.9), 3.6 mM NaCl, and 0.5 ml the crude enzyme. Incubation was performed at 37 ◦ C for 1 h and the reaction was stopped by the addition of 500 ␮l of 6 M HCl. The reaction mixture was then centrifuged for 10 min at 12,000 × g, to pellet the denatured protein. The absorbance was measured at 290 nm before and after incubation. One unit of enzyme activity was defined as an increase of 0.01 in absorbance at 290 nm per minute per mg of protein under assay conditions. Protein concentration was determined using the Bradford assay kit (Bio-Rad, Richmond, VA, USA) and BSA as standard protein according to the manufacturer’s instructions.

2.4. MDA content assay

3. Results

Malondialdehyde content was measured as described by Heath and Packer (1968). The sample of pea leaves (0.3 g) were homogenized in 5 ml of 0.1% trichloroacetic acid and centrifuged at 10,000 × g for 5 min. After centrifugation, 1 ml of supernatant was mixed with 4 ml of 0.5% thiobarbituric acid, and the mixture incubated in boiling water for 30 min, after which it was transferred to an ice bath to stop the reaction. The absorbance was read at 532 nm and adjusted for non-specific absorbance to 600 nm. Malondialdehyde content was estimated by using an extinction coefficient of 155 mmol L−1 cm−1 .

3.1. Changes of endogenous SA level in response to wounding and exogenous JA in pea seedlings

2.5. PPO enzyme assay Polyphenol oxidase was extracted and assayed according to an optimized procedure for banana roots of Sherman et al. (1991). Seived samples of pea leaves (0.5 g) were mixed with 1% (w/v) insoluble polyvinylpolypyrolidone and suspended in 10 ml ice cold 0.2 M sodium phosphate buffer (pH 7.0) containing 0.25% Triton X-100. The samples were centrifuged at 24,000 × g for 30 min at 4 ◦ C. The supernatant was collected and stored at −20 ◦ C until assayed for enzyme activity. For the photometric assay, 10 ␮l of the enzyme extract was mixed with 1 ml of a 5 mM dopamine solution in 50 mM sodium phosphate buffer (pH 7.0) at 27 ◦ C. The change in the optical density of the reaction mixture was immediately recorded at 480 nm for 2 min. The blank consisted of the reaction mixture without the enzyme extract. Polyphenol oxidase activity was determined from the linear part of the reaction curve over time and expressed as the change in optical density per minute and per protein content of the sample (OD480 nm min−1 mg protein−1 ). Protein concentration was determined using the Bradford assay kit (Bio-Rad, Richmond, VA, USA) and bovine serum albumin (BSA) as standard protein according to the manufacturer’s instructions. 2.6. Assay for PAL activity Phenylalanine ammonialyase activity was measured as previously described by Solecka and Kacperska (2003) with some

2.7. Statistical analysis All treatments were repeated at least three times and all samples were analyzed three times. Each replicate consists of 30 seedlings. Student–Newman–Keuls’s test was used to separate the means for P < 0.05.

Changes of endogenous SA content in pea seedling leaves after treatment with wounding and exogenous JA are presented (Fig. 2). After wounding, endogenous free SA level declined immediately and evidently, reached its minimum at 5 h, then recovered to its control level at 24 h (Fig. 2A). Subsequently, a second decrement of free SA content occurred at 36 h, and decreased to its minimum at 48 h. Finally, the level of endogenous free SA recovered to its control level. Correspondingly, the changes in SAG content, a major conjugated form of SA, presented a reverse trend in response to wounding, which showed two inducible peaks above the control level at 5 h and 48 h, respectively (Fig. 2C). Free SA level in response to exogenous JA decreased immediately, begun to increase at 8 h, and finally was close to the control level at 60 h (Fig. 2B). As expected, the level of endogenous SAG increased at the beginning and maintained the same level after 8 h (Fig. 2D). However, when treated with wounding and JA, the total SA content (including free SA and SAG) exhibited an increasing trend after 12 h (Fig. 2E,F). 3.2. Endogenous JA level induced by wounding was inhibited by applied SA in pea A response curve of JA caused by wounding was composed of two phases (Fig. 3A). Once the pea leaves were injured by wounding, endogenous JA content increased rapidly and reached its maximum of accumulation at 1 h, whose value was 200 ng g FW−1 . Then it declined promptly, while followed by the subsequent increment to the second peak at 48 h. After pretreatment with SA before wounding, a peak of endogenous JA accumulation at 1 h was induced and the peak value was only less than one third of that induced by wounding, but the peak at 48 h disappeared (Fig. 3B). PAC was observed to cause a similar result just like SA when it was applied to the plants before wounding (Fig. 3C). Besides, when the seedlings were treated with SA, it was found that the endogenous

H.-R. Yang et al. / Scientia Horticulturae 128 (2011) 166–173

400 Free SA content (ng.g FW-1)

Free SA content (ng.g FW-1)

320 A 280 240 200 160 120

B 300 200 100 0

0

8

16

24

32

40

48

56

64

0

8

Time after treatment (h)

24

32

40

48

56

64

56

64

60

70

Time after treatment (h)

C

SAG content (ng.g FW-1)

SAG content (ng.g FW-1)

16

900

800 700 600 500 400

D 800 700 600 500 400

0

8

16

24

32

40

48

56

64

0

Time after treatment (h)

8

16

24

32

40

48

Time after treatment (h)

1000

1100

Total SA content (ng.g FW-1)

Total SA content (ng.g FW-1)

169

E 900

800

700

F 1000 900 800 700

0

8

16

24

32

40

48

56

Time after treatment (h)

64

0

10

20 30 40 50 Time after treatment (h)

Fig. 2. Changes of endogenous SA after treatment with mechanical wounding (A, C) and 10 ␮M JA (B, D) in pea seedling leaves. Controls (䊉) and leaves from the treated pea seedlings () were obtained as mentioned in Section 2. JA, jasmonic acid; SA, salicylic acid; SAG, salicylic acid 2-O-ˇ-d-glucose. Bars show the standard errors (SE). FW = fresh weight.

JA level decreased below the control level at the beginning and recovered to the control level instantly (Fig. 3D). 3.3. Wounding/JA-inducible MDA content was enhanced by SA application Plants injury caused by various stresses is mainly the result of the action of reactive oxygen species (ROS). The generation of ROS induces lipid peroxidation, which leads to a loss of membrane integrity and tissue necrosis, as well as induction of phytoalexins (Resterucci et al., 1996). Malondialdehyde was the toxic product of lipid peroxidation. Therefore, MDA level was used as an indicator of the ability of defense against wounding in this study. As shown in Fig. 4, if spraying NDGA before wounding, there was an obvious increase in MDA content in contrast to wounding treatment. Surprisingly, MDA content induced by the application of SA prior to wounding was about 86% higher than that induced by wounding, even 12% higher than that resulted from the application of NDGA before wounding. However, after applying a combination of NDGA and SA before wounding, the MDA level showed no significant difference comparing with pretreatment with NDGA or SA,

respectively, which suggested that no synergistic action occurred between the two compounds. Besides, it was noted that the MDA content induced by applied PAC prior to wounding was also significant higher than that caused by wounding. The MDA level induced by JA showed no dramatic difference compared with wounding (Fig. 4). But after the seedlings were sprayed by JA before wounding, MDA level was 19% lower than that induced by wounding. In addition, after a combination of exogenous JA and SA application, MDA content was also significantly higher than that induced by applying JA alone (about 35% higher). Application of PAC combined with JA also obtained similar result. By contrast with wounding, there was also no striking alteration in MDA content after treatment with SA alone. 3.4. Influence of SA on wound/JA-related defense response Polyphenol oxidase generally catalyzes the oxidation of phenolic compounds to quinones. In this experiment, PPO activity was sharply stimulated by wounding (Fig. 5) and wounding plus JA (Fig. 5). Compared with wounding treatment, PPO activity declined significantly after having been sprayed with NDGA before wound-

170

H.-R. Yang et al. / Scientia Horticulturae 128 (2011) 166–173

A

B JA content (ng.g FW-1)

JA content (ng.g FW-1)

80 200 150 100 50 0

60 40 20 0

0

8

16

24

32

40

48

56

64

0

8

Time after treatment (h)

24

32

40

48

56

64

56

64

Time after treatment (h)

60

60 D

C 50

JA content (ng.g FW-1)

JA content (ng.g FW-1)

16

40 30 20 10 0

50 40 30 20 10 0

0

8

16

24

32

40

48

56

64

0

8

Time after treatment (h)

16

24

32

40

48

Time after treatment (h)

Fig. 3. Effect of SA and its inhibitor on JA accumulation induced by wounding. A, Wounding; B, exogenous application of 10 ␮M SA before wounding; C, exogenous application of 150 ␮M PAC before wounding; D, applied 10 ␮M SA. Controls (䊉) and leaves from the treated pea seedlings () were obtained as mentioned in Section 2. Bars show the standard errors (SE). JA, jasmonic acid; SA, salicylic acid; PAC, paclobutrazol. FW = fresh weight.

ing. When applying SA and PAC before wounding, respectively, wound-inducible PPO activity was impaired obviously. The statistical interaction between wounding and SA indicated that their effects on PPO levels were not superimposable: SA alone did not affect PPO activity, but suppressed the PPO activity induced by wounding. PPO activity generally presented the same pattern in the exogenous JA treated seedlings as in the wounded ones.

5

fg

MDA(nmol/g Fw)

e d

3

c

c 2

e

c b

a

1

PPO activity(U.min-1.mg pro-1)

f

100

g

g 4

Phenylalanine ammonialyase catalyzes the deamination of phenylalanine to produce l-trans-cinnamic acid which is the key precursor of the phenylpropanoid biosynthesis pathway involved in the plant’s production of defense-related secondary metabolites, including SA, phytoalexins, and lignin bricks (La Camera et al., 2004). In this study, PAL activity increased intensely in response to wounding and exogenous JA (Fig. 6). But when pretreatment with NDGA was done before wounding, the enhanced PAL activity

e 80

d

d c

60

c

bc 40

a

c

b ab

a

a

20

0

0 CK

W

N+W S+W S+N+W P+W

J

J+W S+J+W S+J

P+J

S

Treatments Fig. 4. Effect of inhibitors and SA application on MDA content induced by wounding and 10 ␮M JA in pea leaves. Leaves from intact pea seedlings were assayed as controls (CK). Malondialdehyde was determined after these treatments: 200 ␮M NDGA (N), 10 ␮M SA (S), 150 ␮M PAC (P), or a combination of both chemicals before wounding (W) and 10 ␮M JA (J). Leaves were sampled immediately for MDA content determination after these treatments were finished. Bars show the standard errors (SE). Different letters indicate a statistical difference at P ≤ 0.05 according to Student–Newman–Keuls test. JA, jasmonic acid; MDA, malondialdehyde; NDGA, nordihydroguaiaretic acid; PAC, paclobutrazol; SA, salicylic acid. FW = fresh weight.

CK

W

N+W S+W S+N+W P+W

J

J+W S+J+W S+J

P+J

S

Treatments Fig. 5. Effect of inhibitors and SA application on PPO activity induced by wounding and 10 ␮M JA. Leaves from intact pea seedlings were assayed as controls (CK). Polyphenol oxidase activity was determined after these treatments: 200 ␮M NDGA (N), 10 ␮M SA (S), 150 ␮M PAC (P), or a combination of both chemicals before treatment with wounding (W) and 10 ␮M JA (J). Leaves were sampled at 36 h for PPO activity assay after these treatments were finished. Bars show the standard errors (SE). Different letters indicate a statistical difference at P ≤ 0.05 according to Student–Newman–Keuls test. JA, jasmonic acid; NDGA, nordihydroguaiaretic acid; PAC, paclobutrazol; PPO, polyphenol oxidase; SA, salicylic acid.

H.-R. Yang et al. / Scientia Horticulturae 128 (2011) 166–173

f ef 30

e

de

d

cd

c 20

b

bc a

a 10

Free SA content (ng.g FW-1)

PAL Activity (U.mg Pro.min-1)

280

g

40

171

A 240 200 160 120

0 CK

W

N+W S+W P+W S+N+W

J

J+W S+J+W S+J

P+J

0

S

8

Treatments

was reduced. Applied SA also had some positive effect on the PAL activity: it relieved the NDGA-caused inhibition of the PAL activity induced by wounding. Additionally, application of PAC before wounding and JA induced lower level of PAL activity compared with wounding and exogenous JA treatments.

It has been known that JA functions as an important wounding signal and could initiate wounding defense response (Wasternack, 2007). When applied proper concentration of JA to pea seedlings, ROS burst immediately (Liu et al., 2005). Reactive oxygen species attack the bio-membrane, leading to increase of MDA content (Fig. 4) and injury of plant tissues; meanwhile, ROS may act as a second messenger and stimulate plant defense response (OrazcoCárdenas et al., 2001), including the activation of antioxidant enzymes to scavenge ROS (Liu et al., 2005). It was found that the MDA level increased when JA biosynthesis was inhibited (Fig. 4). Actually, applied NDGA alone did not decrease MDA content, but had little effect on MDA content compared with intact seedlings (data not shown). In intact plants, JA biosynthesis did not happen; in wounded plants, JA biosynthesis happened and was inhibited by NDGA. In this regard, it was suggested that JA was close related to the resistance to wounding. However, the attenuated MDA level induced by JA was suppressed by pre-treatment with SA (Fig. 4). Thus, it may be concluded that SA impaired the resistance to wounding of pea seedlings by means of suppressing the action of JA. Hereby, there existed the antagonism between JA and SA during the JA-inducible resistance to wounding in pea seedlings. Besides, PPO activity was up-regulated by JA, but not by SA (Fig. 5;

40

48

56

64

56

64

56

64

B 720 660 600 540 480 0

8

16

24

32

40

48

Time after treatment (h)

Total SA content (ng.g FW-1)

4. Discussion

32

780

3.5. Effect of PAC on wound-induced SA content In order to test the effect of PAC on SA biosynthesis induced by wounding, the following experiments were conducted. Comparing with wounding treatment, the free SA content had no significant difference after pretreatment with PAC (Fig. 7A). However, the enhanced SAG content induced by wounding at 48 h was inhibited to some extent by pre-treating with PAC (Fig. 7B), and the increasing trend of total SA content (including free SA and SAG) induced by wounding disappeared (Fig. 7C).

24

Time after treatment (h)

SAG content (ng.g FW-1)

Fig. 6. Effect of inhibitors and SA application on PAL activity induced by wounding and 10 ␮M JA. Leaves from intact pea seedlings were assayed as controls (CK). Phenylalanine ammonialyase activity was determined after these treatments: 200 ␮M NDGA (N), 10 ␮M SA (S), 150 ␮M PAC (P), or a combination of both chemicals before wounding (W) and 10 ␮M JA (J). Leaves were sampled at 48 h for PAL activity assay after these treatments were finished. Bars show the standard errors (SE). Different letters indicate a statistical difference at P ≤ 0.05 according to Student–Newman–Keuls test. JA, jasmonic acid; NDGA, nordihydroguaiaretic acid; PAC, paclobutrazol; PAL, phenylalanine ammonialyase; SA, salicylic acid.

16

920

C

880 840 800 760 720 0

8

16

24

32

40

48

Time after treatment (h) Fig. 7. Effect of PAC on SA content induced by wounding. Leaves from intact seedlings were used as controls (䊉). Leaves treated with wounding () and PAC before wounding ( ) were obtained as mentioned in Section 2. Bars show the standard errors (SE). SA, salicylic acid; PAC, paclobutrazol. FW = fresh weight.

Thaler et al., 2002). However, based on the fact that this woundinduced PPO activity was suppressed by SA (Fig. 5), it could be concluded further that SA had negative effect on JA-related defense response. During evolution process, plants have preserved the SAmediated pathogen-induced signal pathway and the JA-mediated wound-induced signal pathway (De Vos et al., 2005; Koornneef et al., 2008). Plants fine-tune the two pathways by controlling the content of JA and SA to defense against stresses (Beckers and Spoel, 2006). In the present study, it demonstrated that changes of endogenous free SA and JA presented the opposite trend in the wound response of pea seedlings (Figs. 2A–D and 3D), which has also been observed in rice (Felton et al., 1999; Lee et al., 2004).

172

H.-R. Yang et al. / Scientia Horticulturae 128 (2011) 166–173

The second peak of JA accumulation induced by wounding at 48 h, which was due to the JA biosynthesis (Yang et al., 2009), were suppressed by the application of SA prior to wounding (Fig. 3B). It has been reported that SA was considered to act as a competitive inhibitor of allene oxide synthase, a key enzyme related to JA biosynthesis (Norton et al., 2007). Therefore, it could be explained that SA suppressed JA content by means of blocking its biosynthesis. What is interesting is that PAC which conducted as an inhibitor of SA synthesis also inhibited the JA accumulation as SA did (Fig. 3C). It might be the reason why PAC operated more as a chelator of conjugated SA production (SAG) than free SA in some stresses (Liu et al., 2006). Previously, SAG was considered to be as active as free SA in the induction of PR-1 gene expression and the establishment of SAR in tobacco (Hennig et al., 1993), and conjugated SA could also promote thermo-tolerance as effectively as free SA (Liu et al., 2006). In this study, the increasing SAG content corresponded with the declining SA content, leading to a steady value of total SA (Fig. 2E and F), which may indicate the conversion from SA to SAG. However, in the later phase, total SA content increased gradually, which implied that SA synthesis occurred (Fig. 2E and F). And the experiments applying SA inhibitor suggested that the newly synthesized SA was converted to SAG (Fig. 7B and C). In wounded pea seedlings, change of SAG content was consistence with JA content during the later phase (Figs. 2 and 3). Salicylic acid 2-O-ˇ-d-glucose operated oppositely in the wound response of pea in contrast to SA. Inhibition of SAG caused the increase of MDA content (Fig. 4), the decline of PPO activity (Fig. 5) and PAL activity (Fig. 6), which suggested that SAG may play important role in wound defense response. This may be an efficient strategy for plants to defense wounding. Except negative effect of SA on JA-related defense response, synergic action was also observed. It has been reported that the activation of PAL activity could be induced by wounding (Campos et al., 2004). Phenylalanine ammonialyase activity could be induced not only by wounding but also by JA and SA (Fig. 6). When applying SA in a combination with wounding or JA, the enhancement of PAL activity induced by wounding and JA were observed. Besides, SA recovered the reduced PAL activity caused by NDGA after wounding. In this regard, it was considered that JA and SA induced phenolic metabolism related to PAL appears to be induced by the same signals (Campos-Vargas and Saltveit, 2002). In conclusion, SA presented negative effect on JA-related defense response to wounding in pea seedlings: firstly, the trends in change of endogenous level of JA and SA in wound response were opposite; secondly, SA had negative effect on JA-related resistance to wounding in terms of the increase in MDA level and the inhibition of PPO activity. However, synergy between JA and SA also existed in terms of PAL activity. Moreover, what worth to notice is that SAG acted synergistically towards JA in wound defense response. Acknowledgements This work was supported by National Natural Science Foundation of China (No. 30611468) and a major program of Beijing Municipal Science and Technology Commission (No. D07060500160701). The authors would like to thank Dr. Yuanyuan Ma (China Agriculture University, Beijing) for critical reading of this manuscript. References Beckers, G.J.M., Spoel, S.H., 2006. Fine-tuning plant defence signalling: salicylate versus jasmonate. Plant Biol. 8, 1–10. Bostock, R.M., 2005. Signal crosstalk and induced resistance: straddling the line between cost and benefit. Annu. Rev. Phytopathol. 43, 545–580. Bostock, R.M., Karban, R., Thaler, J.S., Weyman, P.D., Gilchrist, D., 2001. Signal interactions in induced resistance to pathogens and insect herbivores. Eur. J. Plant Pathol. 107, 103–111.

Campos, R., Nonogaki, H., Suslow, T., Saltveit, M.E., 2004. Isolation and characterization of a wound inducible phenylalanine ammonia-lyase gene (LsPAL1) from Romaine lettuce leaves. Physiol. Plant. 121, 429–438. Campos-Vargas, R., Saltveit, M.E., 2002. Involvement of putative chemical wound signals in the induction of phenolic metabolism in wounded lettuce. Physiol. Plant. 114, 73–84. Constabel, C.P., Bergey, D.R., Ryan, C.A., 1995. Systemin activates synthesis of woundinducible tomato leaf polyphenol oxidase via the octadecanoid defense signaling pathway. Proc. Natl. Acad. Sci. U.S.A. 92, 407–411. Constabel, C.P., Ryan, C.A., 1998. A survey of wound- and methyl jasmonate-induced leaf polyphenol oxidase in crop plants. Phytochemistry 47, 507–511. Cui, J., Bahrami, A.K., Pringle, E.G., Hernandez-Guzman, G., Bender, C.L., Pierce, N.E., Ausubel, F.M., 2005. Pseudomonas syringae manipulates systemic plant defenses against pathogens and herbivores. Proc. Natl. Acad. Sci. U.S.A. 102, 1791– 1796. De Vos, M., Van Oosten, R., Van Poecke, R.M.P., Van Pelt, J.A., Pozo, M.J., Mueller, M.J., Buchala, A.J., Métraux, J., Van Loon, L.C., Dicke, M., Pieterse, C.M.J., 2005. Signal signature and transcriptome changes of Arabidopsis during pathogen and insect attack. Mol. Plant. Microbe Interact. 18, 923–937. Deng, A.X., Tan, W.M., He, S.P., Liu, W., Nan, T.G., Li, Z.H., Li, Q.X., Wang, B.M., 2008. Monoclonal antibody-based enzyme linked immunosorbent assay for the analysis of jasmonates in plants. J. Integr. Plant Biol. 50, 1–7. Felton, G.W., Donato, K., Broadway, R.M., Duffey, S.S., 1992. Impact of oxidized plant phenolics on the nutritional quality of dietar protein to a noctuid herbivore Spodoptera exigua. J. Insect. Physiol. 38, 277–285. Felton, G.W., Korth, K.L., Bi, J.L., Wesley, S.V., Huhman, D.V., Mathews, M.C., Murphy, J.B., Lamb, C., Dixon, R.A., 1999. Inverse relationship between systemic resistance of plants to microorganisms and to insect herbivory. Curr. Biol. 9, 317–320. Grant, M.R., Lamb, C., 2006. Systemic immunity. Curr. Opin. Plant Biol. 9, 414–420. Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125, 189–198. Hennig, J., Malamy, J., Grynkiewicz, G., Indulski, J., Klessig, D.F., 1993. Interconversion of the salicylic acid signal and its glucoside in tobacco. Plant J. 4, 593–600. Koornneef, A., Leon-Reyes, A., Ritsema, T., Verhage, A., Den Otter, F.C., Van Loon, L.C., Pieterse, C.M.J., 2008. Kinetics of salicylate-mediated suppression of jasmonate signaling reveal a role for redox modulation. Plant Physiol. 147, 1358–1368. La Camera, S., Gouzert, G., Dhondt, S., Hoffmann, L., Fritig, B., Legrand, M., Heitz, T., 2004. Metabolic reprogramming in plant innate immunity: the contributions of phenylpropanoid and oxylipins pathways. Immunol. Rev. 198, 267–281. Lee, A., Cho, K., Jang, S., Rakwal, R., Iwahashi, H., Agrawal, G.K., Shim, J., Han, O., 2004. Inverse correlation between jasmonic acid and salicylic acid during early wound response in rice. Biochem. Biophys. Res. Commun. 318, 734–738. Leon, J., Yalpani, N., Lawton, M.A., Raskin, I., Shulaev, V., 1995. Benzoic acid 2-hydroxylase, a soluble oxygenase from tobacco, catalyzes salicylic acid biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 92, 10413–10417. Liu, H.T., Liu, Y.Y., Pan, Q.H., Yang, H.R., Zhan, J.C., Huang, W.D., 2006. Novel interrelationship between salicylic acid, abscisic acid, and PIP2 -specific phospholipase C in heat acclimation-induced thermotolerance in pea leaves. J. Exp. Bot. 57, 3337–3347. Liu, Y., Huang, W.D., Zhan, J.C., Pan, Q.H., 2005. Systemic induction of H2 O2 in pea seedlings pretreated by wounding and exogenous jasmonic acid. Sci. China Ser. C 48, 202–212. Mahanil, S., Attajarusit, J., Stout, M.J., Thipyapong, P., 2008. Overexpression of tomato polyphenol oxidase increases resistance to common cutworm. Plant Sci. 174, 456–466. Mur, L.A., Kenton, P., Atzorn, R., Miersch, R., Wasternack, C., 2006. The outcomes of concentration-specific interactions between salicylate and jasmonate signalling include synergy, antagonism, and oxidative stress leading to cell death. Plant Physiol. 140, 249–262. Norton, G., Pappusamy, A., Yusof, F., Pujade-Renaud, V., Perkins, M., Griffiths, D., Jones, H., 2007. Characterisation of recombinant Hevea brasiliensis allene oxide synthase: effects of cycloxygenase inhibitors, lipoxygenase inhibitors and salicylates on enzyme activity. Plant Physiol. Biochem. 45, 129–138. Orazco-Cárdenas, M.L., Narváez-Vásquez, J., Ryan, C.A., 2001. Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate. Plant Cell 13, 179–191. ˜ Pena-Cortés, H., Albrecht, T., Prat, S., Weiler, E.W., Willmitzer, L., 1993. Aspirin prevents wound-induced gene expression in tomato leaves by blocking jasmonic acid biosynthesis. Planta 191, 123–128. Rasmussen, J.B., Hammerschmidt, R., Zook, M.N., 1991. Systemic induction of salicylic acid accumulation in cucumber after inoculation with Pseudomonas syringae pv Syringae. Plant Physiol. 97, 1342–1347. Resterucci, C., Stallaert, V., Milat, M.L., Pugin, A., Ricci, P., Blein, J.P., 1996. Relationship between active oxygen species, lipid peroxidation, necrosis, and phytoalexin production induced by elicitins in Nicotiana. Plant Physiol. 111, 885–891. Schenk, P.M., Kazan, K., Wilson, I., Anderson, J.P., Richmond, T., Somervile, S.C., Manners, J.M., 2000. Coordinated plant defense responses in Arabidopsis revealed by microarray analysis. Proc. Natl. Acad. Sci. U.S.A. 97, 11655–11660. Sherman, T., Vaughn, K., Duke, S., 1991. A limited survey of the phylogenetic distribution of polyphenol oxidase. Phytochemistry 30, 2499–2506. Spoel, S.H., Johnson, J.S., Dong, X., 2007. Regulation of tradeoffs between plant defenses against pathogens with different lifestyles. Proc. Natl. Acad. Sci. U.S.A. 104, 18842–18847. Solecka, D., Kacperska, A., 2003. Phenylpropanoid deficiency affects the course of plant acclimation to cold. Physiol. Plant. 119, 253–262.

H.-R. Yang et al. / Scientia Horticulturae 128 (2011) 166–173 Southerton, S.G., Deverall, B.J., 1990. Changes in phenolic acid levels in wheat leaves expressing resistance to Puccinia recondite f. sp Tritici. Physiol. Mol. Plant Pathol. 37, 437–450. Stout, M.J., Thaler, J.S., Thomma, B.P.H.J., 2006. Plant-mediated interactions between pathogenic microorganisms and herbivorous arthropods. Annu. Rev. Entomol. 51, 663–689. Thaler, J.S., Karban, R., Ullman, D.E., Boege, K., Bostock, R.M., 2002. Cross-talk between jasmonate and salicylate plant defense pathways: effects on several plant parasites. Oecologia 131, 227–235. Thaler, J.S., Owen, B., Higgins, V.J., 2004. The role of the jasmonate response in plant susceptibility to diverse pathogens with a range of lifestyles. Plant Physiol. 135, 530–538.

173

Van Wees, S.C.M., De Swart, E.A.M., Van Pelt, J.A., Van Loon, L.C., Pieterse, C.M.J., 2000. Enhancement of induced disease resistance by simultaneous activation of salicylate- and jasmonate-dependent defense pathways in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U.S.A. 97, 8711–8716. Von Dahl, C.C., Baldwin, I.T., 2007. Deciphering the role of ethylene in plant–herbivore interactions. J. Plant Growth Regul. 26, 201–209. Wasternack, C., 2007. Jasmonates: an update on biosynthesis, signal transduction and action in plant stress response, growth and development. Ann. Bot. 100, 681–697. Yang, H.R., Tang, K., Liu, H.T., Pan, Q.H., Huang, W.D., 2009. Jasmonic acid is induced in a biphasic manner in response of pea seedlings to wounding. J. Integr. Plant Biol. 51, 562–573.