changes in activity of ornithine decarboxylase in

ZESZYTY PROBLEMOWE POSTĘPÓW NAUK ROLNICZYCH 2008 z. 524: 401-408

CHANGES IN ACTIVITY OF ORNITHINE DECARBOXYLASE IN WINTER TRITICALE SEEDLINGS STRESSED BY GRAIN APHID ATTACK Cezary Sempruch, Agnieszka Wójcicka, Marek Makosz, Bogumił Leszczyński Department of Biochemistry and Molecular Biology, University of Podlasie, Siedlce

Introduction Ornithine decarboxylase (ODC; EC 4.1.1.17) is a key enzyme of polyamines biosynthesis. Direct product of the reaction catalysed by ODC is putrescine, that is metabolized to spermidine and spermine. Polyamines participate in several important physiological processes involving replication, transcription, translation, cell division, and plant morphogenesis [BOUCHEREAU et al. 1999; KAKKAR et al. 2000]. Moreover, these substances play an important role in the protection of plants against environmental factors caused by different abiotic and/or biotic stresses [TANG, NEWTON 2005; KUZNETSOV et al. 2007].

Free polyamines and their hydroxycinnamic acid amide derivatives (HCAAs) participate in plant reaction towards various pathogens [WALTERS 2003; BAKER et al. 2005]. However, their importance for induced plant resistance against herbivores is still not clear. Our earlier studies showed changes in the content of polyamines and activity of some enzymes involved in their biosynthesis as a result of the cereal aphids feeding [CIEPIELA, SEMPRUCH 2002; SEMPRUCH et al. 2004; SEMPRUCH, CIEPIELA 2005; SEMPRUCH 2005, 2006]. Many plant HCAAs are structurally similar to polyamine toxins present in venoms of spiders and wasp [FIXON-OWOO et al. 2003]. It suggests that plant derived phenolic polyamines are natural bioinsecticides [KLOSE et al. 2002].

The present paper reports on the influence of the grain aphid (Sitobion avenae F.) feeding on the ODC activity within tissues of winter triticale seedlings.

Material and methods Plant and aphids Two cultivars of winter triticale (Triticosecale, WITTM. ex A. CAMUS) obtained from Plant Breeding and Acclimatization Institute (IHAR) at Strzelce near Łódź: cvs Tornado and Witon were used in the experiments. The grain aphid individuals used in the experiments came from a stock culture at University of Podlasie. Acceptability of the triticale by grain aphid Population tests were conducted in the environmental chamber (24°C at day and 18°C at night, 70% relative humidity and photoperiod 16 h : 8 h) at the Department of Biochemistry and Molecular Biology, University of Podlasie in Siedlce. The

C. Sempruch et al.

402

prereproductive period and daily fecundity were estimated on the basis of daily observations. An intrinsic rate of natural increase (rm) and mean time of generation development (T) were calculated using equations by WYATT and WHITE [1977]. Feeding behaviour recording Feeding behaviour of grain aphid on the studied triticale cultivars was monitored by electrical penetration graphs (EPG) with the method according to LESZCZYŃSKI and TJALLINGII [1994]. The duration of the following aphid activities was determined: nonprobing (Np pattern), total pathways (ABC), salivation into sieve elements (E1), phloem sap ingestion (E2) and xylem sap ingestion (G). Ornithine decarboxylase assay Seven day old seedlings of the studied cultivars were artificially infested with five wingless females and which were allowed to feed for two weeks. Infested and control (without aphids) seedlings were collected for analysis after 24 h, 48 h, 1 week and 2 weeks of the grain aphid feeding and the number of aphids on studied plants was determined. The ODC activity was analysed within fresh plant material according to NGO et al. 3 [1987] method. One gram of the seedlings was homogenized in 10 cm of 0.2 M phosphate buffer pH 8.2 with the addition of 2.5 mM of β-mercaptoethanol and 1.5 mM of ethylenediaminetetracetic acid (EDTA). The obtained suspension was filtered through cheese cloth and centrifuged at 18 000 x g for 20 min at 5°C, and the supernatant was used to determine the enzyme activity. 0.5 cm3 of the enzyme extract was mixed with 0.8 cm3 of substrate (10 mM solution of ornithine in phosphate buffer pH 8.2 with 75 nM of pirydoxal-5-phosphate, PLP). The reaction mixture was incubated at 30°C for 30 min and stopped by adding 0.2 cm3 of 10% trichloroacetic acid (TCA). Next 1 cm3 of 4 M NaOH and 2 cm3 99% 1-penthanol were added to 0.5 cm3 of the obtained solution. These components were mixed for 20 s. and centrifuged at 582 x g for 5 min. 1 cm3 of the separated organic (upper) layer was transferred to a glass tube with 1 cm3 of 10 mM trinitrobenzosulfonic acid (TNBS) in 99% 1-pentanol and 2 cm3 99.9% dimethyl sulfoxide (DMSO) and intensively mixed. The mixture was than centrifuged at 600 x g for 5 min and the absorbance of the upper layer was measured with UV-VIS spectrophotometer Hewlett Packard 8453 type at 426 nm. The putrescine content was quantified on the basis of standard curve prepared for the commercial reagent. ODC activity was expressed in µM of putrescine⋅mg-1 of protein⋅hour-1. The estimation of protein quantity within the enzymatic extracts was performed according to LOWRY et al. [1951] method.

Results and disscussion The results obtained in the experiments showed that individuals of S. avenae performed on cv. Witon were characterized by a longer prereproductive period, higher mean time of generation development and lower values of daily fecundity and intrinsic rate of natural increase in comparison to the insects occurred on cv. Tornado (Tab. 1). Table 1; Tabela 1 Values of population parameters of the grain aphid on the studied winter triticale cultivars

CHANGES IN ACTIVITY OF ORNITHINE DECARBOXYLASE ...

403

Wartości parametrów populacyjnych mszycy zboŜowej na badanych odmianach pszenŜyta ozimego Cultivar Odmiana

Preproductive period (days) Okres przedreprodukcyjny (dni) (_±SE)

Daily fecundity per female Płodność dzienna na jedną samicę (_±SE)

Mean time of generation development (T) (days) Średni czas rozwoju pokolenia (T) (dni) (_±SE)

Intrinsic rate of natural increase (rm) Względny wskaźnik wzrostu populacji (rm) (_±SE)

Tornado

7.88±0.21

4.02±0.31**

10.68±0.29

0.3201±0.06**

Witon

8.32±0.21

2.61±0.20

11.28±0.29

0.2727±0.02

**

values statistically higher at P = 0.01 (Student’s t-test); wartości istotne róŜne przy p ≤ 0,01 (test tStudenta) 1,4 Tornado Witon

Duration of aphid activity (hours)

1,2

1

0,8

0,6

0,4

0,2

0 Np

ABC

E1

E2

G

Np - no probing; brak penetracji ABC - total pathways; penetracja tkanek peryferyjnych E1 - salivation into sieve elements; wydzielanie śliny do elementów floemu E2 - phloem sap ingestion; pobieranie soku floemowego G - xylem sap ingestion; pobieranie soku ksylemowego *

differences between studied cultivars significant at P = 0.05 (Student’s t-test); róŜnice między badanymi odmianami istotne przy p ≤ 0,05 (test t-Studenta)

Fig. 1.

Feeding activity (h) of the grain aphid on the studied winter triticale cultivars, during 10 h of the EPG recordings Rys. 1.Czas Ŝerowania (godz.) mszycy zboŜowej na badanych odmianach pszenŜyta ozimego podczas 10 godz. rejestracji EPG

Longer duration of aphid development and especially lower fecundity and rm value on cv. Witon as compared to cv. Tornado suggest, that S. avenae was faced at least partial antibiosis on the seedlings of the cv. Witon. On the other hand CIEPIELA et al. [1999] claimed that antibiotic properties, as an ability of host plant to reduce growth and development rate of the insect are an important mechanism of triticale resistance to grain aphid. EPG recordings shoved that grain aphid females fed on cv. Witon seedlings were characterized by a longer duration of no probing (Np pattern), total pathways (ABC) and xylem sap ingestion (G) and shorter time of salivation into sieve elements (E1) and phloem sap ingestion (E2) than the individuals fed on the cv. Tornado (Fig. 1). Similar results were obtained by CICHOCKA and LESZCZYŃSKI [1999] for Aphis fabae SCOP. that on better host showed longer feeding activity in sieve elements and shorter duration of pathways. Moreover WÓJCICKA et al. [1999] stated that S. avenae on less accepted

C. Sempruch et al.

404

triticale cv. RAH 116 spent more time on no probing and penetration of peripheral tissues and reduced salivation and ingestion from sieve elements than on plants of susceptible cv. RAH 366. An aerial parts of the control and infested seedlings of the cv. Witon were characterized by much higher mean activity of ornithine decarboxylase than analogical parts of the cv. Tonado (Tab. 2). Such differences were not observed in roots of studied plants. ODC activity within the aerial parts of cv. Tornado decreased during the first week of grain aphid feeding and increased later (Fig. 2). Similar results were obtained for the aerial parts of cv. Witon seedlings but changes in the enzyme activity were more visible than within seedlings of cv. Tornado, especially after 24 h and 2 weeks. Moreover, stress induced by pest also caused decrease of the ODC activity during the first week of aphid feeding and an increase after 2 weeks in roots of both studied triticale cultivars (Fig. 2). However, the systemic changes in ODC activity were more evident in case of the cv. Tornado. Table 2; Tabela 2 -1

-1

Mean activity of the ornithine decarboxylase (µg putrescine⋅h ⋅mg protein) within studied parts of winter triticale seedlings Średnia aktywność dekarboksylazy ornityny (µM putrescyny⋅godz.-1⋅mg-1 białka) w analizowanaych częściach siewek pszenŜyta ozimego Seedling parts Część siewki

Cultivar; Odmiana Tornado

t4

P

Witon

Control plants; Rośliny kontrolne Aerial parts; Części nadziemne

61.41±9.37

94.00±4.52

2.37

5.58⋅10-2

Roots; Korzenie

75.86±11.84

68.21±9.18

0.51

0.63

Plants attacked by aphids; Rośliny zaatakowane przez mszyce Aerial parts; Części nadziemne

64.96±2.80

80.95±2.09

4.58

3.76⋅10-3

Roots; Korzenie

66.84±4.15

69.30±3.61

0.45

0.67

Student’s t-test, comparing of mean ornithine decarboxylase activity in studied winter triticale cultivars (t4 and P values in the Table); Test t-Studenta, porównanie średniej aktywności dekarboksylazy ornityny w badanych odmianach pszenŜyta ozizmego (wartości t4 o P podano w tabeli)


405

The results obtained in the experiments suggest that ODC may participate in mechanisms of winter triticale resistance against biotic stress caused by S. avenae attack. Changes in the activity of ornithine decarboxylase were dependent on triticale cultivar, duration of aphid feeding and on the pest number. An increase of the enzyme activity in aerial parts of both studied cultivars was clearly manifested after 2 weeks of the grain aphid feeding. Similar changes were observed within tissues of plants infected by non-insect pathogens [WALTERS 2000, 2003; SUDHA, RAVISHANKAR 2002; BAKER et al. 2005]. Moreover KUMAR et al. [1997] found that in natural conditions about 90% of polyamines occur in the form of HCAAs within plant tissues. Some of these substances are structurally similar to polyamine toxins found in the venoms of spiders and wasp [FIXON-OWOO et al. 2003]. They were acted as highly selective and potent ligands for specific ionotropic receptors, particularly certain glutamate receptors subtype, as well as nicotinic acetylcholine receptors [STRŘMGAARD et al. 2005]. For example N1-coumaroyl spermidine reduced excitatory postsynaptic potentials in the muscles of Drosophila [KLOSE et al. 2002]. This suggest that polyamines might be important for defense responses not only against pathogens but also herbivorous insects. In conclusions, we can suggest that the stress caused by the grain aphid feeding induced the polyamines biosynthesis within the triticale tissues. It seems to be an important part of the aphid induced defence mechanism, that appears within the infested triticale. Such mechanism might have some further consequences since the stress reaction is systemic and transferred to the root tissues.

References BAKER C.J., WHITAKER B.D., ROBERTS D.P., MOCK N.M., RICE C.P., DEAHL K.L., AVER’YANOV A. 2005. Induction of redox sensitive extracellular phenolics during plant-

bacterial interactions. Physiol. Mol. Plant Pathol. 66: 90-98. BOUCHEREAU A., AZIZ A., LARHER F., MARTIN-TANGUY J. 1999. Polyamines and environ-

mental challenges : recent development. Plant Sci. 140: 103-125. CICHOCKA E., LESZCZYŃSKI B. 1999. The use of EPG technique in assessing of susceptibility of the broad bean to black bean aphid (Aphis fabae Scop.). Aphids and Other Homopterous Insects 7: 29-36. CIEPIELA A.P., SEMPRUCH C. 2002. Zmiany w aktywności dekarboksylazy ornitynowej w wybranych gatunkach i odmianach zbóŜ zaatakowanych przez mszycę zboŜową (Sitobion avenae F.). Zesz. Nauk. AR w Krakowie 387: 237-239. CIEPIELA A.P., SEMPRUCH C., SPRAWKA I., CHRZANOWSKI G. 1999. Evaluation of antibiotic properties and tolerance of winter triticale cultivars to grain aphid in Central-Eastern Poland. Aphids & Other Homopterous Insects 7: 187-193. FIXON-OWOO S., LAVASSEUR F., WILLIAMS K., SABADO T.N., LOWE M., KLOSE M., MERCIER A.J., FIELDS P., ATKINSON J. 2003. Preparation and biological assessment of hydroxy-

cinnamic acid amides of polyamines. Phytochem. 63: 315-334. KAKKAR R.K., NAGAR P.K., AHUJA P.S., RAI V.K. 2000. Polyamines and plant morphogenesis. Biol. Plant. 43: 1-11. KLOSE M.K., ATKINSON J.K., MERCIER A.J. 2002. Effect of hydroxy-cinnamoyl conjugate of spermidine on arthropod neuromuscular junctions. J. Comp. Physiol. 187: 945-952. KUMAR A., ALTABELLA T., TAYLOR M.A., TIBURCIO A.F. 1997. Recent advances in polyamine research. Trends Plants Sci. 2: 124-130. KUZNETSOV V., SHORINA M., ARONOVA E., STETSENKO L., RAKITIN V., SHEVYAKOVA N. 2007.

406

C. Sempruch et al.

NaCl and ethylene dependent cadaverine accumulation and its possible protective role in the adaptation of the common ice plant to salt stress. Plant Sci. 172: 363-370. LESZCZYŃSKI B., TJALLINGII W.F. 1994. Przewodnik do elektronicznej rejestracji Ŝerowania owadów w tkankach roślin. Wyd. WSRP Siedlce: 83 p. LOWRY J.O.H., ROSEBROUGH N.J., FARR A.L., RANDAL R.J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 256-277. NGO T.T., BRILLHART K.L., DAVIS R.H., WOMG R.C., BOVAIRD J.H., DIGANGI J.J., RISOV J.L., MARSH J.L., PHAN A.P.H., LENHOFF H.M. 1987. Spectrophotometric assay for ornithine

decarboxylase. Anal. Biochem. 160: 290-293. SEMPRUCH C. 2005. The participation of lysine decarboxylase in interactions between

winter triticale and grain aphid (Sitobion avenae F.). Aphids & Other Hemipterous Insects 11: 163-168. SEMPRUCH C. 2006. The participation of chosen amino acids decarboxylases in constitutive resistance of winter triticale to cereal aphids. Biotechnology 2006 (Ed. By V Řehout) Scientific Pedagogical Publishing, Č. Budejovice: 724-726. SEMPRUCH C., CIEPIELA A.P. 2005. The participation of polyamines in mechanism of winter triticale resistance to grain aphid (Sitobion avenae F.). IOBC wprs Bull. 28: 107-112. SEMPRUCH C., CIEPIELA A.P., SYTYKIEWICZ H., SZYMALSKA M. 2004. Activity of ornithine decarboxylase in winter triticale attacked by grain aphid (Sitobion avenae /F./). Aphid & Other Hemipterous Insects 10: 103-110. STRŘMGAARD K., JENSEN L.S., VOGENSEN S.B. 2005. Polyamine toxins: development of selective ligands for ionotropic receptors. Toxicon 45: 249-254. SUDHA G., RAVISHNAKAR G.A. 2002. Involvement and interaction of various signaling compounds on the plant metabolic evens during defense response, resistance to stress factors, formation of secondary metabolites and their molecular aspects. Plant Cell Tiss. Org. Cult. 71: 181-212. TANG W., NEWTON R.J. 2005. Polyamines reduce salt-induced oxidative damage by increasing the activities of antioxidant enzymes and decreasing lipid peroxidation in Virginia pine. Plant Growth Regul. 46: 31-43. WALTERS D.R. 2000. Polyamines in plant-microbe interactions. Physiol. Mol. Plant Pathol. 57: 137-146. WALTERS D.R. 2003. Polyamines and plant disease. Phytochem. 64: 97-107. WÓJCICKA A., STASZEWSKI Z., WARZECHA E., TJALLINGII F.W., SZYNKARCZYK S., LESZCZYŃSKI B. 1999. Effect of surface waxes of triticale on feeding behaviour of grain aphid,

Sitobion avenae F. Aphids & Other Homopterous Insects 7: 161-168. WYATT I.J., WHITE P.F. 1977. Simple estimation of intrinsic rates for aphids and tetra-

nychid mite. J. Appl. Ecol. 14: 757-766. Key words:

grain aphid, insect-host-plant interactions, ornithine decarboxylase, polyamines, winter triticale Summary

The aim of the present work was to determine an influence of grain aphid, Sitobion avenae F. feeding on ornithine decarboxylase (ODC) activity within tissues of winter triticale. Experiments were carried out on aerial parts and roots of seedlings of two triticale cultivars: Tornado - more susceptible to S. avenae and Witon - less susceptible. The obtained results showed that ODC activity in shoots of cv. Witon


407

decreased during the first week of grain aphid feeding and increased after 2 weeks. However, in the roots of this cultivar the enzyme activity decreased after 48 h and increased after 2 weeks. When susceptible cv. Tornado was studied the grain aphid attack reduced the ODC activity after 24 h of feeding. Moreover, the grain aphid attack reduced the ODC activity during the first week and increased after 2 weeks in the shoots and roots of cv. Tornado. The importance of the ODC activity and polyamines in stress reaction and defense mechanisms of winter triticale to S. avenae is discussed. ZMIANY W AKTYWNOŚCI DEKARBOKSYLAZY ORNITYNY W SIEWKACH PSZENśYTA OZIMEGO POD WPŁYWEM STRESU WYWOŁANEGO śEROWANIEM MSZYCY ZBOśOWEJ Cezary Sempruch, Agnieszka Wójcicka, Marek Makosz, Bogumił Leszczyński Katedra Biochemii i Biologii Molekularnej, Akademia Podlaska w Siedlcach Słowa kluczowe:

mszyca zboŜowa, interakcje owad-roślina Ŝywicielska, dekarboksylaza ornityny, poliaminy, pszenŜyto ozime Streszczenie

Określono wpływ Ŝerowania mszycy zboŜowej (Sitobion avenae F.) na aktywność dekarboksylazy ornityny (ODC) w siewkach pszenŜyta ozimego. Badania przeprowadzono z wykorzystaniem części nadziemnych i korzeni siewek dwóch odmian pszenŜyta: Tornado - bardziej podatna na S. avenae i odmiana Witon - mniej podatna. Wykazano, Ŝe aktywność ODC w pędach odmiany Witon obniŜała się w trakcie pierwszego tygodnia Ŝerowania mszycy zboŜowej i wzrastała po dwóch tygodniach. W korzeniach tej odmiany stwierdzono natomiast spadek aktywności enzymu po 48 godz. i po dwóch tygodniach. Ponadto atak mszycy zboŜowej powodował takŜe redukcję aktywności ODC w pierwszym tygodniu oraz jej wzrost po 2 tygodniach w korzeniach i pędach odmiany Tornado. W pracy przedstawiono znaczenie zmian w aktywności ODC jako reakcji na stres wywołany Ŝerowaniem owada oraz w mechanizmach obronnych pszenŜyta w stosunku do mszycy zboŜowej. Dr Cezary Sempruch Katedra Biochemii i Biologii Molekularnej Akademia Podlaska ul. Prusa 12 08-110 SIEDLCE e-mail: [email protected]

aerial parts; części nadziemne Tornado Enzyme activity in control plants Enzyme activity in infested plants Aphid number 70

150

60

120

50 90

**

*

40

**

Activity of ornithine decarboxylase (µg putrescine/mg protein/h)

20 30

10 0

0

korzenie; roots Tornado 70

150 120

**

60

**

**

90

50 40

**

30

60

20 30

10 0

0 24 h

48 h

1 week

2 week

Number of aphids on analysed plants (number of individuals/blade)

30

**

60

aerial parts; części nadziemne Witon 150

70 60

120

50 90

40

60

30 Number of aphids on analysed plants (number of individuals/blade)

20

Activity of ornithine decarboxylase (µg putrescine/mg protein/h)

30

10

0

0

korzenie; roots Witon

150

70 60

120

50 90

40

60

30 20

30

10

0

0 24 h

48 h

1 week

2 weeks

* **

differences between control and infested plants significant at P ≤ 0.05 (Student’s t-test) differences significant at P ≤ 0.01; róŜnice między roślinami zaatakowanymi i kontrolnymi istotne przy p≤ 0,01 (test t-Studenta)

Fig. 2. Influence of the grain aphid feeding on activity of the ornithine decarboxylase within tissues of seedlings of the studied triticale cultivars Rys. 2.Wpływ Ŝerowania mszycy zboŜowej na aktywność dekarboksylazy ornityny w tkankach siewek badanych odmian pszenŜyta ozimego