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infected potato tubers
Biochimica et Biophysica Acta 1483 (2000) 294^300 www.elsevier.com/locate/bba
Di¡erential formation of octadecadienoic acid and octadecatrienoic acid products in control and injured/infected potato tubers P. Srinivas Reddy, T. Charles Kumar, M. Narsa Reddy, C. Sarada, P. Reddanna * School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India Received 17 August 1999; received in revised form 12 October 1999; accepted 17 November 1999
Abstract Lipoxygenases in plants have been implicated in the activation of defense responses against injury/infection. Pathogenderived polyunsaturated fatty acids, such as arachidonic acid, eicosapentaenoic acid and their metabolites have been shown to elicit defense responses against pathogen infection in plants. However, not much is known about the role of host-derived fatty acids and their metabolites in plant defense responses. In this study, isolation and characterisation of endogenous lipoxygenase metabolites formed in potato tubers in response to injury/infection was undertaken. While 9-hydroperoxyoctadecadienoic acid (9-HPODE), derived from octadecdienoic acid (linoleic acid) is the major lipoxygenase product formed in control potato tubers, 9-hydroperoxyoctadecatrienoic acid (9-HPOTrE), derived from octadecatrienoic acid (Klinolenic acid) is the major lipoxygenase product formed in potato tubers in response to injury or infection with Rhizoctonia bataticola. As a result, the relative ratio of 9-HPODE to 9-HPOTrE showed a shift from 4:1 in control to 1:2 and 1:4.5 in injured and infected potato tubers respectively. From this study, it is proposed that lipoxygenase metabolites of octadecadienoic acid may be involved in physiological responses under control conditions, while octadecatrienoic acid metabolites are mediating the defense responses. This forms the first report on the differential formation of endogenous lipoxygenase products in potato tubers under control and stress conditions. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Lipoxygenase; Potato tuber; Octadecadienoic acid; Octadecatrienoic acid; Injury; Infection
1. Introduction Lipoxygenases (LOX, EC 1.13.11.12) are a group of enzymes involved in the oxygenation of polyunsaturated fatty acids (PUFAs) containing cis, cis-1,4pentadiene structures and are ubiquitous both in plants and animals. The immediate oxygenation products of PUFAs by LOX are hydroperoxy conjugated dienes, which are transformed into a spectrum
* Corresponding author. Fax: +91-40-301-0120; E-mail: [email protected]
of biologically active compounds, such as leukotrienes in animals and jasmonic acid, traumatic acid and alkenals in plants. In plants, LOX products have been shown to play an important role in growth and development, senescence, and defense responses [1]. LOX products have been involved in early signal transduction that occurs during plant^microbe interaction [2]. Though 10 di¡erent potato LOX sequences have been recorded in the databases, three of them have been well characterised which are organ and substrate speci¢c: LOX-1 being highly expressed in tubers and roots, LOX-2 in leaves and LOX-3 in leaves and roots [3]. It is also demonstrated that
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wounding induced the expression of both LOX-2 and LOX-3 in potato leaves [3]. In potato tubers, LOX has been implicated in the elicitor-activation of defense-related responses against Phytophthora infestans, probably through its action on arachidonic acid (AA) and eicosapentaenoic acid (EPA), the PUFAs derived from the fungus [4^6]. The facts that LOX activity is induced in elicitor-treated potato tubers and LOX inhibitors suppress AA-induced phytoalexin accumulation [7,8] suggest that LOX participates in the coupling of elicitor reception and activation of defense responses. Thus most of the studies on defense responses and role of LOXs in potato tubers were concentrated on AA and/or EPA either added directly or derived from the fungus [9^11]. However, none of the LOX metabolites of AA were e¡ective elicitors of hypersensitive responses in potato tubers [12], indicating that LOX has an alternative role other than acting on AA. Moreover, there are no reports on the role of endogenous fatty acids, such as octadecadienoic acid (ODE, 18:2-g-6) and octadecatrienoic acid (OTrE, 18:3-g-3) in LOX-mediated defense responses in potato tubers. Hence the present investigation was undertaken to study the type of LOX metabolites formed from endogenous fatty acids during injury and infection of potato tubers. To our knowledge, this forms the ¢rst report on the di¡erential formation of ODE and OTrE products in control and injured/infected potato tubers. 2. Materials and methods 2.1. Materials Octadecadienoic acid and octadecatrienoic acids were purchased from Sigma, USA. Potato tubers (Solanum tuberosum L. cultivar of kufri jyothi) were obtained from local markets. Strains of Rhizoctonia bataticola were kindly provided by Dr. M.V. Reddy, Legume Pathology Unit, ICRISAT, Hyderabad. 2.2. Methods 2.2.1. Treatment of potato tubers Fresh potato tubers obtained from local markets
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were divided into three groups. The ¢rst group of tubers, kept at laboratory conditions without any treatment, was considered as control. The second group of potato tubers, injured with a sterile knife, was considered as injured. The third group of tubers was infected with a sterile knife dipped in a liquid culture of R. bataticola. The time of injury/infection was noted as zero time. The potato tubers were taken at intervals of 2, 4, 6, 8, 10 and 12 h after injury/ infection for experimentation, cut into pieces and then homogenised in Waring blender. Lipoxygenase activity in the homogenates was measured on an oxygraph as described below. 2.2.2. Lipoxygenase extraction Potato tubers were homogenised (20% w/v) in 100 mM potassium phosphate bu¡er, pH 6.3 containing 2 mM sodium metabisul¢te, 1 mM EDTA and 1 mM ascorbic acid. The homogenate was passed through four layers of cheese cloth and centrifuged at l0 000Ug for 30 min. The supernatant obtained was used as the enzyme source for the assay as well as for endogenous product analysis. 2.2.3. Lipoxygenase assay Lipoxygenase activity was measured polarographically using a Clark type oxygen electrode in YSI model-5300 biological oxygen monitor as described earlier [13]. Typical reaction mixture contained 2.9 ml of bu¡er and 100 Wl of enzyme. The reaction was initiated by the addition of ODE/OTrE (133 WM in ¢nal concentration). The enzyme activity was expressed as U/g tissue, where one unit is de¢ned as Wmol of O2 consumed/min. 2.2.4. Analysis of fatty acids Lipids from potatoes were extracted with chloroform:methanol (2:1 v/v) containing 0.02% butylated hydroxy toluene (BHT) and the lipid fractions were then separated by TLC over silica gel G using nhexane:diethyl ether:acetic acid in a ratio of 80: 20:1 [14]. The phospholipids and fatty acids were scraped o¡ into 10-ml culture tubes separately for methylation. Methyl esters were prepared using boron tri£uoride methanol reagent. Care was taken to maintain a nitrogen atmosphere during all steps of the extraction and fractionation. The fatty acid esters were analysed on a Tracor 540 GC Unit with a £ame
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ionisation detector under the following conditions: inlet temperature 200³C, detector 220³C, separating column 2 m long and 2 mm i.d. packed with 10% silar 10C on Chromosorb W, 80/100 mesh, and connected to an automatic integrator. 2.2.5. Analysis of endogenous lipoxygenase products Endogenous LOX products were extracted from the homogenates of control, injured and infected potato tubers, twice with hexane:ether (50:50 v/v) separately. The pooled organic phase was evaporated to dryness under vacuum and redissolved in HPLC solvent (hexane:propanol:acetic acid, 1000:32:1). The LOX-products were separated on straight phase HPLC (Shimadzu LC 6 AD equipped with SPD-6 AV UV/VIS detector) using Shimadzu CLCSIL (4.6U250 mm) column [15]. The products were monitored at 235 nm. The peaks with characteristic conjugated diene spectrum were identi¢ed based on the retention times, co-chromatography with authentic standards and GC-MS analysis. The identi¢ed compounds were quanti¢ed based on peak heights and peak areas obtained on HPLC integrator. 2.2.6. GC-MS analysis GC-MS analysis of the endogenous LOX products was done at The Pennsylvania State University, USA with the facilities of Dr. C. Channa Reddy, using Hewlett Packard 5890 series II gas chromatograph coupled to a Hewlett Packard 5971 mass spectrometer [16]. The LOX products were reduced with sodium borohydride, methylated and silylated using BSTFA of Supleco, Bellefonte, PA, before GC-MS analysis. The separation conditions were: 15 m fused silica column, 0.20 mm i.d. with 0.20 Wm ¢lm thickness, temperature program 3 min/70³C, then 10³C/min to 240³C. The gas carrier was helium, 2 ml/min. 3. Results 3.1. Fatty acid analysis The fatty acid analysis of potato tubers showed that ODE (g-6) is the predominant fatty acid followed by OTrE (g-3) in both bound and free lipid fractions. The relative distribution of ODE:OTrE
Fig. 1. Measurements of lipoxygenase activity in potato tubers at di¡erent time-intervals after injury or infection with Rhizoctonia bataticola. LOX activity was measured using a Clark type oxygen electrode YSI model-5300 biological oxygen monitor. The typical reaction mixture contained 2.9 ml of phosphate bu¡er and 100 Wl of enzyme and reaction was initiated by the addition of ODE/OTrE (133 WM ¢nal concentration). The LOX activity was expressed as U/g tissue, where one unit is de¢ned as 1 Wmol oxygen consumed/min.
in bound form is 43.6:3.87% and free form is 15.15:8.16%. 3.2. Lipoxygenase activity Lipoxygenase activity in injured/infected potato tubers was measured, for every 2 h after the respective treatments, on an oxygraph. As shown in Fig. 1 LOX activity increased rapidly in potato tubers in response to injury, reaching a maximum of 3-fold after 6 h of injury with a later stabilisation. Infection with R. bataticola, on the other hand, resulted in a rapid induction of LOX activity reaching maximum within 10 h (10-fold) after infection, with a decline at later periods. 3.3. Endogenous LOX products Endogenous LOX products were extracted from control, injured and infected potatoes and separated on SP-HPLC. As shown in Fig. 2A, the endogenous
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Fig. 2. Analysis of LOX products formed endogenously in control (A), injured (B) and infected (C) potato tubers. Endogenous LOX products from the potato homogenates, extracted into hexane:ether (50:50 v/v), were evaporated to dryness under vacuum and redissolved in HPLC solvent (hexane:propanol:acetic acid 1000:32:1). The LOX products were separated on straight phase HPLC (Shimadzu LC 6AD equipped with SPD-6-AV UV/VIS detector) using Shimadzu CLC-SIL (4.6U250 mm) column. The products were monitored at 235 nm.
LOX products from control potatoes showed a major peak with RT 15.67 min and a minor peak with RT 17.56 min on SP-HPLC, when the products were monitored at 235 nm. Both the peaks showed typical conjugated diene spectra with absorption maxima at 235 nm. Further identi¢cation of the peaks was done based on GC-MS analysis. The peaks eluted at 15.67 and 17.56 min were reduced with sodium borohydride, methylated and trimethyl silylated before taking for GC/MS analysis. As shown in Fig. 3A, the peak with RT 15.67 min showed prominent ions at m/e 382 (M ), 292 (M 390) and 225 [M 3157, loss of ^CH2 ^(CH2 )6 ^COOCH3 ]. Based on the above fragmentation pattern and on comparison with the mass spectra of authentic standard, the peak with RT 15.67 min was identi¢ed as 9-HPODE. Similarly, the peak with RT 17.56 min was subjected to GCMS analysis after reduction, methylation and derivatisation (Fig. 3B). As shown in the ¢gure, the spectrum showed prominent ions at m/e 380 (M ), 290 (M 390) and 223 [M 3157, loss of ^CH2 ^(CH2 )6 ^ COOCH3 ]. Based on these and on comparison with
the mass spectra of authentic standard, the peak with RT 17.56 min was identi¢ed as 9-HPOTrE. Similarly, peaks I and II (Fig. 2B,C) eluted in injured and infected potato tubers were identi¢ed as 9-HPODE and 9-HPOTrE, respectively, based on mass spectral analysis (data not shown). Peak III in Fig. 2B,C was identi¢ed as 9-HOTrE, the reduced product of 9HPOTrE. The relative ratio of 9-HPODE to 9-HPOTrE in control tubers was 4:1. In injured potatoes tubers (Fig. 2B), there is a shift in the relative ratio of 9HPODE to 9-HPOTrE from 4:1 to 1:2. HPLC analysis of the products extracted from infected potato tubers, Fig. 2C, revealed a major peak with RT of 17.78 min and a minor peak with RT of 14.98 min, which were identi¢ed as 9-HPOTrE and 9-HPODE, respectively. Similar to injured potato tubers, 9HPOTrE is the major LOX product formed in the infected potato tubers also (Fig. 2C). As a result the relative ratio of 9-HPODE to 9-HPOTrE was changed from 4:1 in control to 1:4.5 in infected tubers.
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Fig. 3. GC-MS analysis of LOX products (peak I, A; peak II, B) separated on HPLC. Peaks I and II were reduced with sodium borohydride, methylated and silylated before analysis on GC-MS using 15 m fused silica column.
4. Discussion The present results show a 3-fold increase in lipoxygenase activity within 6 h of injury and a 10-fold increase after 10 h of infection of the potato tubers. This up-regulation of LOX activity in injured/infected potato tubers may be at genetic or epigenetic levels. The delayed increase in LOX activity observed in the present study indicates that up-regulation is not an immediate e¡ect, suggesting transcriptional and/or translational regulatory processes. Royo et
al. have shown increased lipoxygenase transcript levels in discs from stored potato tubers in response to wounding [3]. This observation supports the ¢rst possibility. Lipoxygenase in potato tubers was implicated in elicitation of defense responses against pathogen infections by AA and EPA, the fatty acids derived from the pathogen [4^6]. Although AA derived from pathogen is a substrate for LOX, none of the products of AA was shown to be an elicitor of LOX induction [12]. This observation suggests an alternative role for LOX other than acting on AA.
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The fatty acid analysis of potato tubers revealed that ODE is the major PUFA either in free pool (15%) or in bound form (44%) with OTrE being 4% in bound and 8% in free form. The endogenous LOX product analysis of control potato tubers revealed the presence of 9-HPODE as the major product with 9HPOTrE as a minor product, indicating that in control conditions, potato LOX prefers ODE, g-6 fatty acid. Substrate speci¢city studies have also revealed that potato LOX exhibits higher activity towards ODE when compared to AA and OTrE [17]. In the present study, the lipoxygenase products formed in potato tubers predominantly are 9-HPODE and 9HPOTrE. This observation is quite opposite to the lipoxygenase products formed in soybean and other legumes, where in 13-HPODE and 13-HPOTrE are the major products [15,18]. This variation in the stereospeci¢c oxygenation by potato lipoxygenase is due to (32) rearrangement of the fatty acid radical as against (+2) rearrangement observed in soybean and other legume lipoxygenases [18,19]. The endogenous product pro¢les of injured as well as infected potato tubers, however, were quite di¡erent from those of control tubers, in that the major product formed being 9-HPOTrE. As a result the relative ratio of 9-HPODE to 9-HPOTrE formed were changed from 4:1 in control to 1:2 in injured and 1:4.5 in infected potato tubers. These observations indicate a preferential shift in LOX speci¢city towards OTrE, from g-6 to g-3 fatty acid. This could be possible due to the speci¢c release of OTrE from membrane phospholipids in response to injury/infection in potato tubers or increased LOX speci¢city towards OTrE. Studies of Mueller et al. [20] showing the release of OTrE from membrane lipids in response to elicitor treatment support the ¢rst possibility. Recently, Ryu and Wang reported an increase in both free ODE and OTrE in wounded castor bean leaves indicating the release of both fatty acids [21]. Another possibility could be di¡erential expression of LOX genes in response to injury and infection as shown in leaves of soybean [22], and even in potato [3], which might be involved in the biosynthesis of jasmonic acid. Interestingly, Royo et al. [3] did not identify the expression of any new LOX isoforms except the constitutive LOX-1 in potato tubers, refuting this possibility. Also,
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9-HPOTrE, the OTrE product of potato tuber LOX formed in response to injury and infection, is not a precursor of either jasmonic acid (JA) or traumatic acid (TA), further questioning the necessity of di¡erential synthesis of LOX products upon injury and infection. Jasmonic acid and traumatic acid, the main regulatory molecules formed through lipoxygenase pathway, are mainly formed from 13-HPODE or 13-HPOTrE in plant systems. In this connection, it is interesting to note that the OTrE product of potato tuber LOX, 9-HPOTrE was more anti-fungal than 9-HPODE towards R. bataticola (data not shown), which might possibly explain the importance of these compounds during injury and infection. Kato et al. have shown that the LOX products of OTrE from infected leaves of tomato are antimicrobial, supporting such a possibility [23]. The excess 9HPOTrE formed during injury/infection could be transformed into 9-HOTrE by peroxidase pathway [24], which is formed in the present study also in smaller quantities in injured/infected potato tubers. Further studies, however, are required to test whether 9-HPOTrE is involved in the activation of defense responses directly or through its metabolites. Thus 9-HPOTrE is the major LOX product formed in response to injury/infection in potato tubers, which is neither a precursor of jasmonic acid nor traumatic acid, indicating the operation of a new pathway of LOX-mediated defense responses in potato tubers. From these results it is suggested that LOX metabolites of ODE may be involved in mediating the physiological responses, while OTrE metabolites may be mediating defense responses under stress conditions in potato tubers. Similar results were obtained in pigeon pea seedlings infected with Fusarium udum, where in 13-HPOTrE, LOX product of OTrE, is the major product formed in infected seedlings compared to 13-HPODE, LOX product of ODE, formed in uninfected control seedlings (data not shown), suggesting that it could be a general phenomenon in plants. Further studies, however, are required on other plant systems to universalise this statement. To our knowledge, as there is no earlier report on the di¡erential formation of endogenous products under control and stress conditions, this forms the ¢rst report and is worth probing thoroughly.
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Acknowledgements The authors gratefully acknowledge Dr. C. Channa Reddy of The Pennsylvania State University, USA for extending GC-MS facilities and Dr. M.V. Reddy, ICRISAT, Hyderabad for providing Rhizoctonia bataticola strains. This work was supported by grants from the Department of Biotechnology, Government of India (BT/18/06/96-PID). References [1] J.N. Siedow, Annu. Rev. Plant Physiol. Plant Mol. Biol. 42 (1991) 145^188. [2] R.M. Bostock, B.A. Stermer, Annu. Rev. Phytopathol. 27 (1989) 353^371. [3] J. Royo, G. Vancanney, A.G. Perez, C. Sanz, K.S. St. Formann, J. Rosahl Sanchez-Serrano, J. Biol. Chem. 271 (1996) 21012^21019. [4] R.M. Bostock, J.A. Kuc, R.A. Laine, Science 212 (1981) 67^ 69. [5] T. Schewe, S. Nigam, in: Sinzinger (Ed.), Recent Advances in Prostaglandin, Thromboxane, and Leukotriene Research, Plenum Press, New York, 1998, pp. 221^226. [6] C.L. Preisig, J.A. Kuc, Plant Physiol. 72 (1983) S158. [7] D.A. Stelzig, R.D. Allen, S.K. Bhatia, Plant Physiol. 72 (1983) 746^749. [8] C.L. Preisig, J.A. Kuc, Plant Physiol. 84 (1987) 891^894. [9] R.M. Bostock, D.A. Scha¡er, R. Hammerschmidt, Physiol. Mol. Plant Pathol. 29 (1986) 349^360.
[10] D.A. Davis, W.W. Currier, Physiol. Mol. Plant Pathol. 29 (1986) 431^441. [11] D.A. Davis, W.W. Currier, Physiol. Mol. Plant Pathol. 33 (1988) 105^114. [12] K.E. Ricker, R.M. Bostock, Physiol. Mol. Plant Pathol. 44 (1994) 65^80. [13] P. Reddanna, J. Whelan, K.R. Maddipati, C.C. Reddy, Methods Enzymol. 187 (1990) 268^277. [14] A. Jayadeep, P. Reddanna, S. Sailesh, U.N. Das, G. Ramesh, K. Vijay Kumar, V.P. Menon, Prostaglandins Leukotrienes Essent. Fatty Acids 52 (1995) 235^239. [15] Y.V. Kiran Kumar, S. Sailesh, M. Prasad, P. Reddanna, Biochem. Arch. 8 (1992) 17^22. [16] G. Ramakrishna Reddy, P. Reddanna, C. Channa Reddy, Curtis Wayne, Biochem. Biophys. Res. Commun. 189 (3) (1992) 1349^1352. [17] T. Shimizu, Z.I. Handa, I. Miki, Y. Seyana, T. Izuni, O. Radmark, B. Samuelsson, Methods Enzymol. 187 (1990) 296^306. [18] V. Nikolaev, P. Reddanna, J. Whelan, G. Hildenbrandt, C.C. Reddy, Biochem. Biophys. Res. Commun. 170 (1990) 491^496. [19] P. Chaitidis, T. Schewe, M. Sutherland, H. Kuhn, S. Nigam, FEBS Lett. 434 (1998) 437^441. [20] M.J. Mueller, W. Brodschelm, E. Spannagl, M.H. Zenk, Proc. Natl. Acad. Sci. USA 90 (1993) 7490^7494. [21] S.B. Ryu, X. Wang, Biochim. Biophys. Acta 1393 (1998) 193^202. [22] D. Saravitz, J.N. Siedow, Plant Physiol. 110 (1996) 287^ 299. [23] T. Kato, Y. Maeda, T. Hirukawa, T. Naimai, N. Yoshioka, Biosci. Biotechnol. Biochem. 56 (1992) 373^375. [24] E. Thee, J. Joyard, Plant Physiol. 110 (1996) 445^454.
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Di¡erential formation of octadecadienoic acid and octadecatrienoic acid products in control and injured/infected potato tubers P. Srinivas Reddy, T. Charles Kumar, M. Narsa Reddy, C. Sarada, P. Reddanna * School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India Received 17 August 1999; received in revised form 12 October 1999; accepted 17 November 1999
Abstract Lipoxygenases in plants have been implicated in the activation of defense responses against injury/infection. Pathogenderived polyunsaturated fatty acids, such as arachidonic acid, eicosapentaenoic acid and their metabolites have been shown to elicit defense responses against pathogen infection in plants. However, not much is known about the role of host-derived fatty acids and their metabolites in plant defense responses. In this study, isolation and characterisation of endogenous lipoxygenase metabolites formed in potato tubers in response to injury/infection was undertaken. While 9-hydroperoxyoctadecadienoic acid (9-HPODE), derived from octadecdienoic acid (linoleic acid) is the major lipoxygenase product formed in control potato tubers, 9-hydroperoxyoctadecatrienoic acid (9-HPOTrE), derived from octadecatrienoic acid (Klinolenic acid) is the major lipoxygenase product formed in potato tubers in response to injury or infection with Rhizoctonia bataticola. As a result, the relative ratio of 9-HPODE to 9-HPOTrE showed a shift from 4:1 in control to 1:2 and 1:4.5 in injured and infected potato tubers respectively. From this study, it is proposed that lipoxygenase metabolites of octadecadienoic acid may be involved in physiological responses under control conditions, while octadecatrienoic acid metabolites are mediating the defense responses. This forms the first report on the differential formation of endogenous lipoxygenase products in potato tubers under control and stress conditions. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Lipoxygenase; Potato tuber; Octadecadienoic acid; Octadecatrienoic acid; Injury; Infection
1. Introduction Lipoxygenases (LOX, EC 1.13.11.12) are a group of enzymes involved in the oxygenation of polyunsaturated fatty acids (PUFAs) containing cis, cis-1,4pentadiene structures and are ubiquitous both in plants and animals. The immediate oxygenation products of PUFAs by LOX are hydroperoxy conjugated dienes, which are transformed into a spectrum
* Corresponding author. Fax: +91-40-301-0120; E-mail: [email protected]
of biologically active compounds, such as leukotrienes in animals and jasmonic acid, traumatic acid and alkenals in plants. In plants, LOX products have been shown to play an important role in growth and development, senescence, and defense responses [1]. LOX products have been involved in early signal transduction that occurs during plant^microbe interaction [2]. Though 10 di¡erent potato LOX sequences have been recorded in the databases, three of them have been well characterised which are organ and substrate speci¢c: LOX-1 being highly expressed in tubers and roots, LOX-2 in leaves and LOX-3 in leaves and roots [3]. It is also demonstrated that
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wounding induced the expression of both LOX-2 and LOX-3 in potato leaves [3]. In potato tubers, LOX has been implicated in the elicitor-activation of defense-related responses against Phytophthora infestans, probably through its action on arachidonic acid (AA) and eicosapentaenoic acid (EPA), the PUFAs derived from the fungus [4^6]. The facts that LOX activity is induced in elicitor-treated potato tubers and LOX inhibitors suppress AA-induced phytoalexin accumulation [7,8] suggest that LOX participates in the coupling of elicitor reception and activation of defense responses. Thus most of the studies on defense responses and role of LOXs in potato tubers were concentrated on AA and/or EPA either added directly or derived from the fungus [9^11]. However, none of the LOX metabolites of AA were e¡ective elicitors of hypersensitive responses in potato tubers [12], indicating that LOX has an alternative role other than acting on AA. Moreover, there are no reports on the role of endogenous fatty acids, such as octadecadienoic acid (ODE, 18:2-g-6) and octadecatrienoic acid (OTrE, 18:3-g-3) in LOX-mediated defense responses in potato tubers. Hence the present investigation was undertaken to study the type of LOX metabolites formed from endogenous fatty acids during injury and infection of potato tubers. To our knowledge, this forms the ¢rst report on the di¡erential formation of ODE and OTrE products in control and injured/infected potato tubers. 2. Materials and methods 2.1. Materials Octadecadienoic acid and octadecatrienoic acids were purchased from Sigma, USA. Potato tubers (Solanum tuberosum L. cultivar of kufri jyothi) were obtained from local markets. Strains of Rhizoctonia bataticola were kindly provided by Dr. M.V. Reddy, Legume Pathology Unit, ICRISAT, Hyderabad. 2.2. Methods 2.2.1. Treatment of potato tubers Fresh potato tubers obtained from local markets
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were divided into three groups. The ¢rst group of tubers, kept at laboratory conditions without any treatment, was considered as control. The second group of potato tubers, injured with a sterile knife, was considered as injured. The third group of tubers was infected with a sterile knife dipped in a liquid culture of R. bataticola. The time of injury/infection was noted as zero time. The potato tubers were taken at intervals of 2, 4, 6, 8, 10 and 12 h after injury/ infection for experimentation, cut into pieces and then homogenised in Waring blender. Lipoxygenase activity in the homogenates was measured on an oxygraph as described below. 2.2.2. Lipoxygenase extraction Potato tubers were homogenised (20% w/v) in 100 mM potassium phosphate bu¡er, pH 6.3 containing 2 mM sodium metabisul¢te, 1 mM EDTA and 1 mM ascorbic acid. The homogenate was passed through four layers of cheese cloth and centrifuged at l0 000Ug for 30 min. The supernatant obtained was used as the enzyme source for the assay as well as for endogenous product analysis. 2.2.3. Lipoxygenase assay Lipoxygenase activity was measured polarographically using a Clark type oxygen electrode in YSI model-5300 biological oxygen monitor as described earlier [13]. Typical reaction mixture contained 2.9 ml of bu¡er and 100 Wl of enzyme. The reaction was initiated by the addition of ODE/OTrE (133 WM in ¢nal concentration). The enzyme activity was expressed as U/g tissue, where one unit is de¢ned as Wmol of O2 consumed/min. 2.2.4. Analysis of fatty acids Lipids from potatoes were extracted with chloroform:methanol (2:1 v/v) containing 0.02% butylated hydroxy toluene (BHT) and the lipid fractions were then separated by TLC over silica gel G using nhexane:diethyl ether:acetic acid in a ratio of 80: 20:1 [14]. The phospholipids and fatty acids were scraped o¡ into 10-ml culture tubes separately for methylation. Methyl esters were prepared using boron tri£uoride methanol reagent. Care was taken to maintain a nitrogen atmosphere during all steps of the extraction and fractionation. The fatty acid esters were analysed on a Tracor 540 GC Unit with a £ame
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ionisation detector under the following conditions: inlet temperature 200³C, detector 220³C, separating column 2 m long and 2 mm i.d. packed with 10% silar 10C on Chromosorb W, 80/100 mesh, and connected to an automatic integrator. 2.2.5. Analysis of endogenous lipoxygenase products Endogenous LOX products were extracted from the homogenates of control, injured and infected potato tubers, twice with hexane:ether (50:50 v/v) separately. The pooled organic phase was evaporated to dryness under vacuum and redissolved in HPLC solvent (hexane:propanol:acetic acid, 1000:32:1). The LOX-products were separated on straight phase HPLC (Shimadzu LC 6 AD equipped with SPD-6 AV UV/VIS detector) using Shimadzu CLCSIL (4.6U250 mm) column [15]. The products were monitored at 235 nm. The peaks with characteristic conjugated diene spectrum were identi¢ed based on the retention times, co-chromatography with authentic standards and GC-MS analysis. The identi¢ed compounds were quanti¢ed based on peak heights and peak areas obtained on HPLC integrator. 2.2.6. GC-MS analysis GC-MS analysis of the endogenous LOX products was done at The Pennsylvania State University, USA with the facilities of Dr. C. Channa Reddy, using Hewlett Packard 5890 series II gas chromatograph coupled to a Hewlett Packard 5971 mass spectrometer [16]. The LOX products were reduced with sodium borohydride, methylated and silylated using BSTFA of Supleco, Bellefonte, PA, before GC-MS analysis. The separation conditions were: 15 m fused silica column, 0.20 mm i.d. with 0.20 Wm ¢lm thickness, temperature program 3 min/70³C, then 10³C/min to 240³C. The gas carrier was helium, 2 ml/min. 3. Results 3.1. Fatty acid analysis The fatty acid analysis of potato tubers showed that ODE (g-6) is the predominant fatty acid followed by OTrE (g-3) in both bound and free lipid fractions. The relative distribution of ODE:OTrE
Fig. 1. Measurements of lipoxygenase activity in potato tubers at di¡erent time-intervals after injury or infection with Rhizoctonia bataticola. LOX activity was measured using a Clark type oxygen electrode YSI model-5300 biological oxygen monitor. The typical reaction mixture contained 2.9 ml of phosphate bu¡er and 100 Wl of enzyme and reaction was initiated by the addition of ODE/OTrE (133 WM ¢nal concentration). The LOX activity was expressed as U/g tissue, where one unit is de¢ned as 1 Wmol oxygen consumed/min.
in bound form is 43.6:3.87% and free form is 15.15:8.16%. 3.2. Lipoxygenase activity Lipoxygenase activity in injured/infected potato tubers was measured, for every 2 h after the respective treatments, on an oxygraph. As shown in Fig. 1 LOX activity increased rapidly in potato tubers in response to injury, reaching a maximum of 3-fold after 6 h of injury with a later stabilisation. Infection with R. bataticola, on the other hand, resulted in a rapid induction of LOX activity reaching maximum within 10 h (10-fold) after infection, with a decline at later periods. 3.3. Endogenous LOX products Endogenous LOX products were extracted from control, injured and infected potatoes and separated on SP-HPLC. As shown in Fig. 2A, the endogenous
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Fig. 2. Analysis of LOX products formed endogenously in control (A), injured (B) and infected (C) potato tubers. Endogenous LOX products from the potato homogenates, extracted into hexane:ether (50:50 v/v), were evaporated to dryness under vacuum and redissolved in HPLC solvent (hexane:propanol:acetic acid 1000:32:1). The LOX products were separated on straight phase HPLC (Shimadzu LC 6AD equipped with SPD-6-AV UV/VIS detector) using Shimadzu CLC-SIL (4.6U250 mm) column. The products were monitored at 235 nm.
LOX products from control potatoes showed a major peak with RT 15.67 min and a minor peak with RT 17.56 min on SP-HPLC, when the products were monitored at 235 nm. Both the peaks showed typical conjugated diene spectra with absorption maxima at 235 nm. Further identi¢cation of the peaks was done based on GC-MS analysis. The peaks eluted at 15.67 and 17.56 min were reduced with sodium borohydride, methylated and trimethyl silylated before taking for GC/MS analysis. As shown in Fig. 3A, the peak with RT 15.67 min showed prominent ions at m/e 382 (M ), 292 (M 390) and 225 [M 3157, loss of ^CH2 ^(CH2 )6 ^COOCH3 ]. Based on the above fragmentation pattern and on comparison with the mass spectra of authentic standard, the peak with RT 15.67 min was identi¢ed as 9-HPODE. Similarly, the peak with RT 17.56 min was subjected to GCMS analysis after reduction, methylation and derivatisation (Fig. 3B). As shown in the ¢gure, the spectrum showed prominent ions at m/e 380 (M ), 290 (M 390) and 223 [M 3157, loss of ^CH2 ^(CH2 )6 ^ COOCH3 ]. Based on these and on comparison with
the mass spectra of authentic standard, the peak with RT 17.56 min was identi¢ed as 9-HPOTrE. Similarly, peaks I and II (Fig. 2B,C) eluted in injured and infected potato tubers were identi¢ed as 9-HPODE and 9-HPOTrE, respectively, based on mass spectral analysis (data not shown). Peak III in Fig. 2B,C was identi¢ed as 9-HOTrE, the reduced product of 9HPOTrE. The relative ratio of 9-HPODE to 9-HPOTrE in control tubers was 4:1. In injured potatoes tubers (Fig. 2B), there is a shift in the relative ratio of 9HPODE to 9-HPOTrE from 4:1 to 1:2. HPLC analysis of the products extracted from infected potato tubers, Fig. 2C, revealed a major peak with RT of 17.78 min and a minor peak with RT of 14.98 min, which were identi¢ed as 9-HPOTrE and 9-HPODE, respectively. Similar to injured potato tubers, 9HPOTrE is the major LOX product formed in the infected potato tubers also (Fig. 2C). As a result the relative ratio of 9-HPODE to 9-HPOTrE was changed from 4:1 in control to 1:4.5 in infected tubers.
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Fig. 3. GC-MS analysis of LOX products (peak I, A; peak II, B) separated on HPLC. Peaks I and II were reduced with sodium borohydride, methylated and silylated before analysis on GC-MS using 15 m fused silica column.
4. Discussion The present results show a 3-fold increase in lipoxygenase activity within 6 h of injury and a 10-fold increase after 10 h of infection of the potato tubers. This up-regulation of LOX activity in injured/infected potato tubers may be at genetic or epigenetic levels. The delayed increase in LOX activity observed in the present study indicates that up-regulation is not an immediate e¡ect, suggesting transcriptional and/or translational regulatory processes. Royo et
al. have shown increased lipoxygenase transcript levels in discs from stored potato tubers in response to wounding [3]. This observation supports the ¢rst possibility. Lipoxygenase in potato tubers was implicated in elicitation of defense responses against pathogen infections by AA and EPA, the fatty acids derived from the pathogen [4^6]. Although AA derived from pathogen is a substrate for LOX, none of the products of AA was shown to be an elicitor of LOX induction [12]. This observation suggests an alternative role for LOX other than acting on AA.
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The fatty acid analysis of potato tubers revealed that ODE is the major PUFA either in free pool (15%) or in bound form (44%) with OTrE being 4% in bound and 8% in free form. The endogenous LOX product analysis of control potato tubers revealed the presence of 9-HPODE as the major product with 9HPOTrE as a minor product, indicating that in control conditions, potato LOX prefers ODE, g-6 fatty acid. Substrate speci¢city studies have also revealed that potato LOX exhibits higher activity towards ODE when compared to AA and OTrE [17]. In the present study, the lipoxygenase products formed in potato tubers predominantly are 9-HPODE and 9HPOTrE. This observation is quite opposite to the lipoxygenase products formed in soybean and other legumes, where in 13-HPODE and 13-HPOTrE are the major products [15,18]. This variation in the stereospeci¢c oxygenation by potato lipoxygenase is due to (32) rearrangement of the fatty acid radical as against (+2) rearrangement observed in soybean and other legume lipoxygenases [18,19]. The endogenous product pro¢les of injured as well as infected potato tubers, however, were quite di¡erent from those of control tubers, in that the major product formed being 9-HPOTrE. As a result the relative ratio of 9-HPODE to 9-HPOTrE formed were changed from 4:1 in control to 1:2 in injured and 1:4.5 in infected potato tubers. These observations indicate a preferential shift in LOX speci¢city towards OTrE, from g-6 to g-3 fatty acid. This could be possible due to the speci¢c release of OTrE from membrane phospholipids in response to injury/infection in potato tubers or increased LOX speci¢city towards OTrE. Studies of Mueller et al. [20] showing the release of OTrE from membrane lipids in response to elicitor treatment support the ¢rst possibility. Recently, Ryu and Wang reported an increase in both free ODE and OTrE in wounded castor bean leaves indicating the release of both fatty acids [21]. Another possibility could be di¡erential expression of LOX genes in response to injury and infection as shown in leaves of soybean [22], and even in potato [3], which might be involved in the biosynthesis of jasmonic acid. Interestingly, Royo et al. [3] did not identify the expression of any new LOX isoforms except the constitutive LOX-1 in potato tubers, refuting this possibility. Also,
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9-HPOTrE, the OTrE product of potato tuber LOX formed in response to injury and infection, is not a precursor of either jasmonic acid (JA) or traumatic acid (TA), further questioning the necessity of di¡erential synthesis of LOX products upon injury and infection. Jasmonic acid and traumatic acid, the main regulatory molecules formed through lipoxygenase pathway, are mainly formed from 13-HPODE or 13-HPOTrE in plant systems. In this connection, it is interesting to note that the OTrE product of potato tuber LOX, 9-HPOTrE was more anti-fungal than 9-HPODE towards R. bataticola (data not shown), which might possibly explain the importance of these compounds during injury and infection. Kato et al. have shown that the LOX products of OTrE from infected leaves of tomato are antimicrobial, supporting such a possibility [23]. The excess 9HPOTrE formed during injury/infection could be transformed into 9-HOTrE by peroxidase pathway [24], which is formed in the present study also in smaller quantities in injured/infected potato tubers. Further studies, however, are required to test whether 9-HPOTrE is involved in the activation of defense responses directly or through its metabolites. Thus 9-HPOTrE is the major LOX product formed in response to injury/infection in potato tubers, which is neither a precursor of jasmonic acid nor traumatic acid, indicating the operation of a new pathway of LOX-mediated defense responses in potato tubers. From these results it is suggested that LOX metabolites of ODE may be involved in mediating the physiological responses, while OTrE metabolites may be mediating defense responses under stress conditions in potato tubers. Similar results were obtained in pigeon pea seedlings infected with Fusarium udum, where in 13-HPOTrE, LOX product of OTrE, is the major product formed in infected seedlings compared to 13-HPODE, LOX product of ODE, formed in uninfected control seedlings (data not shown), suggesting that it could be a general phenomenon in plants. Further studies, however, are required on other plant systems to universalise this statement. To our knowledge, as there is no earlier report on the di¡erential formation of endogenous products under control and stress conditions, this forms the ¢rst report and is worth probing thoroughly.
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Acknowledgements The authors gratefully acknowledge Dr. C. Channa Reddy of The Pennsylvania State University, USA for extending GC-MS facilities and Dr. M.V. Reddy, ICRISAT, Hyderabad for providing Rhizoctonia bataticola strains. This work was supported by grants from the Department of Biotechnology, Government of India (BT/18/06/96-PID). References [1] J.N. Siedow, Annu. Rev. Plant Physiol. Plant Mol. Biol. 42 (1991) 145^188. [2] R.M. Bostock, B.A. Stermer, Annu. Rev. Phytopathol. 27 (1989) 353^371. [3] J. Royo, G. Vancanney, A.G. Perez, C. Sanz, K.S. St. Formann, J. Rosahl Sanchez-Serrano, J. Biol. Chem. 271 (1996) 21012^21019. [4] R.M. Bostock, J.A. Kuc, R.A. Laine, Science 212 (1981) 67^ 69. [5] T. Schewe, S. Nigam, in: Sinzinger (Ed.), Recent Advances in Prostaglandin, Thromboxane, and Leukotriene Research, Plenum Press, New York, 1998, pp. 221^226. [6] C.L. Preisig, J.A. Kuc, Plant Physiol. 72 (1983) S158. [7] D.A. Stelzig, R.D. Allen, S.K. Bhatia, Plant Physiol. 72 (1983) 746^749. [8] C.L. Preisig, J.A. Kuc, Plant Physiol. 84 (1987) 891^894. [9] R.M. Bostock, D.A. Scha¡er, R. Hammerschmidt, Physiol. Mol. Plant Pathol. 29 (1986) 349^360.
[10] D.A. Davis, W.W. Currier, Physiol. Mol. Plant Pathol. 29 (1986) 431^441. [11] D.A. Davis, W.W. Currier, Physiol. Mol. Plant Pathol. 33 (1988) 105^114. [12] K.E. Ricker, R.M. Bostock, Physiol. Mol. Plant Pathol. 44 (1994) 65^80. [13] P. Reddanna, J. Whelan, K.R. Maddipati, C.C. Reddy, Methods Enzymol. 187 (1990) 268^277. [14] A. Jayadeep, P. Reddanna, S. Sailesh, U.N. Das, G. Ramesh, K. Vijay Kumar, V.P. Menon, Prostaglandins Leukotrienes Essent. Fatty Acids 52 (1995) 235^239. [15] Y.V. Kiran Kumar, S. Sailesh, M. Prasad, P. Reddanna, Biochem. Arch. 8 (1992) 17^22. [16] G. Ramakrishna Reddy, P. Reddanna, C. Channa Reddy, Curtis Wayne, Biochem. Biophys. Res. Commun. 189 (3) (1992) 1349^1352. [17] T. Shimizu, Z.I. Handa, I. Miki, Y. Seyana, T. Izuni, O. Radmark, B. Samuelsson, Methods Enzymol. 187 (1990) 296^306. [18] V. Nikolaev, P. Reddanna, J. Whelan, G. Hildenbrandt, C.C. Reddy, Biochem. Biophys. Res. Commun. 170 (1990) 491^496. [19] P. Chaitidis, T. Schewe, M. Sutherland, H. Kuhn, S. Nigam, FEBS Lett. 434 (1998) 437^441. [20] M.J. Mueller, W. Brodschelm, E. Spannagl, M.H. Zenk, Proc. Natl. Acad. Sci. USA 90 (1993) 7490^7494. [21] S.B. Ryu, X. Wang, Biochim. Biophys. Acta 1393 (1998) 193^202. [22] D. Saravitz, J.N. Siedow, Plant Physiol. 110 (1996) 287^ 299. [23] T. Kato, Y. Maeda, T. Hirukawa, T. Naimai, N. Yoshioka, Biosci. Biotechnol. Biochem. 56 (1992) 373^375. [24] E. Thee, J. Joyard, Plant Physiol. 110 (1996) 445^454.
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