- Email: [email protected]
Differential elicitation of proteases and protease inhibitors in two different genotypes of chickpea (Cicer arietinum) by salicylic acid and spermine
ARTICLE IN PRESS Journal of Plant Physiology 166 (2009) 1015—1022
www.elsevier.de/jplph
Differential elicitation of proteases and protease inhibitors in two different genotypes of chickpea (Cicer arietinum) by salicylic acid and spermine Shamrao Rajua, Senigala K. Jayalakshmib, Kuruba Sreeramulua, a
Department of Biochemistry, Gulbarga University, Gulbarga 585106, India Krishi Vignan Kendra, Agriculture Research Station, Gulbarga 585106, India
b
Received 14 July 2008; received in revised form 14 December 2008; accepted 15 December 2008
KEYWORDS Chickpea (Cicer arietinum); Protease; Protease inhibitors; Salicylic acid; Spermine
Summary Elicitation of proteases and protease inhibitors (PIs) by salicylic acid (SA) and spermine (Spm) was investigated in roots and shoots of two different genotypes of chickpea cultivars ICCV10 and L550, which were resistant and susceptible to wilt disease, respectively. SA and Spm were found to suppress the elicitation of proteases in the resistant cv, whereas they induce it in susceptible cv. Elicitation of new trypsin and chymotrypsin inhibitors was observed in the roots and shoots of resistant cv treated with SA and Spm. However, no such elicitation was observed in susceptible cv. These results show for the first time that SA and Spm could elicit synthesis of new PIs capable of inhibiting the proteases of insect Helicoverpa armigera and Fusarium oxysporum, wilt causing pathogen. Antifungal property of root extract of resistant cv increased following treatment of seedling with SA and Spm compared with susceptible cv. & 2008 Elsevier GmbH. All rights reserved.
Introduction The proteolytic enzymes of plants play an important and diverse role in defense against Abbreviations: BApNA, N-benzoyl DL-arginine p-nitroanilide HCl; CTI, chymotrypsin inhibitor; FOX, Fusarium oxysporum f. sp. ciceri; HGP, Helicoverpa armigera gut protease; PIs, protease inhibitors; SA, salicylic acid; Spm, spermine; TEE, L-tyrosine ethyl ester; TI, trypsin inhibitor. Corresponding author. Tel.: +91 8472 263289; fax: +91 8472 263202. E-mail address: [email protected] (K. Sreeramulu).
pathogens (Xia et al., 2004). They could contribute to defense in different ways, acting at the levels of perception, signaling and execution (Vander Hoorn and Jones, 2004). Plant protease inhibitors (PIs) are known to play an important role in plants defense against insect pests and pathogens as well as in regulation of endogenous proteinases. They are one of the major storage proteins in seeds (Moslov et al., 1976; Birk, 2003; Shewry, 2003) and are of interest as potential sources of resistance against pests and pathogens (Konarev et al., 2002; Korsinczky et al., 2004). Induction of trypsin and
0176-1617/$ - see front matter & 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2008.12.005
ARTICLE IN PRESS 1016 chymotrypsin inhibitory activities by aphid infestation, mechanical wounding, abscisic acid and jasmonic acid was studied in barley leaves (Casaretto et al., 2004). However, they appear to play key roles in the regulation of biological processes in plants, such as the recognition of pathogens and pests and the induction of effective defense responses. Salicylic acid and spermine (Spm) have been implicated in the signal transduction pathway leading to plant resistance to various pathogens (Yamakawa et al., 1998; van Loon et al., 2006). Metraux et al. (1990) found that foliar or drench application of iso-nicotinic acid (INA) induces systemic resistance in cucumber against Colletotrichum lagenarium. Since then, resistance induced by salicylic acid and INA was demonstrated in a variety of plant pathogen interactions. Polyamines, Spm and spermidine are found in a wide range of organisms from bacteria to plants and animals. They are basically small molecules implicated in the promotion of plant growth and development by activating the synthesis of nucleic acids (Walters, 2003). It has been reported that Spm is an endogenous signal molecule for the accumulation of acidic and basic PR proteins; Spm and SA act independently for PR protein induction (Yamakawa et al., 1998). However, the role of SA and Spm in the development of systemic acquired resistance (SAR) has been well documented in chickpea (Raju et al., 2007), but their effects on the induction of protease and PIs are not reported. Therefore, in the present investigation, an attempt has been made to examine the effects of SA and Spm on induction of proteases and PIs in two different genotypes of chickpea cultivars, which are susceptible and resistant to wilt disease caused by Fusarium oxysporum f. sp. ciceri. Further, these PIs were found to be potent inhibitors of proteases of Helicoverpa armigera and F. oxysporum.
Materials and methods Seeds of chickpea (Cicer arietinum L.) cv L-550 and ICCV-10, susceptible and resistant to wilt disease, respectively, were procured from the Agricultural Research Station, Gulbarga, Karnataka, India. Trypsin, chymotrypsin, BApNA and TEE were purchased from Sigma Chemicals Co. (St. Louis, USA). X-ray films are of Kodak make. Other reagents were of analytical grade. Seed treatments with SA, Spm and germination experiments were performed as described in our earlier report (Raju et al., 2007).
S. Raju et al.
Extraction of protease and protease inhibitors from chickpea plants The roots and shoots were ground in a pre-chilled pestle and mortar in an ice-cold 50 mM phosphate buffer pH (7.8). The extract was centrifuged at 4 1C for 25 min at 10,000g. The resulting supernatant was used as an enzyme source for further determination of protease activities. Trypsin and chymotrypsin inhibitors were extracted by the modified method of Casaretto et al. (2004). Protein in the supernatant was estimated by Lowry et al. (1951).
Extraction of HGPs and FOX proteases Third instar larvae of Helicoverpa armigera were dissected to isolate the midgut tissue, which was immediately frozen in liquid nitrogen and stored at 70 1C. The midgut tissue was homogenized and mixed with 0.1 M Tris–HCl buffer, pH (8.8), for 2 h at 10 1C. The suspension was centrifuged at 4 1C for 20 min at 10,000g and the resulting supernatant was used as a source of HGP. HGP solution was prepared fresh before use by extracting the frozen midgut (Giri et al., 2003). The isoforms of HGP were separated by electroelution from the preparative gels. FOX was grown for 4–5 d at 28 1C in 250 mL Erlenmeyer flask containing 50 mL of Sabouraud’s dextrose broth. The fungal cells were separated by filtration and the filtrate was used as enzyme source, and assayed with casein as substrate. The isoforms of FOX were separated by electroelution from the preparative gels.
Protease and PI activity assay Protease activity was measured by the modified Kunitz caesinolytic assay (Belew and Porath, 1970). One unit of protease activity was defined as an increase in absorbance by one optical density (OD) of TCA soluble casein hydrolysis products liberated by protease action at 280 nm/min at 27 1C under given assay conditions. Trypsin activity was measured using the synthetic substrate BApNA as described by Erlanger et al. (1964). One unit of trypsin activity was defined as the amount of enzyme that increases absorbance by 0.01 OD at 410 nm at 27 1C under given assay conditions. Chymotrypsin activity was determined using the synthetic substrate TEE by the method described by Schwert and Takenaka (1955). One unit of enzyme activity is equal to the hydrolysis of 1 mM of TEE at 235 nm/min at 27 1C. For the inhibitor assay, a suitable volume of PI extract from chickpea was mixed with commercial bovine
ARTICLE IN PRESS Elicitation of protease and protease inhibitors in chickpea trypsin (15 mg)/chymotrypsin (5 mg) or with HGP/FOX protease (20 mg) and incubated for 15 min. Protease activity was measured by excluding PI extract from the above steps. One PI unit is defined as the amount of inhibitor that inhibits 1 unit of protease activity.
Electrophoretic visualization of proteases and PI Root and shoot extracts of chickpea seedlings treated with SA and Spm or HGP or proteases of FOX were electrophoresed on a vertical slab gel using a discontinuous buffer system by Davis (1964). For protease activity the gel was equilibrated with 0.2 M Tris–HCl buffer pH 7.8 for 10 min, and overlaid on an undeveloped X-ray film for 1 h at 37 1C. The films were then washed with warm water, and protease activity bands were visualized as hydrolyzed gelatin. For visualization of HGPs and proteases of FOX, the gel was equilibrated in 0.2 M glycine/NaOH buffer, pH (10.0) for 10–15 min and then overlaid on X-ray film for 40–45 min. TI and CTIs were visualized after native PAGE using X-ray film contact print technique of Pichare and Kachole (1994). In brief, the native gels were equilibrated in 0.1 M Tris–HCl buffer, pH (7.8) for 10–15 min, followed by incubation in the trypsin solution (0.1 mg/mL) or chymotrypsin (20 mg/ mL) or HGP (20 mg) or FOX protease (20 mg) for 15 min at 37 1C in a shaking water bath. The gels were then washed with the same buffer and placed on a piece of undeveloped X-ray film for 3–5 min; the films were then washed with water, and inhibitor activity bands were visualized as unhydrolyzed gelatin on X-ray films. The X-ray films were developed using Kodak 163 DA developer.
Bioassay for the determination of antifungal activity FOX growth inhibition was studied using a hyphal extension inhibition assay of Roberts and Selitrennikoff (1986). Sterile filter paper discs with filter-sterilized inhibitor extracts of roots and shoots treated with water, SA and Spm, were placed on the periphery of the advancing fungal mycelium. The plates were further incubated at 28 1C for 24 h and observed for crescents of retarded mycelial growth.
Results Protease activity It was found that the protease activity was inhibited by 32% and 4.8% in roots and 60% and
1017
Table 1. Protease activity in roots and shoots of resistant and susceptible cultivars of chickpea treated with SA and Spm for 8 d. Cultivar
Protease activity (mg
1
tissue)
Control
SA
Spm
ICCV10 Roots Shoots
4.3470.11 5.9270.17
2.9570.11 2.3270.13***
4.1374.09 2.5570.19**
L-550 Roots Shoots
2.5275.7 5.2570.20
4.870.23*** 5.8277.26
3.570.17 5.3470.11
Each value in the table represents the mean7S.E from three independent experiments. Differences from control values were significant at Po0.05, **Po0.01, ***Po0.001 from control, according to ANOVA variance for regression.
56% in shoots of resistant cv when treated with SA and Spm, respectively. On the other hand, the protease activities were increased by 47% and 28% in roots, 9.7% and 1.6% in shoots of the susceptible cv L-550 when treated with the said chemicals, respectively (Table 1). There were three isoforms of proteases found in roots of the resistant cv treated with water (control) and Spm. However, in SA-treated seedlings, isoforms of protease 1 and 3 were inhibited (Figure 1A). There were four isoforms of proteases in control shoots; however, on treatment with SA and Spm complete inhibition of protease 1 and 4 was seen, while partial inhibition of protease 2 and 3 was observed (Figure 1B). In contrast, the banding pattern of proteases in roots of susceptible cv reveals two isoforms in control, and induction of new isoforms of protease 3 and 4 was observed in seedlings treated with SA and Spm (Figure 1C). Similarly, protease profile of shoots reveals four isoforms in control and further their activities were induced by SA and Spm (Figure 1D).
Trypsin and chymotrypsin inhibitory assay PI extract from resistant cv roots and shoots exposed to SA showed increased trypsin inhibitory activity of 9871.73 UI mg 1 protein (39.7% over control) and 9072.3 UI mg 1 protein (39% over control), respectively, followed by 6172.88 UI mg 1 protein (38% over control) and 5270.57 UI mg 1 protein (28.8% over control) on Spm treatment compared with their respective controls. On the other hand, PI extract of roots of susceptible cv treated with SA and Spm, respectively, resulted in an increased trypsin inhibitory activity of
ARTICLE IN PRESS 1018
S. Raju et al. 6071.75 UI mg 1 protein (20% over control) and 6672.88 UI mg 1 protein (27% over control), whereas PI extract of shoots showed increased trypsin inhibitory activity of 6075.8 UI mg 1 protein (30% over control) and 4972.30 UI mg 1 protein (14% over control) compared with their respective controls (Figure 2A). Similarly the chymotrypsin inhibitory activity of PI extract of resistant cv treated with SA and Spm, respectively, was found to be increased by 9271.15 UI mg 1 protein (52% over control), 7272.30 UI mg 1 protein (38% over control) in roots and 7973.46 UI mg 1 protein (63% over control), 6671.73 UI mg 1 protein (57% over control) in shoots compared with their respective controls. In contrast, PI extract of susceptible cv roots and shoots showed 20–30% increase in chymotrypsin inhibitory activity over their respective controls (Figure 2B).
Activity (UI µg-1 protein)
Figure 1. Detection of isoforms of protease activity by gel X-ray film contact method. (A) Roots and (B) shoots of resistant cv; (C) roots and (D) shoots of susceptible cv. Small arrow indicates newly synthesized protease isoforms. Other experimental details are given in Materials and methods. 120 100
*** **
Control
80
SA
**
60
** **
**
Root
Shoot
*
40 20 0 Root
Shoot
120 100 80
Control
***
SA
Spm
*** ***
***
60 40
* * * *
*
20 0 Root
Shoot
ICCV10
Root
Activity of HGP was inhibited by around 62% and 66% in roots and 67% and 57% in shoot extracts of PI of resistant cv. In contrast, only 44% and 41% in roots and about 40% and 38% in shoot of extracts PI of susceptible cv seedlings treated with SA and Spm. The FOX protease activity was inhibited by about 65% and 68% in roots and 52% and 48% in shoot extracts of PI of resistant cv, whereas inhibition was by about 49% and 34% in roots and 44% and 35% in shoots extract of PI of susceptible cv treated with SA and Spm (data not shown).
Visualization of TIs and CTIs by X-ray film contact print method
L-550
ICCV10 Activity (UI µg-1 protein)
Spm
HGP and FOX protease inhibitory assay
Shoot L-550
Figure 2. Accumulation of trypsin inhibitory (A) and chymotrypsin inhibitory (B) activity. PIs were extracted from 8-d-old chickpea seedlings, cv ICCV10 and L-550 treated with SA and Spm and assayed. Each bar represents the mean of three measurements7s.e. Asterisks denote significant difference at *Po0.05; **Po0.01 ***Po0.001 from control, according to ANOVA variance for regression.
The banding pattern of TI reveals four TI isoforms in roots and five in shoots of water-treated control plants of resistant cv. Further, induction of newly synthesized TI-5 and TI-6 isoforms in roots was observed after treatment with SA and Spm (Figure 3A(a)). Similarly, TI profile of shoots reveals induction of three new isoforms (TI-5, TI-6 and TI-7) and two isoforms (TI-6 and TI-7), respectively, in shoots of resistant cv treated with SA and Spm (Figure 3A(b)). In contrast, TI profile of susceptible cv reveals three isoforms in control roots, while five isoforms were observed on treatment with SA and Spm. Among these, two (TI-1 and TI-5) were newly synthesized ones after treatment with SA and Spm (Figure 3A(c)), whereas two isoforms were observed in control shoots and five upon treatment with SA. However, no significant change in the banding pattern was observed even after Spm treatment (Figure 3A(d)). The banding
ARTICLE IN PRESS Elicitation of protease and protease inhibitors in chickpea
1019
Figure 4. Electrophoretic separation and visualization of HGP (A) and FOX protease (B). HGP or FOX protease resolved gel was incubated with C (water), SA- or Spmtreated root (a) and shoot (b) extract of PIs of resistant cv; root (c) and shoot (d) extract of PIs of susceptible cv. The gel strips were washed in buffer and overlaid on X-ray film for 45 min.
In vitro activity of HGPs and FOX proteases
Figure 3. Detection of TIs (A) and CTIs (B) by X-ray film contact print method. Roots (a) and shoots (b) of resistance cv; roots (c) and shoots (d) of susceptible cv, treated with water (control), SA and Spm. Small arrow indicates newly synthesized protease inhibitors. Other experimental details are given in Materials and methods.
pattern of CTI reveals two isoforms (CTI-3 and CTI-5) in control, five in SA and four in Spm-treated roots of resistant cv (Figure 3B(a)). Similarly, the pattern in shoots reveals two isoforms in control, while five and three were observed, respectively, in shoots of resistant cv treated with SA and Spm (Figure 3B(b)). In contrast, the CTI profile of susceptible cv reveals four isoforms in control roots. However, a newly synthesized isoform (CTI-2) was observed after treatment with SA and Spm (Figure 3B(c)), whereas two isoforms were noticed in control shoots (Figure 3B(d)). Further, the intensities of these isoforms were increased after treatment with SA and Spm.
It is known that several proteases of diverse properties are present in insect gut and pathogenic organisms. Figure 4A shows the activity of HGP (a–d) and FOX proteases 4B(a–d) are distributed in at least two major isoforms. We have observed that protease 1 of both the organisms is partially inhibited, whereas isoform protease 2 of HGP is not affected by native PI extract of both the cultivars. However, isoforms of protease 1 and 2 of both the organisms are inhibited maximally by PI extract of resistant cv treated with SA and Spm compared with susceptible cv. Further PI extract of the resistant cv is resolved in the gel and incubated with the protease 1 and 2 separately. It was observed that the PI extract of the resistant cv treated with SA and Spm inhibited the isoforms of protease 1 and 2 of both the organism compared with control PI extract (Figure 5). This experiment clearly shows that the resistant cv seedlings upon treatment with SA and Spm synthesize new PIs which are highly effective against HGP and FOX protease isoforms. On the other hand, the PI
ARTICLE IN PRESS 1020
S. Raju et al.
Figure 5. Inhibition of protease 1 and 2 of HGP (A–D) and FOX (E–H) by PIs extract of resistant cv. PIs extract was separated electrophoretically as described in Materials and methods and incubated the gel with proteases. Lanes (A and C) – water treated and lanes (B and D) – SA treated roots extract of resistant cv against HGP-1 and -2; lanes (E and G) – water treated and lanes (F and H) – SAtreated roots extract of resistant cv against FOX protease 1 and 2.
extract of susceptible cv did not exert any effect on protease 2 of both the organisms (data not shown).
Figure 6. Bioassay of FOX. In vitro plate assay for antifungal activity of roots PI extract of both the cultivars towards FOX. C – water-treated control; SA and Spm – PI extract of roots treated with SA and Spm. Other details are given in Materials and methods.
Bioassay of F. oxysporum Crude PI extract of both the cultivars was checked for their ability to inhibit fungal mycelial growth. FOX mycelial growth was inhibited more effectively by SA- and Spm-treated root extracts of resistant cv than that of susceptible cv. The inhibition of fungal growth is negligible in control roots of both the varieties. The activity of PIs may be a result of inhibition of FOX proteases, which are responsible for growth and development of FOX (Figure 6).
Discussion Proteases may have a direct role in hydrolyzing proteins secreted by invading pathogens thereby preventing pathogenesis. The obvious increase in protease activity after SA treatment in susceptible cv could be due to the induction of new proteins during cell death/necrosis which occurs in pathogenesis-related or hypersensitive response. It is known that the pathogenesis-related proteins belonging to different classes are induced in plants during infection (Carr and Klessig, 1989). The results of gel X-ray protease assay clearly reveal protease induction upon salicylic acid treatment in susceptible cv, which may be due to the colonization of the pathogen and its proliferation inside the host system. However, no induction of proteases is seen upon treatment with SA and Spm in resistant cv. This means that SA-mediated acquired resistance is provided by a critical balance of protease
and PIs. Protease 1 and 3 in root (Figure 1A) and protease 1 and 4 (Figure 1B) in shoots were not observed on SA treatment, suggesting that SAmediated signaling events inhibit the proteases. These observations further led to suggest that SA signaling might be responsible for the inhibition of protease activity as a result of activation of specialized PIs in the resistant cv. Increased trypsin inhibitory activity was observed in the resistant maize seedlings compared with the susceptible one, on treatment with SA or Fusarium moniliform (Molodchenkova et al., 2002). Similar changes in trypsin inhibitor activity were observed in seedlings of winter wheat and spring barley grown in salicylic acid or infected by F. graminearum (Adamovskaya et al., 1999). These studies indicate the involvement of various PIs in the regulation of defense-associated cell death. In relation to the above results, the data presented here reveal the induction of new TIs (Figure 3A(a,b)) and CTIs (Figure 3B(a,b)) in resistant cv compared with their corresponding susceptible cv (Figure 3A(c,d) and B (c,d)) in response to SA and Spm. These results suggest that SA and Spm are involved directly in the synthesis of different PIs in both the genotypes of chickpea differentially. The inhibition of HGPs by bitter gourd proteinase inhibitor was reported by Telang et al. (2003). Although considerable data are available on native proteinase inhibitor of chickpea against H. armigera, no information is available on PIs
ARTICLE IN PRESS Elicitation of protease and protease inhibitors in chickpea induced by SA and Spm, since these inhibitors are synthesized as part of the plant defense response in developing roots and shoots against phytopathogenic organisms. Our results may constitute the first report on the synthesis of chickpea PIs in response to SA and Spm. In the present study, efforts were made to analyze insect proteases and their behavior with PI extracts of chickpea before and after treatment with SA and Spm. The induced PIs in resistant cv are the potent inhibitors of HGP compared with their respective controls (Figure 4A(a,b)), whereas proteinase inhibitor from susceptible cv shows partial inhibition of HGP-1 and no effect at all on HGP-2 isoform (Figure 4A(c,d)). Our results suggest that considerable variability exists among the iso-proteinases of H. armigera gut with respect to chickpea genotype. PIs are of particular interest for engineering resistance because they are a part of the plant’s native defense proteins against pests and pathogens. There are few reports stating increased resistance to a particular insect in transgenic plants expressing high levels of heterologous PIs (Jouanin et al., 1998; Schuler et al., 1998). In some studies it has been reported that insects can grow normally on PI-expressing transgenic plants (Jongsma and Boulter, 1997; Cloutier et al., 2000). This is probably because of low inhibition potential of the selected inhibitor against the pest proteinases or low levels of expression of the PIs in the target tissues. In such cases the expressed PIs are of little use in protecting the plant from the pest. The induction of new PIs by SA and Spm in this study provides a new insight toward developing a rationalized strategy for long resistance to H. armigera. Although PIs show high sequence homology of over 80% among themselves, they can still vary considerably in their potential to bring and inhibit a particular protease. Small variation near the active domain of the inhibitor proteins may potentially alter their biological property and specificity (Zhao et al., 1996). Many phytopathogenic microorganisms produce active extracellular proteases that along with other enzymes play an important role in pathogenesis (Ball et al., 1991; Mosolov and Valueva, 2004). PIs in plants are able to suppress enzymatic activity of phytopathogenic microorganism. Trypsin and chymotrypsin inhibitor from soybean seeds and also from potato tubers is able to suppress activity of protease secreted by phytopathogenic fungus, F. solani (Mosolov et al., 1976). However, in the present study complete inhibition of protease 2 of FOX was observed in roots of resistant cv treated with SA and Spm (Figure 4B(a,b)), while partial inhibition was observed in water-treated roots and
1021
shoots as well as shoots treated with SA and Spm. However, FOX-1 remained unaffected. FOX protease resolved gel strips, when incubated in PIs extract of susceptible cv, reveal moderate inhibition in roots extract, while no major changes were observed in shoots extract. These results suggest that the inhibition of FOX protease 2 may be due to increased accumulation of proteinase inhibitors. It also suggests that the expression of PIs is more in resistant cv than in susceptible ones. This shows that these PIs may interfere with proteases of FOX and protect the plant from pathogen invasion. We have observed that resistant cv contained constitutive expression of proteases which were inhibited upon SA and Spm treatment. This suggests that SA might be responsible significantly for the inhibition of protease activity as a result of activation of specialized PIs. It also suggests the role of intricate SA-mediated signaling pathway in trigger-induced resistance by setting a critical balance of the proteases and PIs, which decide the ultimate fate of cell in programmed cell death during pathogenesis, resulting in the development of acquired resistance. However, induction of proteases in susceptible cv by SA and Spm suggests that it causes cell death as a susceptible response. Further, our data on the interaction between chickpea PIs with H. armigera and FOX proteases provide the basis for a new biotechnological approach to strengthen chickpea defense against insect and pathogen. Changes in trypsin and chymotrypsin activity induced by SA or Spm confirmed the involvement of these chemicals in the induction of new trypsin and chymotrypsin inhibitors in chickpea. The PIs as an antifungal protein (Lorito et al., 1994) are expressed constitutively at low levels and accumulated in specific tissues in response to fungal attack or to other inducers. In the present study, it was observed that the fungal growth was inhibited by the root extract of the resistant cv following treatment of the seedlings with SA and Spm (Figure 6). Few antifungal proteins have been isolated and shown to inhibit various phytopathogenic fungi (Vernerkar et al., 1999). It can be concluded that ICCV10 is a resistant cv as it showed high response to SA and Spm and produced new PIs which were effective against HGP and FOX, whereas L-550 is a susceptible cv as it is unable to produce PIs and inhibit the growth of FOX.
References Adamovskaya VG, Molodchenkova OO, Tsiselskaya LI. Trypsin inhibitor activity in seedlings of winter wheat
ARTICLE IN PRESS 1022 and spring barley grown in salicylic acid and infected with Fusarium graminearum. J Fiziol Biokhim Kult Rast 1999;5:366–71. Ball AM, Ashby AM, Daniels MJ, Ingram DS, Johnstone K. Evidence for requirement of extracellular protease in the pathogenic interaction of Pyrenopeziza brassicae with oil seed rape. Physiol Mol Plant Pathol 1991;38:147–61. Belew M, Porath J. Extracellular proteinase from Penicillium notatum. In: Perimann GE, Lorand L, editors. Methods in enzymology, vol. 19. New York, USA: Academic Press; 1970. p. 576–81. Birk Y. Plant protease inhibitors: significance in nutrition, plant protection, cancer prevention and genetic engineering. Berlin: Springer; 2003 (p. 170–9). Carr JP, Klessig DF. The pathogenesis related proteins of plants. In: Setlow JK, Holleander A, editors. Genetic engineering, principles and methods, vol. 11. New York: Plenum Press; 1989. p. 65–109. Casaretto JA, Zuniga GE, Corcuera LJ. Abscisic acid and jasmonic acid affect protease inhibitor activities in barley leaves. J Plant Physiol 2004;161:389–96. Cloutier C, Jean C, Fournier M, Yelle S, Michaud D. Adult Colorodo potato beetles, Leptinotarsa decemilineata compensate for nutritional stress on oryzacystatin I-transgenic potato plants by hypertrophic behaviour and overproduction of insensitive proteases. Arch Insect Biochem Physiol 2000;44:69–81. Davis BJ. Disc electrophoresis II. Methods and application to human serum. Ann N Y Acad Sci 1964;121:404–29. Erlanger BF, Kokowesky N, Cohen W. The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 1964;95:227–78. Giri AP, Abhay M, Harsulkar, Ku MSB, Gupta VS, Deshpande VV, et al. Identification of potent inhibitors of Helicoverpa armigera gut proteinases from winged bean seeds. Phytochemistry 2003;63:523–32. Jongsma MA, Boulter CJ. The adaptation of insect to plant protease inhibitors. J Insect Physiol 1997;43: 885–95. Jouanin L, Bonade-Bottino M, Girard C, Morrot G, Giband M. Transgenic plants for insect resistance. Plant Sci 1998;131:1–11. Konarev AV, Tomooka N, Vaughan D. Proteinase inhibitor polymorphism in genus Vigna subgenus Ceratotropis and its biosystamatic implications. Euphytica 2002; 123:165–77. Korsinczky MLJ, Schirra JH, Craik DJ. Sunflower trypsin inhibitor-1. Curr Protein Pept Sci 2004;5:351–9. Lorito M, Broadway RM, Hayes CK, Woo SL, Noviello C, Williams DL, et al. Protease inhibitors from plants as a novel class of fungicides. Mol Plant Microbe Interact 1994;7:525–7. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with folin-phenol reagent. J Biol Chem 1951;193:265–75. Metraux JP, Signer H, Ryals J, Ward E, Wyss-Benz M, Gaudin J, et al. Increase in salicylic acid at the onset of systemic acquired resistance in cucumber. Science 1990;250:1004–6.
S. Raju et al. Molodchenkova OO, Adamovskaya VG, Levitskij YA, Gontarenko OV, Sokolov VM. Maize response to salicylic acid and Fusarium moniliforme. Appl Biochem Microbiol 2002;38:381–5. Mosolov VV, Valueva TA. Role of inhibitors of proteolytic enzymes in plant defense against phytopathogenic micro-organisms. Biochemistry 2004;69:1305–9. Mosolov VV, Loginova MD, Fedurkina NV, Benken II. The biological significance of proteinase inhibitors in plants. Plant Sci Lett 1976;7:77–80. Pichare MM, Kachole MS. Detection of electrophoretically separated protease inhibitors using X-ray film. J Biochem Biophys Methods 1994;28:215–24. Raju S, Jayalakshmi SK, Usharani S, Sreeramulu K. Induction of systemic acquired resistance in susceptible and resistant cultivars of chickpea (Cicer arietinum L.) genotypes: a comparative study in response to salicylic acid and spermine. Physiol Mol Biol Plants 2007;13:27–36. Roberts WK, Selitrennikoff CP. Plant proteins that inactivate foreign ribosomes. Biochem Biophys Acta 1986;880:161–70. Schuler TH, Poppy GM, Kerry BR, Denholm I. Insect resistant transgenic plants. Trends Biotechnol 1998; 16:168–75. Schwert GW, Takenaka Y. A spectrophotometric determination of trypsin and chymotrypsin. Biochem Biophys Acta 1955;16:570–5. Shewry PR. Tuber storage proteins. Ann Bot 2003;91:755–69. Telang M, Srinivasan A, Patankar A, Harsulkar A, Joshi V, Damle A, et al. Bitter gourd proteinase inhibitors potential growth inhibitors of Helicoverpa armigera and Spodoptera litura. Phytochemistry 2003;63:643–52. Vander Hoorn RA, Jones JD. The plant proteolytic machinery and its role in defense. Curr Opin Plant Biol 2004;7:400–7. van Loon LC, Rep M, Pieterse CMJ. Significance of inducible defense-related proteins in infected plants. Annu Rev Phytopathol 2006;44:135–62. Vernerkar JV, Ghatge MS, Deshpande VV. Alkaline protease inhibitor. A novel class of antifungal proteins against phytopathogenic fungi. Biochem Biophys Res Commun 1999;262:702–7. Walters DR. Polyamines and plant disease. Phytochemistry 2003;62:97–107. Xia Y, Suzuki H, Borevitz J, Blount J, Gou Z, Patel K, et al. An extracellular aspartic protease functions in Arabidopsis disease resistance signaling. EMBO J 2004;23: 980–8. Yamakawa H, Kamada H, Satoh M, Ohashi Y. Spermine is a salicylic acid-independent endogenous inducer for both tobacco acidic pathogenesis related proteins and resistance against tobacco mosaic virus infection. Plant Physiol 1998;118:1213–22. Zhao Y, Botella MA, Subramanyam L, Niu X, Nielsen SS, Bressan RA, et al. Two wound inducible soybean cysteine proteinase inhibitors have greater insect digestive proteinase inhibitor activities than a constitutive homolog. Plant Physiol 1996;111:1296–9.
www.elsevier.de/jplph
Differential elicitation of proteases and protease inhibitors in two different genotypes of chickpea (Cicer arietinum) by salicylic acid and spermine Shamrao Rajua, Senigala K. Jayalakshmib, Kuruba Sreeramulua, a
Department of Biochemistry, Gulbarga University, Gulbarga 585106, India Krishi Vignan Kendra, Agriculture Research Station, Gulbarga 585106, India
b
Received 14 July 2008; received in revised form 14 December 2008; accepted 15 December 2008
KEYWORDS Chickpea (Cicer arietinum); Protease; Protease inhibitors; Salicylic acid; Spermine
Summary Elicitation of proteases and protease inhibitors (PIs) by salicylic acid (SA) and spermine (Spm) was investigated in roots and shoots of two different genotypes of chickpea cultivars ICCV10 and L550, which were resistant and susceptible to wilt disease, respectively. SA and Spm were found to suppress the elicitation of proteases in the resistant cv, whereas they induce it in susceptible cv. Elicitation of new trypsin and chymotrypsin inhibitors was observed in the roots and shoots of resistant cv treated with SA and Spm. However, no such elicitation was observed in susceptible cv. These results show for the first time that SA and Spm could elicit synthesis of new PIs capable of inhibiting the proteases of insect Helicoverpa armigera and Fusarium oxysporum, wilt causing pathogen. Antifungal property of root extract of resistant cv increased following treatment of seedling with SA and Spm compared with susceptible cv. & 2008 Elsevier GmbH. All rights reserved.
Introduction The proteolytic enzymes of plants play an important and diverse role in defense against Abbreviations: BApNA, N-benzoyl DL-arginine p-nitroanilide HCl; CTI, chymotrypsin inhibitor; FOX, Fusarium oxysporum f. sp. ciceri; HGP, Helicoverpa armigera gut protease; PIs, protease inhibitors; SA, salicylic acid; Spm, spermine; TEE, L-tyrosine ethyl ester; TI, trypsin inhibitor. Corresponding author. Tel.: +91 8472 263289; fax: +91 8472 263202. E-mail address: [email protected] (K. Sreeramulu).
pathogens (Xia et al., 2004). They could contribute to defense in different ways, acting at the levels of perception, signaling and execution (Vander Hoorn and Jones, 2004). Plant protease inhibitors (PIs) are known to play an important role in plants defense against insect pests and pathogens as well as in regulation of endogenous proteinases. They are one of the major storage proteins in seeds (Moslov et al., 1976; Birk, 2003; Shewry, 2003) and are of interest as potential sources of resistance against pests and pathogens (Konarev et al., 2002; Korsinczky et al., 2004). Induction of trypsin and
0176-1617/$ - see front matter & 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2008.12.005
ARTICLE IN PRESS 1016 chymotrypsin inhibitory activities by aphid infestation, mechanical wounding, abscisic acid and jasmonic acid was studied in barley leaves (Casaretto et al., 2004). However, they appear to play key roles in the regulation of biological processes in plants, such as the recognition of pathogens and pests and the induction of effective defense responses. Salicylic acid and spermine (Spm) have been implicated in the signal transduction pathway leading to plant resistance to various pathogens (Yamakawa et al., 1998; van Loon et al., 2006). Metraux et al. (1990) found that foliar or drench application of iso-nicotinic acid (INA) induces systemic resistance in cucumber against Colletotrichum lagenarium. Since then, resistance induced by salicylic acid and INA was demonstrated in a variety of plant pathogen interactions. Polyamines, Spm and spermidine are found in a wide range of organisms from bacteria to plants and animals. They are basically small molecules implicated in the promotion of plant growth and development by activating the synthesis of nucleic acids (Walters, 2003). It has been reported that Spm is an endogenous signal molecule for the accumulation of acidic and basic PR proteins; Spm and SA act independently for PR protein induction (Yamakawa et al., 1998). However, the role of SA and Spm in the development of systemic acquired resistance (SAR) has been well documented in chickpea (Raju et al., 2007), but their effects on the induction of protease and PIs are not reported. Therefore, in the present investigation, an attempt has been made to examine the effects of SA and Spm on induction of proteases and PIs in two different genotypes of chickpea cultivars, which are susceptible and resistant to wilt disease caused by Fusarium oxysporum f. sp. ciceri. Further, these PIs were found to be potent inhibitors of proteases of Helicoverpa armigera and F. oxysporum.
Materials and methods Seeds of chickpea (Cicer arietinum L.) cv L-550 and ICCV-10, susceptible and resistant to wilt disease, respectively, were procured from the Agricultural Research Station, Gulbarga, Karnataka, India. Trypsin, chymotrypsin, BApNA and TEE were purchased from Sigma Chemicals Co. (St. Louis, USA). X-ray films are of Kodak make. Other reagents were of analytical grade. Seed treatments with SA, Spm and germination experiments were performed as described in our earlier report (Raju et al., 2007).
S. Raju et al.
Extraction of protease and protease inhibitors from chickpea plants The roots and shoots were ground in a pre-chilled pestle and mortar in an ice-cold 50 mM phosphate buffer pH (7.8). The extract was centrifuged at 4 1C for 25 min at 10,000g. The resulting supernatant was used as an enzyme source for further determination of protease activities. Trypsin and chymotrypsin inhibitors were extracted by the modified method of Casaretto et al. (2004). Protein in the supernatant was estimated by Lowry et al. (1951).
Extraction of HGPs and FOX proteases Third instar larvae of Helicoverpa armigera were dissected to isolate the midgut tissue, which was immediately frozen in liquid nitrogen and stored at 70 1C. The midgut tissue was homogenized and mixed with 0.1 M Tris–HCl buffer, pH (8.8), for 2 h at 10 1C. The suspension was centrifuged at 4 1C for 20 min at 10,000g and the resulting supernatant was used as a source of HGP. HGP solution was prepared fresh before use by extracting the frozen midgut (Giri et al., 2003). The isoforms of HGP were separated by electroelution from the preparative gels. FOX was grown for 4–5 d at 28 1C in 250 mL Erlenmeyer flask containing 50 mL of Sabouraud’s dextrose broth. The fungal cells were separated by filtration and the filtrate was used as enzyme source, and assayed with casein as substrate. The isoforms of FOX were separated by electroelution from the preparative gels.
Protease and PI activity assay Protease activity was measured by the modified Kunitz caesinolytic assay (Belew and Porath, 1970). One unit of protease activity was defined as an increase in absorbance by one optical density (OD) of TCA soluble casein hydrolysis products liberated by protease action at 280 nm/min at 27 1C under given assay conditions. Trypsin activity was measured using the synthetic substrate BApNA as described by Erlanger et al. (1964). One unit of trypsin activity was defined as the amount of enzyme that increases absorbance by 0.01 OD at 410 nm at 27 1C under given assay conditions. Chymotrypsin activity was determined using the synthetic substrate TEE by the method described by Schwert and Takenaka (1955). One unit of enzyme activity is equal to the hydrolysis of 1 mM of TEE at 235 nm/min at 27 1C. For the inhibitor assay, a suitable volume of PI extract from chickpea was mixed with commercial bovine
ARTICLE IN PRESS Elicitation of protease and protease inhibitors in chickpea trypsin (15 mg)/chymotrypsin (5 mg) or with HGP/FOX protease (20 mg) and incubated for 15 min. Protease activity was measured by excluding PI extract from the above steps. One PI unit is defined as the amount of inhibitor that inhibits 1 unit of protease activity.
Electrophoretic visualization of proteases and PI Root and shoot extracts of chickpea seedlings treated with SA and Spm or HGP or proteases of FOX were electrophoresed on a vertical slab gel using a discontinuous buffer system by Davis (1964). For protease activity the gel was equilibrated with 0.2 M Tris–HCl buffer pH 7.8 for 10 min, and overlaid on an undeveloped X-ray film for 1 h at 37 1C. The films were then washed with warm water, and protease activity bands were visualized as hydrolyzed gelatin. For visualization of HGPs and proteases of FOX, the gel was equilibrated in 0.2 M glycine/NaOH buffer, pH (10.0) for 10–15 min and then overlaid on X-ray film for 40–45 min. TI and CTIs were visualized after native PAGE using X-ray film contact print technique of Pichare and Kachole (1994). In brief, the native gels were equilibrated in 0.1 M Tris–HCl buffer, pH (7.8) for 10–15 min, followed by incubation in the trypsin solution (0.1 mg/mL) or chymotrypsin (20 mg/ mL) or HGP (20 mg) or FOX protease (20 mg) for 15 min at 37 1C in a shaking water bath. The gels were then washed with the same buffer and placed on a piece of undeveloped X-ray film for 3–5 min; the films were then washed with water, and inhibitor activity bands were visualized as unhydrolyzed gelatin on X-ray films. The X-ray films were developed using Kodak 163 DA developer.
Bioassay for the determination of antifungal activity FOX growth inhibition was studied using a hyphal extension inhibition assay of Roberts and Selitrennikoff (1986). Sterile filter paper discs with filter-sterilized inhibitor extracts of roots and shoots treated with water, SA and Spm, were placed on the periphery of the advancing fungal mycelium. The plates were further incubated at 28 1C for 24 h and observed for crescents of retarded mycelial growth.
Results Protease activity It was found that the protease activity was inhibited by 32% and 4.8% in roots and 60% and
1017
Table 1. Protease activity in roots and shoots of resistant and susceptible cultivars of chickpea treated with SA and Spm for 8 d. Cultivar
Protease activity (mg
1
tissue)
Control
SA
Spm
ICCV10 Roots Shoots
4.3470.11 5.9270.17
2.9570.11 2.3270.13***
4.1374.09 2.5570.19**
L-550 Roots Shoots
2.5275.7 5.2570.20
4.870.23*** 5.8277.26
3.570.17 5.3470.11
Each value in the table represents the mean7S.E from three independent experiments. Differences from control values were significant at Po0.05, **Po0.01, ***Po0.001 from control, according to ANOVA variance for regression.
56% in shoots of resistant cv when treated with SA and Spm, respectively. On the other hand, the protease activities were increased by 47% and 28% in roots, 9.7% and 1.6% in shoots of the susceptible cv L-550 when treated with the said chemicals, respectively (Table 1). There were three isoforms of proteases found in roots of the resistant cv treated with water (control) and Spm. However, in SA-treated seedlings, isoforms of protease 1 and 3 were inhibited (Figure 1A). There were four isoforms of proteases in control shoots; however, on treatment with SA and Spm complete inhibition of protease 1 and 4 was seen, while partial inhibition of protease 2 and 3 was observed (Figure 1B). In contrast, the banding pattern of proteases in roots of susceptible cv reveals two isoforms in control, and induction of new isoforms of protease 3 and 4 was observed in seedlings treated with SA and Spm (Figure 1C). Similarly, protease profile of shoots reveals four isoforms in control and further their activities were induced by SA and Spm (Figure 1D).
Trypsin and chymotrypsin inhibitory assay PI extract from resistant cv roots and shoots exposed to SA showed increased trypsin inhibitory activity of 9871.73 UI mg 1 protein (39.7% over control) and 9072.3 UI mg 1 protein (39% over control), respectively, followed by 6172.88 UI mg 1 protein (38% over control) and 5270.57 UI mg 1 protein (28.8% over control) on Spm treatment compared with their respective controls. On the other hand, PI extract of roots of susceptible cv treated with SA and Spm, respectively, resulted in an increased trypsin inhibitory activity of
ARTICLE IN PRESS 1018
S. Raju et al. 6071.75 UI mg 1 protein (20% over control) and 6672.88 UI mg 1 protein (27% over control), whereas PI extract of shoots showed increased trypsin inhibitory activity of 6075.8 UI mg 1 protein (30% over control) and 4972.30 UI mg 1 protein (14% over control) compared with their respective controls (Figure 2A). Similarly the chymotrypsin inhibitory activity of PI extract of resistant cv treated with SA and Spm, respectively, was found to be increased by 9271.15 UI mg 1 protein (52% over control), 7272.30 UI mg 1 protein (38% over control) in roots and 7973.46 UI mg 1 protein (63% over control), 6671.73 UI mg 1 protein (57% over control) in shoots compared with their respective controls. In contrast, PI extract of susceptible cv roots and shoots showed 20–30% increase in chymotrypsin inhibitory activity over their respective controls (Figure 2B).
Activity (UI µg-1 protein)
Figure 1. Detection of isoforms of protease activity by gel X-ray film contact method. (A) Roots and (B) shoots of resistant cv; (C) roots and (D) shoots of susceptible cv. Small arrow indicates newly synthesized protease isoforms. Other experimental details are given in Materials and methods. 120 100
*** **
Control
80
SA
**
60
** **
**
Root
Shoot
*
40 20 0 Root
Shoot
120 100 80
Control
***
SA
Spm
*** ***
***
60 40
* * * *
*
20 0 Root
Shoot
ICCV10
Root
Activity of HGP was inhibited by around 62% and 66% in roots and 67% and 57% in shoot extracts of PI of resistant cv. In contrast, only 44% and 41% in roots and about 40% and 38% in shoot of extracts PI of susceptible cv seedlings treated with SA and Spm. The FOX protease activity was inhibited by about 65% and 68% in roots and 52% and 48% in shoot extracts of PI of resistant cv, whereas inhibition was by about 49% and 34% in roots and 44% and 35% in shoots extract of PI of susceptible cv treated with SA and Spm (data not shown).
Visualization of TIs and CTIs by X-ray film contact print method
L-550
ICCV10 Activity (UI µg-1 protein)
Spm
HGP and FOX protease inhibitory assay
Shoot L-550
Figure 2. Accumulation of trypsin inhibitory (A) and chymotrypsin inhibitory (B) activity. PIs were extracted from 8-d-old chickpea seedlings, cv ICCV10 and L-550 treated with SA and Spm and assayed. Each bar represents the mean of three measurements7s.e. Asterisks denote significant difference at *Po0.05; **Po0.01 ***Po0.001 from control, according to ANOVA variance for regression.
The banding pattern of TI reveals four TI isoforms in roots and five in shoots of water-treated control plants of resistant cv. Further, induction of newly synthesized TI-5 and TI-6 isoforms in roots was observed after treatment with SA and Spm (Figure 3A(a)). Similarly, TI profile of shoots reveals induction of three new isoforms (TI-5, TI-6 and TI-7) and two isoforms (TI-6 and TI-7), respectively, in shoots of resistant cv treated with SA and Spm (Figure 3A(b)). In contrast, TI profile of susceptible cv reveals three isoforms in control roots, while five isoforms were observed on treatment with SA and Spm. Among these, two (TI-1 and TI-5) were newly synthesized ones after treatment with SA and Spm (Figure 3A(c)), whereas two isoforms were observed in control shoots and five upon treatment with SA. However, no significant change in the banding pattern was observed even after Spm treatment (Figure 3A(d)). The banding
ARTICLE IN PRESS Elicitation of protease and protease inhibitors in chickpea
1019
Figure 4. Electrophoretic separation and visualization of HGP (A) and FOX protease (B). HGP or FOX protease resolved gel was incubated with C (water), SA- or Spmtreated root (a) and shoot (b) extract of PIs of resistant cv; root (c) and shoot (d) extract of PIs of susceptible cv. The gel strips were washed in buffer and overlaid on X-ray film for 45 min.
In vitro activity of HGPs and FOX proteases
Figure 3. Detection of TIs (A) and CTIs (B) by X-ray film contact print method. Roots (a) and shoots (b) of resistance cv; roots (c) and shoots (d) of susceptible cv, treated with water (control), SA and Spm. Small arrow indicates newly synthesized protease inhibitors. Other experimental details are given in Materials and methods.
pattern of CTI reveals two isoforms (CTI-3 and CTI-5) in control, five in SA and four in Spm-treated roots of resistant cv (Figure 3B(a)). Similarly, the pattern in shoots reveals two isoforms in control, while five and three were observed, respectively, in shoots of resistant cv treated with SA and Spm (Figure 3B(b)). In contrast, the CTI profile of susceptible cv reveals four isoforms in control roots. However, a newly synthesized isoform (CTI-2) was observed after treatment with SA and Spm (Figure 3B(c)), whereas two isoforms were noticed in control shoots (Figure 3B(d)). Further, the intensities of these isoforms were increased after treatment with SA and Spm.
It is known that several proteases of diverse properties are present in insect gut and pathogenic organisms. Figure 4A shows the activity of HGP (a–d) and FOX proteases 4B(a–d) are distributed in at least two major isoforms. We have observed that protease 1 of both the organisms is partially inhibited, whereas isoform protease 2 of HGP is not affected by native PI extract of both the cultivars. However, isoforms of protease 1 and 2 of both the organisms are inhibited maximally by PI extract of resistant cv treated with SA and Spm compared with susceptible cv. Further PI extract of the resistant cv is resolved in the gel and incubated with the protease 1 and 2 separately. It was observed that the PI extract of the resistant cv treated with SA and Spm inhibited the isoforms of protease 1 and 2 of both the organism compared with control PI extract (Figure 5). This experiment clearly shows that the resistant cv seedlings upon treatment with SA and Spm synthesize new PIs which are highly effective against HGP and FOX protease isoforms. On the other hand, the PI
ARTICLE IN PRESS 1020
S. Raju et al.
Figure 5. Inhibition of protease 1 and 2 of HGP (A–D) and FOX (E–H) by PIs extract of resistant cv. PIs extract was separated electrophoretically as described in Materials and methods and incubated the gel with proteases. Lanes (A and C) – water treated and lanes (B and D) – SA treated roots extract of resistant cv against HGP-1 and -2; lanes (E and G) – water treated and lanes (F and H) – SAtreated roots extract of resistant cv against FOX protease 1 and 2.
extract of susceptible cv did not exert any effect on protease 2 of both the organisms (data not shown).
Figure 6. Bioassay of FOX. In vitro plate assay for antifungal activity of roots PI extract of both the cultivars towards FOX. C – water-treated control; SA and Spm – PI extract of roots treated with SA and Spm. Other details are given in Materials and methods.
Bioassay of F. oxysporum Crude PI extract of both the cultivars was checked for their ability to inhibit fungal mycelial growth. FOX mycelial growth was inhibited more effectively by SA- and Spm-treated root extracts of resistant cv than that of susceptible cv. The inhibition of fungal growth is negligible in control roots of both the varieties. The activity of PIs may be a result of inhibition of FOX proteases, which are responsible for growth and development of FOX (Figure 6).
Discussion Proteases may have a direct role in hydrolyzing proteins secreted by invading pathogens thereby preventing pathogenesis. The obvious increase in protease activity after SA treatment in susceptible cv could be due to the induction of new proteins during cell death/necrosis which occurs in pathogenesis-related or hypersensitive response. It is known that the pathogenesis-related proteins belonging to different classes are induced in plants during infection (Carr and Klessig, 1989). The results of gel X-ray protease assay clearly reveal protease induction upon salicylic acid treatment in susceptible cv, which may be due to the colonization of the pathogen and its proliferation inside the host system. However, no induction of proteases is seen upon treatment with SA and Spm in resistant cv. This means that SA-mediated acquired resistance is provided by a critical balance of protease
and PIs. Protease 1 and 3 in root (Figure 1A) and protease 1 and 4 (Figure 1B) in shoots were not observed on SA treatment, suggesting that SAmediated signaling events inhibit the proteases. These observations further led to suggest that SA signaling might be responsible for the inhibition of protease activity as a result of activation of specialized PIs in the resistant cv. Increased trypsin inhibitory activity was observed in the resistant maize seedlings compared with the susceptible one, on treatment with SA or Fusarium moniliform (Molodchenkova et al., 2002). Similar changes in trypsin inhibitor activity were observed in seedlings of winter wheat and spring barley grown in salicylic acid or infected by F. graminearum (Adamovskaya et al., 1999). These studies indicate the involvement of various PIs in the regulation of defense-associated cell death. In relation to the above results, the data presented here reveal the induction of new TIs (Figure 3A(a,b)) and CTIs (Figure 3B(a,b)) in resistant cv compared with their corresponding susceptible cv (Figure 3A(c,d) and B (c,d)) in response to SA and Spm. These results suggest that SA and Spm are involved directly in the synthesis of different PIs in both the genotypes of chickpea differentially. The inhibition of HGPs by bitter gourd proteinase inhibitor was reported by Telang et al. (2003). Although considerable data are available on native proteinase inhibitor of chickpea against H. armigera, no information is available on PIs
ARTICLE IN PRESS Elicitation of protease and protease inhibitors in chickpea induced by SA and Spm, since these inhibitors are synthesized as part of the plant defense response in developing roots and shoots against phytopathogenic organisms. Our results may constitute the first report on the synthesis of chickpea PIs in response to SA and Spm. In the present study, efforts were made to analyze insect proteases and their behavior with PI extracts of chickpea before and after treatment with SA and Spm. The induced PIs in resistant cv are the potent inhibitors of HGP compared with their respective controls (Figure 4A(a,b)), whereas proteinase inhibitor from susceptible cv shows partial inhibition of HGP-1 and no effect at all on HGP-2 isoform (Figure 4A(c,d)). Our results suggest that considerable variability exists among the iso-proteinases of H. armigera gut with respect to chickpea genotype. PIs are of particular interest for engineering resistance because they are a part of the plant’s native defense proteins against pests and pathogens. There are few reports stating increased resistance to a particular insect in transgenic plants expressing high levels of heterologous PIs (Jouanin et al., 1998; Schuler et al., 1998). In some studies it has been reported that insects can grow normally on PI-expressing transgenic plants (Jongsma and Boulter, 1997; Cloutier et al., 2000). This is probably because of low inhibition potential of the selected inhibitor against the pest proteinases or low levels of expression of the PIs in the target tissues. In such cases the expressed PIs are of little use in protecting the plant from the pest. The induction of new PIs by SA and Spm in this study provides a new insight toward developing a rationalized strategy for long resistance to H. armigera. Although PIs show high sequence homology of over 80% among themselves, they can still vary considerably in their potential to bring and inhibit a particular protease. Small variation near the active domain of the inhibitor proteins may potentially alter their biological property and specificity (Zhao et al., 1996). Many phytopathogenic microorganisms produce active extracellular proteases that along with other enzymes play an important role in pathogenesis (Ball et al., 1991; Mosolov and Valueva, 2004). PIs in plants are able to suppress enzymatic activity of phytopathogenic microorganism. Trypsin and chymotrypsin inhibitor from soybean seeds and also from potato tubers is able to suppress activity of protease secreted by phytopathogenic fungus, F. solani (Mosolov et al., 1976). However, in the present study complete inhibition of protease 2 of FOX was observed in roots of resistant cv treated with SA and Spm (Figure 4B(a,b)), while partial inhibition was observed in water-treated roots and
1021
shoots as well as shoots treated with SA and Spm. However, FOX-1 remained unaffected. FOX protease resolved gel strips, when incubated in PIs extract of susceptible cv, reveal moderate inhibition in roots extract, while no major changes were observed in shoots extract. These results suggest that the inhibition of FOX protease 2 may be due to increased accumulation of proteinase inhibitors. It also suggests that the expression of PIs is more in resistant cv than in susceptible ones. This shows that these PIs may interfere with proteases of FOX and protect the plant from pathogen invasion. We have observed that resistant cv contained constitutive expression of proteases which were inhibited upon SA and Spm treatment. This suggests that SA might be responsible significantly for the inhibition of protease activity as a result of activation of specialized PIs. It also suggests the role of intricate SA-mediated signaling pathway in trigger-induced resistance by setting a critical balance of the proteases and PIs, which decide the ultimate fate of cell in programmed cell death during pathogenesis, resulting in the development of acquired resistance. However, induction of proteases in susceptible cv by SA and Spm suggests that it causes cell death as a susceptible response. Further, our data on the interaction between chickpea PIs with H. armigera and FOX proteases provide the basis for a new biotechnological approach to strengthen chickpea defense against insect and pathogen. Changes in trypsin and chymotrypsin activity induced by SA or Spm confirmed the involvement of these chemicals in the induction of new trypsin and chymotrypsin inhibitors in chickpea. The PIs as an antifungal protein (Lorito et al., 1994) are expressed constitutively at low levels and accumulated in specific tissues in response to fungal attack or to other inducers. In the present study, it was observed that the fungal growth was inhibited by the root extract of the resistant cv following treatment of the seedlings with SA and Spm (Figure 6). Few antifungal proteins have been isolated and shown to inhibit various phytopathogenic fungi (Vernerkar et al., 1999). It can be concluded that ICCV10 is a resistant cv as it showed high response to SA and Spm and produced new PIs which were effective against HGP and FOX, whereas L-550 is a susceptible cv as it is unable to produce PIs and inhibit the growth of FOX.
References Adamovskaya VG, Molodchenkova OO, Tsiselskaya LI. Trypsin inhibitor activity in seedlings of winter wheat
ARTICLE IN PRESS 1022 and spring barley grown in salicylic acid and infected with Fusarium graminearum. J Fiziol Biokhim Kult Rast 1999;5:366–71. Ball AM, Ashby AM, Daniels MJ, Ingram DS, Johnstone K. Evidence for requirement of extracellular protease in the pathogenic interaction of Pyrenopeziza brassicae with oil seed rape. Physiol Mol Plant Pathol 1991;38:147–61. Belew M, Porath J. Extracellular proteinase from Penicillium notatum. In: Perimann GE, Lorand L, editors. Methods in enzymology, vol. 19. New York, USA: Academic Press; 1970. p. 576–81. Birk Y. Plant protease inhibitors: significance in nutrition, plant protection, cancer prevention and genetic engineering. Berlin: Springer; 2003 (p. 170–9). Carr JP, Klessig DF. The pathogenesis related proteins of plants. In: Setlow JK, Holleander A, editors. Genetic engineering, principles and methods, vol. 11. New York: Plenum Press; 1989. p. 65–109. Casaretto JA, Zuniga GE, Corcuera LJ. Abscisic acid and jasmonic acid affect protease inhibitor activities in barley leaves. J Plant Physiol 2004;161:389–96. Cloutier C, Jean C, Fournier M, Yelle S, Michaud D. Adult Colorodo potato beetles, Leptinotarsa decemilineata compensate for nutritional stress on oryzacystatin I-transgenic potato plants by hypertrophic behaviour and overproduction of insensitive proteases. Arch Insect Biochem Physiol 2000;44:69–81. Davis BJ. Disc electrophoresis II. Methods and application to human serum. Ann N Y Acad Sci 1964;121:404–29. Erlanger BF, Kokowesky N, Cohen W. The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 1964;95:227–78. Giri AP, Abhay M, Harsulkar, Ku MSB, Gupta VS, Deshpande VV, et al. Identification of potent inhibitors of Helicoverpa armigera gut proteinases from winged bean seeds. Phytochemistry 2003;63:523–32. Jongsma MA, Boulter CJ. The adaptation of insect to plant protease inhibitors. J Insect Physiol 1997;43: 885–95. Jouanin L, Bonade-Bottino M, Girard C, Morrot G, Giband M. Transgenic plants for insect resistance. Plant Sci 1998;131:1–11. Konarev AV, Tomooka N, Vaughan D. Proteinase inhibitor polymorphism in genus Vigna subgenus Ceratotropis and its biosystamatic implications. Euphytica 2002; 123:165–77. Korsinczky MLJ, Schirra JH, Craik DJ. Sunflower trypsin inhibitor-1. Curr Protein Pept Sci 2004;5:351–9. Lorito M, Broadway RM, Hayes CK, Woo SL, Noviello C, Williams DL, et al. Protease inhibitors from plants as a novel class of fungicides. Mol Plant Microbe Interact 1994;7:525–7. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with folin-phenol reagent. J Biol Chem 1951;193:265–75. Metraux JP, Signer H, Ryals J, Ward E, Wyss-Benz M, Gaudin J, et al. Increase in salicylic acid at the onset of systemic acquired resistance in cucumber. Science 1990;250:1004–6.
S. Raju et al. Molodchenkova OO, Adamovskaya VG, Levitskij YA, Gontarenko OV, Sokolov VM. Maize response to salicylic acid and Fusarium moniliforme. Appl Biochem Microbiol 2002;38:381–5. Mosolov VV, Valueva TA. Role of inhibitors of proteolytic enzymes in plant defense against phytopathogenic micro-organisms. Biochemistry 2004;69:1305–9. Mosolov VV, Loginova MD, Fedurkina NV, Benken II. The biological significance of proteinase inhibitors in plants. Plant Sci Lett 1976;7:77–80. Pichare MM, Kachole MS. Detection of electrophoretically separated protease inhibitors using X-ray film. J Biochem Biophys Methods 1994;28:215–24. Raju S, Jayalakshmi SK, Usharani S, Sreeramulu K. Induction of systemic acquired resistance in susceptible and resistant cultivars of chickpea (Cicer arietinum L.) genotypes: a comparative study in response to salicylic acid and spermine. Physiol Mol Biol Plants 2007;13:27–36. Roberts WK, Selitrennikoff CP. Plant proteins that inactivate foreign ribosomes. Biochem Biophys Acta 1986;880:161–70. Schuler TH, Poppy GM, Kerry BR, Denholm I. Insect resistant transgenic plants. Trends Biotechnol 1998; 16:168–75. Schwert GW, Takenaka Y. A spectrophotometric determination of trypsin and chymotrypsin. Biochem Biophys Acta 1955;16:570–5. Shewry PR. Tuber storage proteins. Ann Bot 2003;91:755–69. Telang M, Srinivasan A, Patankar A, Harsulkar A, Joshi V, Damle A, et al. Bitter gourd proteinase inhibitors potential growth inhibitors of Helicoverpa armigera and Spodoptera litura. Phytochemistry 2003;63:643–52. Vander Hoorn RA, Jones JD. The plant proteolytic machinery and its role in defense. Curr Opin Plant Biol 2004;7:400–7. van Loon LC, Rep M, Pieterse CMJ. Significance of inducible defense-related proteins in infected plants. Annu Rev Phytopathol 2006;44:135–62. Vernerkar JV, Ghatge MS, Deshpande VV. Alkaline protease inhibitor. A novel class of antifungal proteins against phytopathogenic fungi. Biochem Biophys Res Commun 1999;262:702–7. Walters DR. Polyamines and plant disease. Phytochemistry 2003;62:97–107. Xia Y, Suzuki H, Borevitz J, Blount J, Gou Z, Patel K, et al. An extracellular aspartic protease functions in Arabidopsis disease resistance signaling. EMBO J 2004;23: 980–8. Yamakawa H, Kamada H, Satoh M, Ohashi Y. Spermine is a salicylic acid-independent endogenous inducer for both tobacco acidic pathogenesis related proteins and resistance against tobacco mosaic virus infection. Plant Physiol 1998;118:1213–22. Zhao Y, Botella MA, Subramanyam L, Niu X, Nielsen SS, Bressan RA, et al. Two wound inducible soybean cysteine proteinase inhibitors have greater insect digestive proteinase inhibitor activities than a constitutive homolog. Plant Physiol 1996;111:1296–9.