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Volume 15, 2005

PARTICIPATION OF POTATO CELLS cАМP IN THE TRANSFER OF SYSTEMIC SIGNAL IN RING ROT PATHOGENESIS

Lidiya A. Lomovatskaya, Anatoly S. Romanenko, Nadia V. Krivolapova, Valentina N. Kopytchuk
Siberian Institute of Plant Physiology and Biochemistry, Siberian Division of the Russian Academy of Sciences,
P.O. Box 1243, 664033 Irkutsk, Russia, Fax: (3952)510754; ?-mail: phytoimm@sifibr.irk.ru

Abstract

The influence of exopolysacharides (EPS) produced by ring rot pathogen Clavibaсter michiganensis subsp. sepedonicus on the concentration dynamics of cАМP pool and its derivatives in in vitro potato plants of two cultivars, contrasted by resistance to this pathogen, was studied. Immunoenzyme assay (IEA) with primary rabbit antibodies to cAMP, and secondary goat antibodies binded with peroxidase was used. Incubation of plant roots to EPS resulted in the emergence of three waves of cАМP accumulation, which propagated in the direction root-stem and embraced the whole plant. In case of the first, most powerful wave, maximal cАМP concentration exceeding the value of this parameter in control by two orders was observed in the upper part of the stem after 1 minute already. Further two less powerful waves emerged after 120 and 480 minutes respectively, with cАМP concentration being significantly lower. The susceptible cultivar also generated three waves, but after 15, 120 and 480 minutes, with the highest cАМP concentration in the upper part of the stem registered only after 480 minutes. Therefore, in the potato plants cells adenylate cyclase signal system participates in the transfer of systemic signal for generation of protection responses, but the resistant cultivar demonstrates its much quicker response to exogenous metabolite of bacterial pathogen.

Key words: Solanum tuberosum L., Clavibaсter michiganensis subsp. sepedonicus (Spick. et Kotth.) Skaptason et Burk., in vitro plants, cАМP, pathogenesis, exopolysacharides.

Introduction

The existence of adenylate cyclase signal pathway in the plants is commonly acknowledged, different aspects of its functioning are being addressed [1]. Nevertheless, the role of this system in the plant protection from various pathogens remains insufficiently studied; there is only a small number of works dedicated to fungal and viral infections [2,3,4]. That is why we found it necessary to study the influence of exopolysacharides (EPS) of pathogen caused potato ring rot, being a rather distributed and hazardous disease, on the change of cАМP concentration both in the growth medium and in different regions of the two potato cultivars contrasted by resistance to the given pathogen.

The following methods are normally used to determine the cАМP level in the plants: radio-immune, immunoenzyme, Gillman’s method based on the cАМP ability to link with a specific protein, various spectrometric methods [5]. According to Newton with co-authors, most of the enumerated methods, except spectrometric one, produce a summarized estimation of not only cАМP, but also its derivatives, such as 2–deoxyadenosine-3,5–cАМP, 2–О–glutamine–3,5–cАМP, 2–О–asparagine–3,5–cАМP, whose functions are presently not well known [5]. On the basis of these presumptions we found it possible to use in our work the method of immunoenzyme analysis (IEA), which identifies cАМP and its derivatives [5].

Materials and Methods

Objects of investigation. In vitro potato (Solanum tuberosum L.) plants of two cultivars were used in the experiments: Lugovskoi – resistant, and Luk’yanovskii – susceptible to Clavibaсter michiganensis subsp. sepedonicus (Spick. et Kotth.) Skaptason et Burk (Cms). Plants were grown from the cuttings in aseptic conditions on the liquid nutrient medium of Murashige-Skoog [6] with the addition of vitamins and phytohormones [7]. Bacteria of virulent strain 5369 were cultivated as per the method [8]. Exopolysacharides were extracted from the medium and purified by ion-exchange chromatography [8].

Co-incubation of in vitro plants with EPS. EPS in the final concentration of 0.1% were added to the growth medium. The plants kept in the growth medium without EPS addition were used as control. Samples were taken after 1, 15, 120 and 480 min. The plants (5 samples for each variant) were frozen in liquid nitrogen, and then divided into sections: 1 – roots; 2, 3, 4 – stem sections 4 cm long from the base to the top, which were used for determination of cАМP level.

Extraction, purification and immune enzyme analysis (IEA) of cАМP. The plant material was homogenized in the extraction medium of the following composition: 50 mМ Tris-HCl, рН 7,4 + 0,1 mМ theophylline + 1mМ DL-dithiothreitol + 0,5 mg/ml polyvinylpyrrolidon (Sigma, USA). The homogenate was filtered through two layers of kapron and centrifuged for 40 min at 20000 g. In order to divide cАМP and its derivatives from other cyclic nucleotides, the obtained supernatant was passed through the column with neutral aluminum oxide (Merck, Germany). For this purpose the column (7Х1 cm) was filled with 1 g of neutral aluminum oxide and balanced with the extraction medium. 1 ml of supernatant was added, the elution was equilibrated with the extraction medium. The presence of cАМP and its derivatives (further in the text – cAMP) in the ample was controlled with the help of chromatographic equipment (Yvicord, Sweden) with wave length 276 nm, which appeared in the second ml of eluate in one peak. To determine the concentration of the substances under investigation we used the modified immunoenzyme analytical method (IEA). In microwell plate for IEA there were introduced 0.1 ml of the mixture with the following composition: 1 ml of the sample investigated, + 0.08 ml 25% glutar aldehide + 1 mg/ml BSA (Sigma, USA). The samples in microwell plate were incubated for 15 h at 37⁰С and the microwell were three times washed by ТТBS medium (0.02 М Na-phosphate buffer, рН 7.0 + 0.1 М NaCl + 0.3 % tween-20). In each microwell there was added 0.1 ml of horse serum (Microgen, Russia), diluted 1:10 by ТBS (ТTBS without detergent), then the plane-table were kept for 1 h at 37⁰С. Then 0.1 ml of primary rabbit antibodies specific against cАМP (Sigma, USA) were entered in the microwell in the concentration of 60 μg/ml in ТBS. The material was incubated for 2 h at 37⁰С and washed by ТТBS for three times. Then the samples were treated again by horse serum. After this in each microwell there were added 0.1 ml of peroxidase-marked secondary goat antibodies (Sigma, USA) in the dilution 1:800 and, after 1 h of incubation at room temperature, they were three times washed by ТТBS. To reveal peroxidase reaction the following composition was used: 10 ml 0.1 М of phosphate-cytrate buffer, рН 5.3 + 4 mg o-phenylenediamine (Sigma, USA) + 0.25 мл 3% H₂O₂. In each microwell of the plate there was entered 0.1 ml of this mixture. The color developed for 20 min. The reaction was stopped by addition of 0.1 ml/hole of 4N H₂SO₄. Optical density of the solutions was determined on the spectrophotometer SF–56а (Labo, Russia) with wave length 490 nm. Homogenization medium without plant samples was used as control. Caliber curve was constructed on the basis of cАМP (Sigma, USA). cАМP concentrations were calculated per 1 g of the weight substance mass. The experiments were held in four analytical and three biological repetitions. The results were statistically treated with the estimation of standard average error [9].

Results and discussion

In the experiments on in vitro potato plants were obtained the following results. First, there were observed considerable cultivar differences in cАМP content in control. The resistant cultivar had the following values: 1-st section (see “Methods”) – 2.1 nM; 2-nd section – 2.1 nM; 3-d section – 0.08 nM; 4-th – 0.29 nM. The susceptible cultivar – 1.9; 0.6; 0.9 and 5.5 nM, respectively. As seen, the pool of cAMP considerably differed not only through the plant length, but also in the plants of different cultivars. Carrying of 0.1% pathogen EPS in the nutrient medium caused fast and significantly larger compared to control cАМP accumulation in the stem and leaves (table 1). In the direction root-stem there was observed periodic increase and then decrease of this metabolite level, that is its concentration dynamics was of wave-like character. The plants of resistant cultivar showed three waves of cАМP accumulation. During the first, most powerful wave, maximal cАМP concentration exceeding the ones in control by two orders was observed already 1 min after the incubation. Two further weak waves emerged with the intervals of 120 and 480 min, and propagated much slower (table 1). The susceptible cultivar also generated three waves: the first and the second ones – after 15 and 120 min, the third, most intense – in 480 min. This was accompanied by the significant cAMP release to the growth medium (table 1).

Such concentration dynamics of the signal molecule allows to infer that the resistant cultivar has a much higher intercellular speed of signal transduction than the susceptible cultivar, and the increase of extracellular cАМP level, that is release of the excess of signal molecule from the cell, enable the cells to quickly reduce the intensity of alarm signal down to the norm. The susceptible cultivar needs much more time for this.

The data to prove this fact are available in the literature. Thus, in the suspension cells of alfalfa already 4 min after their treatment by fungal elicitor the cАМP level sharply increased, which was accompanied by significant accumulation of phytoalexin [2]. As regards cАМP release to the outer medium, this phenomenon was found in animals and plants at normal and stress conditions [10, 11, 12].

It is still unknown, which mechanism is used by the resistant cultivar to transfer the signal for a long distance. The following variants seem possible: а) cАМP may transfer, along apoplast included, towards both stem and life tissues of the plant in response to the generation of primary signal in the root cells by the pathogen exopolysacharides; b) nitrogen oxide, salicylic or jasmone acids act as mobile secondary signals; c) there should function another, fast propagating signal, which triggered adenylate cyclase system. The first suggestion is indirectly proven by the transfer of large quantities of cАМP from the root cells in the outer space in the presence of pathogen EPS (table 1). Direct cАМP movement along the plant is apparently possible, but this signal molecule is unlikely to be able to move with the speed of 10 -12 cm/min. It may be supposed that after two-three hours some part of cАМP synthesized in the roots may be transported towards the upper part of the plant, but taking into account very high activity of plant phosphoduesterases that converted cАМP into AMP [1], and energy dependence of cАМP transport [11], we find such way of signal transfer for a long distance unlikely. Salicylic acid (SA), most probably, is not a mobile translocation signal, but acts as a target for its perception [13]. Jasmone acid – SA antagonist – is known to be produced by the plant not earlier than 20 minutes after the addition of the elicitor [14].

More probable candidates for the role of early mobile system signal during ring rot pathogenesis are nitrogen oxide [15], as well as disturbance wave arising as a result of membrane potential, ΔрН and ion flows through plasma membrane [1, 16]. In this connection it should be noted that EPS Cms are able to intensely acidify the plants growth medium, and the cells of the potato resistant cultivars – to restore pH-homeostasis more efficiently as compared to the susceptible ones, including those used in the present work [17]. Considering potato plants response to EPS Cms influence, we should bear in mind that in the cell walls and plasma membrane of potato cells there are present receptors to EPS of this pathogen, and their amount in the susceptible cultivar cells is by one order higher [18,19]. The acquired results allow to conclude that with specific interaction of EPS Cms with the receptors of root cells of potato test tube plants, the latter systemically activate adenylate cyclase signal system; this process is more intense in the plants of resistant cultivar, but not the sensitive one, which later activates genetically determined protective mechanisms restricting the development of pathogenic process.

Literature

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Table 1. Influence of ring rot pathogen EPS (0,1%) on the cAMP concentration change in nutrition medium and growth region of in vitro potato plants

cAMP contents, % to control
Root	Stem, sm from root			Nutrition medium
	1-4	4-8	8-12
cv. Lugovskoi (resistant)
Exposition with EPS:
1 min
906±89	2145±22	12000±1190	14000±1370	40±3,8
15 min
200±18	990±93	530±48	139±14	50±4,3
120 min
94±10	384±30	258±23	27±3	197±9,2
480 min
70±6	110±10	1476±130	282±29	117±9,6
cv. Lukianovskii (susceptible)
1 min
464±40	307±25	81±38	51±4	436±19,0
15 min
282±27	338±18	63±5,9	111±9	57±26,8
120 min
304±26	1776±151	267±20	100±8	1560±63,7
480 min
1000±87	6700±593	2980±27	4910±380	244±10,9

LOMOVATSKAYA Lidiya Arnol’dovna, Ph. D Biology, senior scientific researcher. Home address: 664033 Irkutsk, Lermontov str., 321-а, ap.2. Home telephone: 42-89-67, office telephone: 42-50-09. Fax: (3952) 51-07-54 Fax: (3952) 51-07-54. E-mail: phytoimm@sifibr.irk.ru

ROMANENKO Anatoly Sidorovich, D. Sc., Prof. Home address: 664033 Irkutsk, Lermontov str., 275-b, ap. 43. Home telephone: 51-03-43, office telephone: 42-50-09.

KRIVOLAPOVA Nadia Vladimirovna, post-graduate student. Home address: 664027 Irkutsk, General Dovator str., 12, ap.52. Home telephone: 38-69-41, office telephone: 42-50-09.

KOPYTCHUK Valentina Nikolayevna, engineer. Home address: 664033 Irkutsk, Lermontov str., 297-А, ap.164.

Correspondence is to be conducted with L.A. Lomovatskaya.

Technical College - Bourgas,

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