The Catecholamine Biosynthesis Route in Potato Is Affected by Stress

Plant Physiology and Biochemistry 42 (2004) 593–600 www.elsevier.com/locate/plaphy Original article The catecholamine biosynthesis route in potato is affected by stress

Anna S´wiVdrych a, Katarzyna Lorenc-Kukuła a, Aleksandra Skirycz a, Jan Szopa a,b,*

a Institute of Biochemistry and Molecular Biology, University of Wrocław, Przybyszewskiego Street 63/77, 51-148 Wrocław, Poland b Department of Plant Physiology, University of Szczecin, Waa˛ska Street 13, 71-415 Szczecin, Poland

Received 16 April 2004; accepted 6 July 2004 Available online 29 July 2004

Abstract

The catecholamine compounds in potato (Solanum tuberosum L.) leaves and tubers have been identified by gas chromatography coupled to mass spectrometry (GC-MS) measurements. The finding that the catecholamine level is dramatically increased upon tyrosine decarboxylase (TD) overexpression potentiates the investigation on their physiological significance in plants. It was then evidenced that catecholamines play an important role in regulation of starch–sucrose conversion in plants. In this paper we investigated catecholamine biosynthetic pathway in potato plants exposed to the different stress conditions. The activation of TD (EC 4.1.1.25), tyrosine hydroxylase (TH, EC 1.14.18.1) and L-Dopa decarboxylase (DD, EC 4.1.1.25) was a characteristic feature of the potato leaves treated with abscisic acid (ABA). In high salt condition only TD activity was increased and in drought both TH and DD were activated. UV light activated predominantly DD activity. Leaves of plants grown in the dark and in red light circumstances were characterized by significantly decreased activities of all the three enzymes whereas those grown in cold were characterized by the decreased activity of DD only. In all, stress conditions the normetanephrine level and thus catecholamine catabolism was significantly decreased. Increased catecholamine level in TD-overexpressing potato resulted in enhanced pathogen resistance. Our data suggest that plant catecholamines are involved in plant responses towards biotic and abiotic stresses. It has to be pointed out that this is the first report proposing catecholamine as new stress agent compounds in plants. © 2004 Elsevier SAS. All rights reserved.

Keywords: Catecholamines; L-Dopa decarboxylase; Tyrosine decarboxylase; Tyrosine hydroxylase; Solanum tuberosum; Stress response; Transgenic potato plant

1. Introduction metabolism. Mobilization of glycogen is accompanied by inhibition of glycogen synthesis. Such double control pre- Catecholamines are a group of biogenic amines possess- vents futile cycles to take place. The physiological action of ing a 3,4-dihydroxysubstituted-phenyl ring. Dopamine, catecholamines in animal cells is mediated by their interac- norepinephrine, epinephrine and their derivatives are wide- tion with G-protein coupled receptors that stimulate or in- spread in animals and have also been identiﬁed by gas chro- hibit the enzyme adenylyl cyclase (AC). In most animal cells matography coupled to mass spectrometry (GC-MS) in po- cyclic AMP (cAMP) exerts its effect by activating cAMP- tato plant [18]. dependent, serine–threonine protein kinase (PKA). Both epi- The biochemical role of catecholamines in animal cells is nephrine and norepinephrine are synthesized and released well studied. They act as neurotransmitters but the best- from the medulla of the mammalian adrenal gland. understood example of the hormonal action of epinephrine The biosynthesis of catecholamine is initiated onto two and norepinephrine in mammals is regulation of glycogen ways starting from tyrosine. Hydroxylation of tyrosine by tyrosine hydroxylase (TH) or tyrosine decarboxylation by tyrosine decarboxylase (TD) is the initial step followed by dopamine hydroxylation to norepinephrine and subsequent Abbréviations: ABA, abscisic acid; DD, L-Dopa decarboxylase; GC- methylation to epinephrine. It should be pointed out that TD MS, gas chromatography coupled to mass spectrometry; SA, salicylic acid; L TD, tyrosine decarboxylase; TH, tyrosine hydroxylase. is also suggested to conduct -3,4-dihydroxy phenyl alanin * Corresponding author. (L-Dopa) decarboxylation [2]. Catecholamine catabolism E-mail address: [email protected] (J. Szopa). starts via methylation of the hydroxyl group of the catechol

0981-9428/$ - see front matter © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.plaphy.2004.07.002 594 A. S´wiVdrych et al. / Plant Physiology and Biochemistry 42 (2004) 593–600 ring followed by oxidation to homovanillic acid and vanillyl- In this report we have presented the data on catecholamine mandelic acid and ends by secretion via urine [14]. content and key enzyme activities of catecholamine biosyn- The careful analysis of potato plant extract by sensitive thetic pathway under different stress conditions. Also the GC-MS method confirmed the presence of tyramine and data on the catecholamine protection of potato against patho- L-Dopa as well as dopamine and norepinephrine, and lead to gen infection have been presented. identification of the new compound of catecholamine catabolism, named normetanephrine. Epinephrine, homovanillic acid and vanillylmandelic acid were not found possibly due 2. Results to detection limit [18]. In contrast to the vast amount of knowledge concerning Recently we have suggested that catecholamines regulate the role and action of catecholamine in mammals, very little potato starch metabolism [17]. This finding provokes the is known on physiological significance of catecholamine in question about upstream signals responsible for changing plants. The involvement of TD was implicated in plant catecholamine content in plants. In order to answer this, wounding response [11]. The significant increase in TD was catecholamine content and activities of main enzyme impli- detected upon infection of potato leaves with Phytophtora cated in catecholamine biosynthesis were measured in potato infestans and in elicitor-treated parsley cells [6]. The exact leaves under different stress conditions. role of tyramine is however as yet speculative. Several reports suggest that catecholamines are precursors for alka- 2.1. Enzyme activities loids. The best known is the hallucinogen mescaline, identified in several species of cacti, and tetrahydroisoquinoline The leaves exposed to abscisic acid (ABA) treatment alkaloid [5], both derived from dopamine. Norepinephrine is showed significant increase in three enzyme activities. The believed to be a precursor for berberastine, an alkaloid of activities of TH, TD and L-Dopa decarboxylase (DD) in- Hydrastis canadensis [15]. Other reports suggest that cat- creased about twofold, 55% and 50%, respectively (Fig. 1). echolamines may interact with plant hormones [1,12]. The TH and DD were also activated in case of drought. The Several reports pointed out the significance of compounds highest activation of TD was found in leaves incubated in that originated from catecholamine biosynthetic pathway. high salt concentration. The increase in TD was accompanied Studies have [3,10] shown that the biosynthesis of hydroxy- by significant decrease in TH and DD activity. Only slight cinnamic acid amides from tyramine and their subsequent increase in TD and significant decrease in DD was observed polymerization in the cell wall by oxidative enzymes is the integral and ubiquitous component of the plant defense response to pathogen challenge. These amides, together with other cell wall-bound phenolics, are believed to create a barrier against pathogens by reducing the digestibility of the cell wall [4]. Recently a new compound derived from tyrosine has been identified in leaves of potato plant. The glucosyl derivative of tyrosyl residue has been identified as a product of TD action instead of tyramine and the potential storage role of this compound was suggested [7]. In order to study the physiological function of catecholamines in a more detailed way, transgenic plants overexpressing enzyme TD were generated and analyzed [17]. The overexpression of TD, which controls the important step of catecholamine synthesis, increased the tyramine and norepinephrine content in transgenic potato tubers. The increase of tyramine ranged from 15% to more than twofold when compared to control plants. The level of L-Dopa was enhanced in all examined transgenic lines; however, the increase was slight, ranging from 0.3% to 30% of the control value. Thus the data suggested that activation of tyramine synthesis in TD plants occurred by overexpression of cDNA encoding TD. The examination of the plants for catecholamines content revealed dramatic increase in norepinephrine quantity, 10- to Fig. 1. Activities of the TD, TH and DD measured in leaves from potato 15-fold increase was detected depending on transgenic line; plants (S. tuberosum L. cv. Desiree) under different stress conditions. Enzymes activities are given in pkatals (calculated from pmol of tyramine whereas the normetanephrine level was significantly de- for TD, L-Dopa for TH and dopamine for DD·per min) mg–1 of protein creased in all transgenic lines suggesting the inhibition of added. The data are presented as the mean ± S.E. of determinations on six norepinephrine catabolism. individual plants under each stress condition. A. S´wiVdrych et al. / Plant Physiology and Biochemistry 42 (2004) 593–600 595 in leaves exposed to low temperature. The same effect on The decrease in all enzyme activities in leaves stored in enzyme activities was revealed in leaves exposed to red light dark and in infrared conditions resulted in significant de- and to the dark. Under both circumstances significant decrease in norepinephrine content suggesting the involvement crease in all three enzyme activities was detected. The high- of catecholamine in plant adaptation to artificial light condi- est increase in DD activity was measured in leaves treated tion. Interestingly under UV conditions the significant in- with UV, however, accompanied by significant decrease in crease in DD activity was sufficient to cause a very high TH. increase of dopamine but slight decrease in norepinephrine Summing up the characteristic for ABA treatment, is acti- content. vation of both TH and TD initial steps of catecholamine Summing up we suggest that catecholamine are involved synthesis, for drought is TH activation and TD for salt treat- in plant adaptation to drought and UV treatment. Interest- ment. Other analyzed stresses inhibit or do not affect mea- ingly in all analyzed cases the activity of norepinephrine sured enzyme activities. It should be also pointed out that the transmethylase was significantly decreased resulting in the decarboxylation reaction of tyrosine and L-Dopa are differ- lower normetanephrine content. This suggests using norme- entially affected by stress condition suggesting the involve- tanephrine as universal indicator of stressed potato. ment of decarboxylases with substrate specificity in plant stress response. 2.3. Carbohydrate level

2.2. Compounds content Recently we have evidenced that the downstream effect of catecholamine increase is starch mobilization. This is why Increased activities of TH, TD and DD measured in ABA we decided to measure starch and sucrose content in stressed treated leaves resulted in signiﬁcant increase in dopamine potato leaves and then compared the data with norepineph- and norepinephrine content (Fig. 2). Even higher increase in rine content (Fig. 3). In case of ABA and drought the increase dopamine and norepinephrine was detected in leaves ex- in norepinephrine content resulted in slight but still not sig- posed to drought thus suggesting that activation of TH and niﬁcant decrease in starch. In reverse, in case of UV treat- DD is effective enough for stimulation of dopamine and ment the decrease in norepinephrine level resulted in el- norepinephrine biosynthesis. In case of high salt concentra- evated starch level. However, there were also examples like tion the inhibition of TH and DD resulted in decrease in leaves stored in red light condition or in dark where the dopamine content. However, the increase in norepinephrine decrease in norepinephrine content was accompanied by might derive from the increase of TD activity in salt stressed decrease in starch accumulation suggesting that except cat- condition. Leaves exposed to low temperature showed only echolamine action there are other mechanisms participating slight decrease in dopamine content and also slight increase in starch metabolism of potato leaves. of norepinephrine thus suggesting that catecholamine are not affected by cold stress. 2.4. The catecholamines level in leaves extract from TD-overexpressing potato plants

The ﬁnding that catecholamine are involved in plant adaptation to different stress conditions suggested that the ma-

Fig. 3. The level of starch and sucrose measured in leaves under different stress conditions. Leaf material was frozen in liquid nitrogen, examined in Fig. 2. The level of dopamine, norepinephrine and normetanephrine measu- enzymatic assay and compared to the control value (K) obtained for leaves red in leaves under different stress conditions. The values ± S.E. represent incubated on MS medium in standard conditions. The value ± S.E. repre- six determinations for individual plant under each stress condition. sents six determinations for each condition. 596 A. S´wiVdrych et al. / Plant Physiology and Biochemistry 42 (2004) 593–600 nipulation of catecholamine synthesis may protect plant tubers and leaves of potato plants. The exception is the against biotic and abiotic stresses. We generated transgenic tyramine. In opposite to tubers, tyramine content in leaves is potato plants overexpressing TD enzyme. The cDNA from significantly decreased. We speculate that the reduction in parsley under the control of 35S promoter was introduced via tyramine content in leaves may indicate its immediate flux Agrobacterium infection into Solanum tuberosum var. De- into other than catecholamine metabolic pathway. siree. The transformation and selection procedure was described before [17]. The potato tubers from transgenic plants 2.5. The virus infection slightly but changed showed significant increase in catecholamine content [17]. catecholamine metabolism in leaves In order to verify the data obtained for transgenic potato tubers, the level of catecholamine was investigated in leaf The potato virus Y strain 0 (PVY0) virus specifically extract using GC-MS method. All transgenic lines showed infects potato plants causing the reduction of tuber yield. significant decrease in tyramine content that was in opposite Seven days after infection, the PVY0-inoculated plants were to tuber situation. The quantity of L-Dopa was enhanced even characterized by wilted leaf phenotype, visibly more inten- eight times (line TD33) when compared to control plant. The sive in case of nontransformed plant (not shown). The level content of downstream derivatives (dopamine and norepi- of catecholamines in inoculated leaves revealed only very nephrine) was also significantly increased (Fig. 4). Thus, the slight but stable increase in tyramine, L-Dopa, dopamine and data suggest that overexpression of cDNA encoding TD norepinephrine content in all transgenic lines (Fig. 4). The leads to the similar changes in catecholamine content in increase ranged from 15% to 25% compared to the control level. The content of catecholamines in nontransformant was also only very slightly changed. Interestingly in all cases the content of normetanephrine was reasonably decreased thus suggesting that norepinephrine methylating enzyme activity was reduced upon virus infection and confirms the usefulness of this compound measurement for detection of potato stressed condition.

2.6. Level of total salicylic acid in leaves from TD-overexpressing plants

Previous studies strongly suggest that salicylic acid (SA) plays an important role in plant signaling upon pathogen infection. This is why we decided to examine whether there is a correlation between catecholamine and SA levels upon potato infection. Data obtained from SA measurements in leaves of control and transgenic plant are presented in Fig. 5. All examined transgenic lines showed signiﬁcant increase in total level of SA that ranged from 10% to over 50% compared to the control plants. The level of total SA further increased after PVY0 infection and this increase ranged from 10% to 35% when compared to the compound level in infected control leaves (Fig. 5). In order to investigate potential relationship between increased levels of SA and catecholamine measured in leaves of transgenic plants, the correlation coefﬁcient was calcu-

Fig. 4. The level of catecholamine in noninfected (grey bars) and virus infected (black bars) leaves from control and TD-overexpressing potato Fig. 5. The content of total SA (free and glucoside derivative) determined in plants. The value ± S.E. represents three determinations for each TD trans- noninfected leaves from control and TD plants (grey bars) and in leaves genic line (numbered). The control (WT) and transgenic lines are represen- infected with PVY0 (black bars). The control (WT) and transgenic lines are ted by at least three independent leaf samples. Asterisks (*) indicate values represented by at least three independent leaf samples. Asterisks (*) indicate that are signiﬁcantly different from the wild type plants. values that are signiﬁcantly different from the wild type plants. A. S´wiVdrych et al. / Plant Physiology and Biochemistry 42 (2004) 593–600 597

L-Dopa synthesis, respectively [15]. Dopamine hydroxylation leads to norepinephrine that is further methylated to normetanephrine. It is worth pointing out that normetanephrine has been only recently identified in plants [18].As expected the overexpression of cDNA encoding parsley TD resulted in significant increase of tyramine content measured in tubers of all transgenic lines [17]. Fig. 6. Tubers of the control and transgenic plants infected with E. carato- The norepinephrine content in TD tubers was also reason- vora. The diameter (in mm) of the infection zone was measured 6 d after ably increased and the level of normetanephrine was signifi- inoculation. Data represent the mean ± S.E. of determination on 15 indivi- cantly reduced when compared to control plant. Since one of dual tubers from each control and transgenic lines. Asterisks (*) indicate the best-understood examples of the hormonal action of values that are significantly different from the wild type plants. catecholamine in mammals is the control of glycogen mobi- lated. The value of coefficient for SA and tyramine, dopam- lization, the level of starch and soluble sugars in tubers of ine and norepinephrine was –0.70, 0.55 and 0.75. Thus the transgenic plants was analyzed. The obtained data were very calculated data suggest strong negative correlation of the clear, decrease in starch and significant increase in glucose, content of total SA and tyramine and also strong but positive fructose and sucrose content [17] accompanied the increase correlation with the level of catecholamine. The molecular in norepinephrine. Hence it is convincible that catecholamine mechanism of the crosstalk between measured compounds play an important role in regulation of starch–sucrose me- content is however as yet difficult to understand. tabolism in plants. Interestingly similar changes in carbohydrate flux, result- 2.7. Infection of transgenic potato tubers with bacteria ing in decreased level of starch but increased content of Ervinia caratovora sucrose, were also characteristic for potato tubers under stress conditions. In mammalian systems, catecholamine Since the catecholamine content responded to virus infec- serves as stress hormones, displaying a rapid transient in- tion and TD has been proposed to contribute to plant defense crease as a result of stress. In order to see whether or not against pathogens, TD potato tubers were tested for their similar response occurs in plants, leaves of potato plants were sensibility to infection with bacteria E. carotovora var. caro- wounded and catecholamine levels prior to and 5, 10 and tovora (E.c.c). Six days after tuber slices inoculation with 13 min after wounding were determined. Although the data pathogen, the diameter (in mm) of infection zone was mea- varied, there was a consistent increasing trend in concentra- sured. All transgenic plants showed significantly increased tion of dopamine, norepinephrine and normetanephrine [18]. resistance towards pathogen (Fig. 6) and the increase ranged Data presented in this paper clearly suggest that catechola- from 30% for TD13 to above 50% in case of TD55 when mine synthesis is affected not only by wounding but also by compared to control plant. The calculated correlation coeffi- several other stress conditions. Interestingly different stress cient in TD plants for pathogen response and tyramine, conditions affected different enzymes implicated in cat- L-Dopa, norepinephrine and normetanephrine content was echolamine biosynthesis. It was shown that only ABA treat- 0.56, 0.90, 0.96 and –0.76, respectively. Thus the calculated ment activates both initial steps of catecholamine biosynthe- data suggest strong positive correlation of plant protection sis.Activities of TH, TD and DD were significantly increased against pathogen infection and catecholamines, except nega- leading to elevated norepinephrine content. Though the tive correlation in case of normetanephrine compound con- changes in sucrose and starch levels were not significant the tent. decreasing tendency for starch and increasing for sucrose was observed. Activity of TD was also increased in leaves 3. Discussion under high salt, storage in low temperature and under UV illumination. The TH activity increased under water stress In contrast to the vast amount of knowledge concerning conditions while activity of DD was triggered under UV the role and action of catecholamine in mammals, very little treatment and drought. Obtained results strongly suggest that is known on physiological significance of catecholamine in the key catecholamine biosynthetic enzymes operate inde- plants. Since most of the components of animal catechola- pendently and react specifically on the plant growth condi- mine signaling pathway have been also identified in plants tion. It should be pointed out that several reports suggested (G-protein, cAMP, PKA homologs) the catecholamine role the involvement of TD in L-Dopa decarboxylation. However, in primary plant carbon metabolism is highly possible. To based on the result obtained in this paper at least L-Dopa check this hypothesis transgenic plants overexpressing TD, decarboxylating activity of TD is differentially regulated in which controls the important step of catecholamine synthe- response to stress conditions. sis, were created and characterized in respect to catechola- Several reports suggested that TD is involved in the bio- mine and sugar content [17]. synthesis of numerous secondary metabolites and thus takes In plants the first step of catecholamine biosynthesis, can part in plant defense mechanism against pathogen infection be catalyzed by either TD or TH, leading to tyramine or [3,11]. It was thus interesting to investigate whether the 598 A. S´wiVdrych et al. / Plant Physiology and Biochemistry 42 (2004) 593–600 tubers of TD transgenic potato plants will be protected 4.2. Stress conditions against infection with E. carotovora. In fact all the examined tubers showed increased resistant against pathogen infection. Fully developed leaves (third leaf from the top of the The correlation coefficient for pathogen response and plant) from 4-week-old potato plants (S. tuberosum L. cv. tyramine, L-Dopa, norepinephrine and normetanephrine was Desiree) grown in greenhouse, were placed on MS medium calculated. The obtained values clearly indicate that cat- and exposed to different stress conditions. They were incu- echolamines are implicated in potato response to pathogen bated at low temperature (4 °C), under red light (635 nm) and attack. This suggestion is further supported by measurements in the dark for 1 h; the UV treatment was at 312 nm 15 min. of SA level in infected potato. Increase of SA content was The leaves exposed to ABA and high salt were incubated for accompanied by increase of catecholamine level. Obtained 1 h on MS medium containing 100 µM ABA and 3 M NaCl in data strongly suggest that the catecholamine biosynthesis is standard light and temperature conditions (as described in affected by pathogen infection. The molecular mechanism of Section 4.1). For drought stress, leaves were stored for an this increase is however as yet unknown. It will be interesting hour in a glass open jar. Control leaves (K) were incubated on to investigate which cellular processes, except starch me- MS medium in standard growth conditions and for the time tabolism regulation, are affected by catecholamine com- indicated. Immediately after exposure, the leaves were fro- pounds. zen in liquid N2 and stored at –80 °C until use for determina- The expression of the TD under the control of the 35S tion of enzyme activities, catecholamine and sugar content. promoter in transgenic canola and tobacco plants has been reported. This modification led to the increased level of free 4.3. Tyrosine hydroxylase, tyrosine decarboxylase and cell wall-bound tyramine as well as to a decreased and L-Dopa decarboxylase enzyme assays digestibility of cell wall [3]. It was found that the accumulation of hydroxycinnamic acid tyramine amides in cell wall Plant leaf tissues were grounded in liquid N2 and extracted was connected with response of plants against pathogen [13]. (in presence of polyvinyl polypyrrolidone for TD and DD In contrast to this, it was reported that the overproduction of assays) in 200 mM bis–Tris (pH 7.2), 1 mM EDTA and TD in potato plants has not resulted in altered amounts of 28 mM 2-mercaptoethanol. Debris were removed by cen- tyramine in leaves [7]. They have found, however, that in- trifugation (14 000 × g) and the supernatant was desalted on stead of tyramine increase the glucosylated derivative of a NAP-10 column. TH activity was determined by measure- tyrosine is accumulated in leaves. The compound might ments of L-Dopa production. The assay mixtures contained serve as amino acid storage form. 20 mM MES (pH 6.1), 1 mM EDTA, 10 µM DMPH4,28mM In conclusion the content of several compounds of cat- 2-mercaptoethanol, 100 µg catalase (50 mg ml–1 from bovine echolamine biosynthesis pathway is affected under different liver, Sigma), an excess amount of tyrosine and 100 µl of plant growth conditions which might suggest their regulatory protein extract in total volume of 200 µl [19]. TD and DD function in plant response to these stressed situations. Al- activities were determined by measurements of the tyramine though the molecular mechanism of this response is as yet and dopamine production. The assay mixtures contained unknown the manipulation of catecholamine level in plant 50 mM bis–Tris (pH 7.2), 1 mM EDTA, 25 µM pyridoxal might serve as an advantage in plant protection improve- 5-phosphate, 28 mM 2-mercaptoethanol, an excess amount ment. of tyrosine (for TD assay) and L-Dopa (for DD assay) and 100 µl of protein extract in total volume of 200 µl [6]. The reactions were carried out at 25 °C for 30 min. Then the assay 4. Methods mixtures were freeze-dried in speedvac and methanol- extracted for GC-MS analysis as described [17]. TH, TD and –1 4.1. Plant material DD activities were expressed in pkatals mg protein in the extract. The protein content in the extracts from plant tissue TD-overexpressing plants were generated by introduction were determined according to Bradford. of a cDNA encoding TD from Petroselinum crispum, (kindly provided by Dr. I. Somssich; EMBL/GenBank database ac- 4.4. Determination of starch and sucrose content cession number M96071) in vector BinAR under the control of the 35S CaMV promoter and Nos terminator. Potato plant Potato leaves (100 mg) were extracted with 100% (S. tuberosum L. cv. Desiree) transformation via Agrobacte- ethanol—50 mM HEPES–KOH (pH 7.4) at 70 °C. The rium infection and selection of transgenic plants was exactly supernatant was used for enzymatic analysis of the sucrose the same as that described [17]. Plants were grown in the content [16]. For starch measurements, extracted plant mate- greenhouse in soil under a 16 h light (22 °C)–8 h dark (15 °C) rial was homogenized in 0.2 M KOH and, following incuba- regime in individual pots and were watered daily. Tubers tion at 95 °C, adjusted to pH 5.5 with 1 M acetic acid. Starch were harvested 3 months after transfer of the tissue culture was hydrolyzed with amyloglucosidase, and the amount of plants to the greenhouse. released glucose determined enzymatically. A. S´wiVdrych et al. / Plant Physiology and Biochemistry 42 (2004) 593–600 599

4.5. Infection of transgenic potato tubers with bacteria E. An equal volume of 0.2 M sodium acetate buffer (pH 4.5) carotovora was added, and the pH was adjusted to 1–1.5 with HCl before extraction. Free SA was extracted with 2 volumes of Fifteen potato tubers (medium size 60 g) from each trans- cyclopentane/ethyl acetate/isopropanol 50:50:1. The organic genic line and control plant were inoculated with the bacteria phase was dried under nitrogen and analyzed by HPLC as E. carotovora var. carotovora (E.c.c) in concentration of 5 × described [9]. 108 cells per ml–1 of suspension, as described [8]. After infection tubers were incubated for6dat20°Cbefore 4.9.2. Quantitation of SA conjugates measurement of the diameter (in mm) of the infection zone. SA conjugates were indirectly quantitated by acid- The size of the infection zone in transgenic tubers was com- hydrolyzing the compounds that remained in sodium acetate pared with that obtained in control plants. buffer after organic extraction and analyzing the released SA by HPLC. Acid hydrolysis was performed by incubating the 4.6. Tissue extraction for GC-MS samples in boiling water bath at pH 1–1.5 for 30 min and then extracting free SA as above. Frozen leaf tissue (100 mg) from plants 4 weeks after transfer in vitro cultured plants to soil, was powdered in 4.10. Statistics analysis liquid nitrogen, extracted with methanol (4 ml g–1 fresh weight), heated for 15 min at 70 °C, and centrifuged (5 min, Statistics were calculated with the use of t-test. The term 12 000 × g). The samples were diluted with water to 50% in significant is used when P < 0.05 with the t-test. methanol and extracted with chloroform (1:1, v/v). A portion of water phase was dried under vacuum and used for deriva- tization [18]. Ribitol was used as an internal standard added Acknowledgments directly to the sample homogenate (30 µg g–1 fresh weight). We would like to thank Dr. Jerzy Stachowiak (Agricul- 4.7. GC-MS analysis tural University of Poznan´) for catecholamine measurements and Professor Dr. Jacek Hennig (PAS, Warsaw) for help in The dried extracts were dissolved in pyridine/methoxy- SA quantification. This work was supported by grants –1 amine (20 mg ml ) at 37 °C for 90 min and then acidic 3PO6A 01523 from State Research Committee (KBN). protons were derivatized with N-methyl-N-trimethylsilyl- trifluoroacetamide (MSTFA) at 37 °C for 90 min. Two micro- liters of sample were used for analysis in GC-MS. Measure- References ments were conducted exactly as described for tuber extracts [17]. [1] Y.R. Dai, P.J. Michaels, H.E. 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