(Converted)-4 - Journal of Cell and Molecular Biology - Haliç ...

‹STANBUL 

1998 

VOLUME 2 • NO. 1 • 2003 • ISSN 1303-3646 

HAL‹Ç UNIVERSITY 

FACULTY OF ARTS AND SCIENCES 

Journal of Cell and 

Molecular Biology

Haliç University 

Faculty of Arts and Sciences 

Journal of Cell and Molecular Biology 

Founder 

Prof. Dr. Gündüz GED‹KO⁄LU 

President of Board of Trustee 

Rights held by 

Prof. Dr. Ahmet YÜKSEL 

Rector 

Correspondence Address: 

The Editorial Office 


Haliç Üniversitesi, Fen-Edebiyat Fakültesi, 

Ahmet Vefik Pafla Cad., No: 1, 34280, 

F›nd›kzade, ‹stanbul-Turkey 

Phone: 90 212 530 50 24 

Fax: 90 212 530 35 35 

E-mail: jcmb@halic.edu.tr 

Journal of Cell and Molecular Biology is 

indexed in EBSCO database. 

Summaries of all articles in this journal are 

available free of charge from www.halic.edu.tr 

ISSN 1303-3646 

printed at yaflar printing house 

Igor ALEXANDROV, Dubna, Russia 

Çetin ALGÜNEfi, Edirne, Turkey 

Aglaia ATHANASSIADOU, Patros, Greece 

fiehnaz BOLKENT, ‹stanbul, Turkey 

Nihat BOZCUK, Ankara, Turkey 

‹smail ÇAKMAK, ‹stanbul, Turkey 

Adile ÇEV‹KBAfi, ‹stanbul, Turkey 

Beyaz›t ÇIRAKO⁄LU, ‹stanbul, Turkey 

Ayfl›n ÇOTUK, ‹stanbul, Turkey 

Zihni DEM‹RBA⁄, Trabzon, Turkey 

Mustafa DJAMGOZ, London, UK 

Aglika EDREVA, Sofia, Bulgaria 

Advisory Board 


Molecular Biology 

Published by Haliç University 


Editor 

Atilla ÖZALPAN 

Associate Editor 

Narç›n PALAVAN ÜNSAL 

Editorial Board 

Çimen ATAK 

Atok OLGUN 

P›nar ÖZKAN 

Nihal BÜYÜKUSLU 

Kürflat ÖZD‹LL‹ 

Damla BÜYÜKTUNÇER 

Özge EM‹RO⁄LU 

Mehmet Ali TÜFEKÇ‹ 

Merve ALO⁄LU 

Asl› BAfiAR 

Ünal EGEL‹, Bursa, Turkey 

Candan JOHANSEN, ‹stanbul, Turkey 

As›m KADIO⁄LU, Trabzon, Turkey 

Valentine KEFEL‹, Pennsylvania, USA 

Göksel OLGUN, Edirne, Turkey 

U¤ur ÖZBEK, ‹stanbul, Turkey 

Zekiye SULUDERE, Ankara, Turkey 

‹smail TÜRKAN, ‹zmir, Turkey 

Mehmet TOPAKTAfi, Adana, Turkey 

Meral ÜNAL, ‹stanbul, Turkey 

Mustafa YAT‹N, Boston, USA 

Ziya Z‹YLAN, ‹stanbul, Turkey



Volume 2/2003 



‹stanbul-TURKEY




Founder 

Prof. Dr. Gündüz GED‹KO⁄LU 

President of Board of Trustee 

Rights held by 

Prof. Dr. Ahmet YÜKSEL 

Rector 

Correspondence Address: 



Haliç Üniversitesi, Fen-Edebiyat Fakültesi, 

Ahmet Vefik Pafla Cad., No: 1, 34280, 

F›nd›kzade, ‹stanbul-Turkey 

Phone: 90 212 530 50 24 

Fax: 90 212 530 35 35 

E-mail: jcmb@halic.edu.tr 

Journal of Cell and Molecular Biology is 

indexed in EBSCO database. 

Summaries of all articles in this journal are 

available free of charge from www.halic.edu.tr 

ISSN 1303-3646 

printed at yaflar printing house 

Igor ALEXANDROV, Dubna, Russia 

Çetin ALGÜNEfi, Edirne, Turkey 

Aglaia ATHANASSIADOU, Patros, Greece 

fiehnaz BOLKENT, ‹stanbul, Turkey 

Nihat BOZCUK, Ankara, Turkey 

‹smail ÇAKMAK, ‹stanbul, Turkey 

Adile ÇEV‹KBAfi, ‹stanbul, Turkey 

Beyaz›t ÇIRAKO⁄LU, ‹stanbul, Turkey 

Ayfl›n ÇOTUK, ‹stanbul, Turkey 

Zihni DEM‹RBA⁄, Trabzon, Turkey 

Mustafa DJAMGOZ, London, UK 

Aglika EDREVA, Sofia, Bulgaria 

Advisory Board 



Published by Haliç University 


Editor 


Associate Editor 

Narç›n PALAVAN ÜNSAL 

Editorial Board 

Çimen ATAK 

Atok OLGUN 

P›nar ÖZKAN 

Nihal BÜYÜKUSLU 

Kürflat ÖZD‹LL‹ 

Damla BÜYÜKTUNÇER 

Özge EM‹RO⁄LU 

Mehmet Ali TÜFEKÇ‹ 

Merve ALO⁄LU 

Asl› BAfiAR 

Ünal EGEL‹, Bursa, Turkey 

Candan JOHANSEN, ‹stanbul, Turkey 

As›m KADIO⁄LU, Trabzon, Turkey 

Valentine KEFEL‹, Pennsylvania, USA 

Göksel OLGUN, Edirne, Turkey 

U¤ur ÖZBEK, ‹stanbul, Turkey 

Zekiye SULUDERE, Ankara, Turkey 

‹smail TÜRKAN, ‹zmir, Turkey 

Mehmet TOPAKTAfi, Adana, Turkey 

Meral ÜNAL, ‹stanbul, Turkey 

Mustafa YAT‹N, Boston, USA 

Ziya Z‹YLAN, ‹stanbul, Turkey


CONTENTS Volume 2, No.1, 2003 

Dedication 

Review articles 

Polyamines in plants: An overview 

Bitkilerde poliaminler: Genel bir bak›fl 

R. Kaur-Sawhney, A.F. Tiburcio, T. Altabella, A.W. Galston 1-12 

Phenolic cycle in plants and environment 

Bitkilerde fenolik döngü ve çevre 

V. I. Kefeli, M. V. Kalevitch, B. Borsari 13-18 

Research papers 

The short-term effects of single toxic dose of citric acid in mice 

Farelerde sitrik asidin tek toksik dozunun k›sa süreli etkileri 

T. Aktaç, A. Kabo¤lu, E. Bakar, H. Karakafl 19-23 

Characterisation of RRPPPP77 mutant lines of the col-5 ecotype of AArraabbiiddooppssiiss tthhaalliiaannaa 

Arabidopsis thaliana’n›n col-5 ekotipinden elde edilen mutant hatlardan RPP7 

geninin karakterizasyonu 

C. Can, M. Özaslan, E. B. Holub 25-30 

The effect of mmeettaa-topolin on protein profile in radish cotyledons 

Meta-topolinin turp kotiledonlar›nda protein profiline etkisi 

S. Ça¤, N. Palavan-Ünsal 31-34 

The effect of electromagnetic fields on oxidative DNA damage 

Elektromanyetik alan›n oksidatif DNA hasar› üzerindeki etkisi 

S. ‹fller, G. Erdem 35-38 

Chromosomes of a balanced translocation case evaluated with atomic force 

microscopy 

Dengeli translokasyon vakas›nda kromozomlar›n atomik güç mikroskobu ile 

de¤erlendirilmesi 

Z. Y›lmaz, M. A. Ergun, E. Tan 39-42 

Effect of epirubicin on mitotic index in cultured L-cells 

Epirubisinin kültürdeki L-hücrelerinde mitotik indekse etkisi 

G. Özcan Ar›can, M. Topçul 43-48 

Letter to editor 49-51 

Book reviews 53 

Instructions to authors 55-56

This issue is dedicated to 

P rof. Dr. Arthur W. Galston 

for his invaluable contribution to plant biology

Arthur W. Galston, Curriculum Vitae 

Born: April 21, 1920 Eaton Professor of Botany, Emeritus, Department of 

Molecular, Cellular and Developmental Biology, 

Education: B.S. Cornell University, 1940; Yale University, New Haven, CT 06520-8103, 

M.S. University of Illinois, 1942; Ph. D. 1943 Tel. (203) 432-6161; e-mail arthur.galston@yale.edu 

Honors: Elected to Phi Beta Kappa; Phi Kappa Phi; Sigma Xi; American Academy of Arts and Sciences, National 

Sigma Xi Lecturer, 1966; National Phi Beta Kappa Visiting Scholar, 1972-1973; Award of the New York Academy 

of Sciences, 1979; William Clyde De Vane Medal for lifelong teaching and scholarship, Yale University, 1994; 

Honorary LL.D, 1980 Iona; Honorary Ph. D., Hebrew University of Jerusalem, 1992. 

Experience: Plant Physiologist, Emergency Rubber Project, California Institute of Technology 1943-1944; Instuctor 

in Botany, Yale University, 1946-1947; Senior Research California Institute of Technology, 1947-1950; Associate 

Professor of Biology, California Institute of Technology, 1951-1955. Professor of Plant Physiology, Department of 

Botany, Yale University 1955-1961; Chairman, Department of Botany, 1961-1962; Director, Division of Biological 

Sciences, Yale University, 1965-1966; Professor of Biology, 1962-1973; Eaton Professor of Botany, 1973-; 

Chairman, Department of Biology 1985-1988; Eaton Professor Emeritus, 1990. 

Fellow of the John Simon Guggenheim Memorial Foundation, Stockholm and Sheffield, 1950-1951; Fulbright 

Fellow, Canberra, Australia, 1960-1961; National Science Foundation Faculty Fellow, London 1967-1968; Albert 

Einstein Fellow and Visiting Professor, Hebrew University of Jerusalem, 1980; Visiting Fellow Wolfson College, 

Cambridge, England, 1983; Visiting Scientist, RIKEN Institute, Wako, Saitama, Japan, 1988-1989. 

Secretary, American Society of Plant Physiologists, 1955-1957; Vice President, 1957-1958; President, 1962-1963. 

Secretary-Treasurer, International Association for Plant Physiology, 1962-1967. Vice-President 

Botanical Society, 1967-1968; President 1968-1969; Award, 1970. Member, Commitee on Space Biology and 

Medicine, National Research Council; Member Life Sciences Advisory Committee, NASA; also Long Range 

Strategic Planning Committee in Life Sciences Advisory Committee, NASA; Member, NASA Disciplinary Working 

Group for CELLS (Controlled Ecological Life Support Sytem). 

P resent of past Editorial Board Member: Plant Growth Regulation, Pesticide Physiology and Biochemistry, 

Environment, Chemical and Engineering News, Science Year, Plant Physiology, Phsiology, Phytochemistry, 

American Journal of Botany, Lloydia. Formerly regular columnist, Natural History Magazine. 

Former Member: Metabolic and Regulatory Biology Panels, National Science Foundation; Executive Committee, 

Growth Society; Life Science Advisory Committee, NASA; and Governing Boards, Biological Sciences Curriculum 

Study, Commission on Undergraduate Education in the Biological Sciences and AIBS. 

First American scientist to visit the People’s Rebuplic of China, 1971. 

Books: ‘Principles of Plant Physiology’ (with J. Bonner), Freeman, 1952. ‘Life of the Green Plant’, Prentice Hall, 

1961, 2 nd Ed. , 1964, 3 rd Ed. , 1980 (with P. J. Davies and R. L. Satter). ‘Control Mechanisms in Plant Development’, (with 

P. J. Davies), Prentice Hall, 1970. ‘Daily Life in People’s China’, Crowell, 1973; Simon and Schuster, 1975. ‘Green 

Wisdom’ Basic Books, Inc. NY, 1981; Putnam, 1983. ‘Life Processes in Plants’, Freeman (Scientific American 

Library), 1994. ‘New Dimensions in Bioethics’, Arthur W. Galston and Emily G. Shurr, eds. Kluwer Academic 

Publishers, Boston/Dordrecht/London, 2001. 

More than 320 articles in referred scientific journal; approximately 60 general articles on problems of science and 

society.

Journal of Cell and Molecular Biology 2: 1-12, 2003. 

Haliç University, Printed in Turkey. 


Ravindar Kaur-Sawhney 1 *, Antonio F. Tiburcio 2 , Teresa Altabella 2 , and Arthur W. Galston 1 

1 Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, 

06520-8103, USA; 2 Laboratori de Fisiologia Vegetal, Facultad de Farmacia, Universitat de Barcelona, 

Spain (* author for correspondence) 

Received 21 October 2002; Accepted 10 November 2002 

Abstract 

This article presents an overview of the role of polyamines (PAs) in plant growth and developmental processes. The 

PAs, putrescine, spermidine and spermine are low molecular weight cations present in all living organisms. PAs and 

their biosynthetic enzymes have been implicated in a wide range of metabolic processes in plants, ranging from cell 

division and organogenesis to protection against stress. Because the PA pathway has now been molecularly and 

biochemically elucidated, it is amenable to modulation by genetic approaches. Genes for several key biosynthetic 

enzymes namely, arginine decarboxylase, ornithine decarboxylase and S-adenosyl methionine decarboxylase have 

been cloned from different plant species, and antibodies to some genes are now available. Both over-expressed and 

antisense transgenic approaches to PA biosynthetic genes have provided further evidence that PAs are required for 

plant growth and development. However, molecular mechanisms underlying PA effects on these processes remain 

unclear. Analysis of gene expression by using DNA microarray genomic techniques should help determine the precise 

role of these compounds. The potential of proteomics to unravel the role of PAs in particular cellular processes has 

also been examined. The extensive use of the two-hybrid system and other proteomic approaches will provide new 

insights into the role of PAs in signal transduction. Furthermore, there is evidence that proteomics provides an 

excellent tool for determining supramolecular organizations of PA metabolic enzymes which may help in 

understanding homeostatic control of this metabolic pathway. 

KKeeyy wwoorrddss:: Polyamines, mutants, transgenic plants, genomics, proteomics 


Özet 

Bu makalede poliaminlerin (PA) bitki büyüme ve geliflme olaylar›ndaki rolüne genel bir bak›fl yap›lmaktad›r. PA ler 

putresin, spermidin ve spermin, düflük molekül a¤›rl›kl› ve tüm canl› organizmalarda mevcut olan maddelerdir. PA 

lerin ve bunlar›n biyosentetik enzimlerinin bitkileri strese karfl› korumaya yönelik olarak hücre bölünmesinden 

organogeneze kadar de¤iflen genifl bir metabolik olaylar zincirinde yer ald›¤› ortaya konmufltur. Günümüzde PA yolu 

moleküler ve biyokimyasal yönden aç›kl›¤a kavufltu¤u için genetik yaklafl›mlarla düzenlenmeye uygundur. Çeflitli 

anahtar biyosentez enzimleri, arginin dekarboksilaz, ornitin dekarboksilaz ve S-adenozil metiyonin dekarboksilaz›n 

genleri farkl› bitki türlerinde klonlanm›flt›r ve günümüzde baz› genlerin antikorlar›n› elde etmek mümkündür. PA 

biyosentezi genlerine hem over-ekspres ve hem de antisens transgenik yaklafl›mlar PA lerin bitki büyüme geliflmesi 

için gereklili¤ini daha da ortaya koymufltur. Bununla birlikte bu olaylardaki PA etkilerinin moleküler mekanizmas› 

hala aç›kl›¤a kavuflmam›flt›r. DNA mikroarray genom teknikleri kullan›larak yap›lan gen ekspresyon analizleri bu 

bilefliklerin rollerini kesin olarak belirlemeye yard›mc› olacakt›r. PA lerin özellikle hücresel olaylardaki rolünü ortaya 

koymaya yönelik olarak proteomi¤in potansiyeli de araflt›r›lm›flt›r. ‹ki-hibrit sistemi ve di¤er proteomik yaklafl›mlar›n 

yo¤un kullan›m›, PA lerin sinyal iletimindeki rolüne yeni bir bak›fl aç›s› getirecektir. Bundan baflka proteomi¤in, PA 

metabolik yolunun homeostatik kontrolünü anlamaya yard›mc› olabilecek, PA metabolizma enzimlerinin 

supramoleküler organizasyonunun belirlenmesinde çok önemli bir araç oldu¤u konusunda veriler mevcuttur. 

AAnnaahhttaarr ssöözzccüükklleerr:: Poliaminler, mutantlar, transgenik bitkiler, genomik, proteomik 

1

2 Ravindar Kaur-Sawhney et al. 

1. Introduction 

Polyamines (PAs) are low molecular weight 

polycations found in all living organisms (Cohen, 

1998). They are known to be essential for growth and 

development in prokaryotes and eukaryotes (Tabor and 

Tabor, 1984; Tiburcio et al., 1990). In plant cells, the 

diamine putrescine (Put), triamine spermidine (Spd) 

and tetramine spermine (Spm) constitute the major 

PAs. They occur in the free form or as conjugates 

bound to phenolic acids and other low molecular 

weight compounds or to macromolecules such as 

proteins and nucleic acids. As such, they stimulate 

DNA replication, transcription and translation. They 

have been implicated in a wide range of biological 

processes in plant growth and development, including 

senescence, environmental stress and infection by 

fungi and viruses. Their biological activity is attributed 

to their cationic nature. These findings have been 

discussed in several recent review articles (Tiburcio et 

al., 1993; Galston et al.,1997; Bais and Ravishankar, 

2002). 

The use of PA biosynthesis inhibitors has shown a 

causal relationship between changes in endogenous PA 

levels and growth responses in plants. These 

observations led to further studies into undestanding 

the mode of PA action. Some of the important 

observations suggest that PAs can act by stabilizing 

membranes, scavenging free radicals, affecting nucleic 

acids and protein synthesis, RNAse, protease and other 

enzyme activities, and interacting with hormones, 

phytochrome, and ethylene biosynthesis (reviewed in 

Slocum et al., 1984; Galston and Tiburcio, 1991). 

Because of these numerous biological interactions of 

PAs in plant systems, it has been difficult to determine 

their precise role in plant growth and development. 

In recent years, however, investigations into 

molecular genetics of plant PAs have led to isolation of 

a number of genes encoding PA biosynthetic enzymes 

and development of antibodies to some of the genes. 

Furthermore mutants and transgenic plants with altered 

PA metabolism have also been developed. Genomic 

and proteomic approaches are being used to further 

gain an understanding into the role of PAs in plant 

developmental processes. These findings will hopefully 

lead to a better understanding of their specific functions 

in plants. Several useful reviews on these aspects have 

been published (Galston et al., 1997; Walden et al., 

1997; Malmberg et al., 1998; Martin-Tanguy, 2001; 

Bais and Ravishankar, 2002). 

This article presents an overview of the role of PAs 

in plants with particular emphasis on recent 

investigations using molecular and genetic 

approaches. 

2. Polyamine biosynthesis 

The PA biosynthetic pathway in plants has been 

thoroughly investigated and reviewed in detail (Evans 

and Malmberg, 1989; Tiburcio et al., 1990; Slocum, 

1991a; Martin-Tanguy, 2001). Briefly, PAs are 

synthesized from arginine and ornithine by arginine 

decarboxylase (ADC) and ornithine decarboxylase 

(ODC) as illustrated in Figure 1. The intermediate 

agmatine, synthesized from arginine, is converted to 

Put, which is further transformed to Spd and Spm by 

successive transfers of aminopropyl groups from 

decarboxylated S-adenosylmethionine (dSAM) 

catalysed by specific Spd and Spm synthases. The 

aminopropyl groups are derived from methionine, 

which is first converted to S-adenosylmethionine 

(SAM), and then decarboxylated in a reaction 

catalyzed by SAM decarboxylase (SAMDC). The 

resulting decarboxylated SAM is utilized as an 

aminopropyl donor. SAM is a common precursor for 

both PAs and ethylene, and SAMDC regulates both 

biosynthetic pathways as illustrated in Figure 1. 

A number of investigators have used PA inhibitors 

to modulate the cellular PA titer in order to determine 

their role in various plant processes. Four commonly 

used inhibitors of PA synthesis are: 1. 

Difluoromethylornithine (DFMO), an irreversible 

inhibitor of ODC (reviewed in Bey et al., 1987); 2. 

Difluoromethylarginine (DFMA), an irreversible 

inhibitor of ADC (Bitonti et al., 1987); 3. Methylglyoxyl-bis 

guanylhydrazone (MGBG), a competitive 

inhibitor of S-adenosyl-methionine decarboxylase 

(SAMDC) (Williams-Ashman and Schenone,1972); 

and 4. Cyclohexylamine (CHA), a competitive 

inhibitor of spermidine synthase (Hibasami et al., 

1980). Common oxidases are diamine oxidase and 

polyamine oxidase (PAO), as reviewed by Smith and 

Marshall (1988). Each PA has been found to be 

catabolized by a specific oxidase. 

Several investigations have dealt with localization 

of PAs and their biosynthetic enzymes in plants 

(reviewed by Slocum, 1991b). However, paucity of 

information regarding the exact cellular and 

subcellular localization of these entities remains one of

Methionine 

S - adenosylmethionine 

AVG 

ACC 

Ethylene 

SAMDC 

ACC synthase 

ACC oxidase 

MGBG 

the obstacles in understanding their biological role. 

Recent studies have shown that PAs are present in the 

cell wall fractions, vacuole, mitochondria and 

chloroplasts (Torrigiani et al., 1986; Slocum, 1991b; 

Tiburcio et al., 1997). The biosynthetic enzymes, 

ODC, SAMDC, and Spd synthase have been reported 

to be localized in the cytoplasm, whereas ADC is 

localized in the thylakoid membrane of chloroplast 

(Borrell et al., 1996; Tiburcio et al., 1997) and PAO in 

the cell wall (Kaur-Sawhney et al., 1981). ODC 

activity has also been observed in the nucleus 

(Slocum, 1991b). However, these findings have to be 

interpreted with caution because various procedural 

problems can mask the results. Despite these advances 

in understanding the metabolic processes involving 

PAs and their localization in plant cells, the precise 

role of PAs in plant morphogenesis remains elusive. 

DFMO 

Ornithine Arginine 

dSAM 

ODC 

Polyamines in plants 3 

ADC 

DFMA 

Agmatine 

Putrescine 

Spdsynthase 

Spermidine 

Spermine 

Spmsynthase 

Figure 1: Polyamine biosynthetic pathway and its linkage to ethylene biosynthetis. Biosynthetic enzymes are ADC, ODC and 

SAMDC and the inhibitor DFMA, DFMO and MGBG. 

3. Polyamines in plant growth and development 

The availability of specific inhibitors of PA 

biosynthesis has helped in investigating the 

mechanisms involved in PA interactions to some extent, 

providing a partial understanding of their physiological 

role in plant growth and development. Clearly, PAs are 

involved in many plant developmental processes, 

including cell division, embryogenesis, reproductive 

organ development, root growth, tuberization, floral 

initiation and development, fruit development and 

ripening as well as leaf senescence and abiotic stresses 

(reviewed by Evans and Malmberg, 1989; Galston et 

al., 1997; Bais and Ravishankar, 2002; Tiburcio et al., 

2002). Changes in free and conjugated PAs and their 

biosynthetic enzymes, namely ADC, ODC, and 

SAMDC have been found to occur during these 

developmental processes. Earlier experiments had 

shown that increases in PAs and their biosynthetic 

enzymes are associated with rapid cell division in many 

plant systems e.g., carrot embryogenesis (Montague


and Koppenbrink, 1978; Feirer et al., 1984), tomato 

ovaries (Heimer and Mizrahi, 1982), tobacco ovaries 

(Slocum and Galston, 1985), and fruit development 

(reviewed in Kakkar and Rai, 1993). Similar results 

have been reported for many other plant species 

(reviewed in Bais and Ravishankar, 2002). In contrast, 

several other studies have suggested that correlations 

between PAs and their biosynthetic enzymes and plant 

growth processes, especially somatic embryogenesis, 

are not universal and may be species specific (reviewed 

in Evans and Malmberg, 1989; Galston et al., 1997; 

Bais and Ravishankar, 2002). 

In general, cells undergoing division contain high 

levels of free PAs synthesized via ODC, and cells 

undergoing expansion and elongation contain low 

levels of free PAs synthesized via ADC (see review by 

Galston and Kaur-Sawhney, 1995). High levels of 

endogenous PAs and their conjugates have also been 

found in apical shoots and meristems prior to 

flowering (Cabbane et al., 1981) and flower parts of 

many plants (Martin-Tanguy, 1985). Our experiments 

using callus cultures derived from thin layer explants 

of pedicels from tobacco inflorescence show that 

endogenous Spd increased more rapidly than other 

PAs in floral buds than in vegetative buds. Addition of 

CHA, an inhibitor of Spd synthesis, to the culture 

medium reduced flower formation in a dose dependent 

manner and such inhibition was correlated with a 

switch to initiation of vegetative instead of flower 

buds. This inhibition was reversed by the addition of 

exogenous Spd (Kaur-Sawhney et al., 1988). More 

recently, we have found that higher levels of 

endogenous PAs occur in flowers and siliques when 

compared with their levels in leaves and bolts of 

certain strains of Arabidopsis. Addition of the PA 

biosynthetic inhibitors, DFMA and CHA to the culture 

medium, at time of seed germination, inhibited bolting 

and flower formation and this was partially reversed 

by addition of exogenous Spd (Applewhite et al., 

2000). These results clearly show that Spd is involved 

in flower initiation and development. Similar results 

have been reported in other plants also (reviewed by 

Galston et al.,1997; Bais and Ravishankar, 2002). 

Many plant growth and development processes 

known to be regulated by plant hormones, such as 

auxins, 2,4-D, GA and ethylene, have also been 

correlated with changes in PA metabolism. These 

changes occur in both endogenous levels of PAs and 

their biosynthetic enzymes and appear to be tissue 

specific (reviewed by Galston and Kaur- 

Sawhney,1995). Thus, PAs which may or may not be 

mobile in plants (Young and Galston, 1983; Bagni and 

Pistocchi, 1991) can serve as intracellular mediators of 

hormone actions (Galston and Kaur-Sawhney, 1995). 

Supporting evidence for this hypothesis has been 

obtained in experiments using specific inhibitors of PA 

biosynthesis (Bagni et al., 1981; Egea-Cortines and 

Mizrahi, 1991; reviewed in Galston et al., 1997; Bais 

and Ravishankar, 2002). 

Of the major plant hormones, ethylene has been 

most intensively investigated with respect to PA 

metabolism. The two metabolites, PAs and ethylene, 

play antagonistic roles in plant processes. While PAs 

inhibit senescence of leaves (Kaur-Sawhney et al., 

1982), cell cultures of many monocot and dicot species 

(Muhitch et al., 1983) and fruit ripening (Kakkar and 

Rai, 1993), ethylene promotes these processes. The 

most commonly held view is that PAs and ethylene 

regulate each other’s synthesis, either directly or 

through metabolic competition for SAM, a common 

precursor for their biosynthesis (Figure 1). PAs inhibit 

ethylene biosynthesis, perhaps by blocking the 

conversion of SAM to ACC and of ACC to ethylene 

(Apelbaum et al., 1981; Suttle, 1981; Even-Chen et al., 

1982; Furer et al., 1982). Ethylene, on the other hand, 

is an effective inhibitor of ADC and SAMDC, key 

enzymes in PA biosynthetic pathway (Apelbaum et al., 

1985; Icekson et al., 1985). Thus, PAs may affect 

senescence and fruit ripening by modulating PA and 

ethylene biosynthesis. 

Apparently, PAs are essential members of an array 

of internal metabolites required in many plant 

developmental processes, but their precise role in these 

processes has yet to be established. Whereas, specific 

PAs at specific concentrations may be required at 

critical stages of growth and morphogenetic events, no 

definitive data are available to establish their role as 

plant hormones. 

4. Manipulation of the polyamine pathway 

The PA pathway is ubiquitous in living organisms and 

is relatively short (see Section 2) in terms of the 

number of enzymes involved. Most of the genes 

coding for enzymes involved in the pathway have been 

cloned from different sources (Kumar et al., 1997; 

Walden et al., 1997; Galston et al., 1997; Tiburcio et 

al., 1997; Malmberg et al., 1998; Kumar and Minocha, 

1998; Panicot et al., 2002b). Thus, the PA pathway

epresents an excellent model to test various 

hypotheses and to answer fundamental biological 

questions derived from pathway manipulation (Thu- 

Hang et al., 2002; Bhatnagar et al., 2002). 

Initially, approaches to manipulate the PA pathway 

made use of suicide inhibitors, but the effects of 

DFMO and DFMA on ODC and ADC respectively, are 

variable in different plant systems, ranging from 

inhibition to stimulation or no effect and depending on 

the concentration, plant system tested and the 

existence of compensatory mechanisms (Slocum and 

Galston, 1987). Therefore, alternative approaches to 

manipulate polyamine metabolism have been 

developed during the recent years. 

4.1. Mutants 

Mutants deficient in PA biosynthesis have been 

isolated from several biological systems. Hafner et al. 

(1979) isolated PA mutants in Escherichia coli 

showing decreased growth and increased sensitivity to 

paraquat (Milton et al., 1990). Yeast mutants 

presenting ODC as the sole pathway, show reduced 

growth and altered sporulation on PA deficient 

medium (Cohn et al., 1980; Whitney and Morris, 

1978). Chinese hamster ovary cells lacking ODC 

activity do not grow in medium lacking PA (Steglich 

and Scheffler, 1983) and a moderately reduced brood 

size was observed in a Caenorhabditis elegans ODC 

deletion mutant (Macrae et al., 1995). Mutations in 

genes affecting Spd and Spm biosynthesis have also 

been isolated in yeast. The spe3 Spd synthase mutation 

causes a growth arrest, which can be complemented 

with externally added Spd (Hamasaki-Katagiri et al., 

1997), while the yeast spe4 mutant is defective in Spm 

biosynthesis (Hamasaki-Katagiri et al., 1998). 

Less is known about mutants affecting PA 

metabolism in plants. Mutants with high levels of 

ADC activity have been identified in petunia because 

of their abnormal morphology (Geerats et al., 1988), 

but the basis of the mutation is still not known. 

Screening for resistance to the SAMDC inhibitor 

MGBG (Malmberg and Rose, 1987) or to inhibitory 

concentrations of Spm (Mirza et al., 1997), yielded 

mutants that showed reduced sensitivity to the 

respective agent, but these mutants have not been 

further exploited for the analysis of PA function. 

Watson et al. (1998) isolated EMS mutants of A. 

thaliana that are reduced in ADC activity. The mutants 

fall into two complementation groups, spe1 and spe2, 

which may correspond to the two gene copies 

encoding ADC, ADC1 and ADC2 (Watson et al., 

1998). The mutations have not been mapped and 

therefore it cannot be excluded that other functions, 

i.e. regulatory elements, are affected (Soyka and 

Heyer, 1999). More recently, Hanzawa et al. (2000) 

reported that the inactivation of the Arabidopsis 

ACAULIS5 (ACL5) gene causes a defect in the 

elongation of stem internodes by reducing cell 

expansion. It was suggested that ACL5 encodes a Spm 

synthase, but the possibility that ACL5 may exhibit 

broad amine substrate specificities and be involved in 

the synthesis of other polyamines could not be 

excluded (Hanzawa et al., 2000). 

Thus far the only well characterized plant 

polyamine biosynthetic mutant has been generated by 

using reverse genetics. The availability of mutant 

collections generated either by transposon or T-DNA 

tagging now facilitates the identification of knockouts 

in any gene of interest using PCR-based mutant 

screening techniques (Ferrando et al., 2002). By using 

these techniques, Soyka and Heyer (2000) isolated an 

Arabidopsis thaliana mutant line carrying an insertion 

of the En-1 transposable element at the ADC2 locus 

which should be regarded as a complete loss-offunction 

or knockout mutation. The ADC2 knockout 

mutant shows no obvious phenotype change under 

normal growth conditions, but is completely devoid of 

ADC induction by osmotic stress. As ADC1 gene 

expression was not affected in the mutant, it was 

concluded that ADC2 is the gene responsible for 

induction of ADC and PA biosynthesis under osmotic 

stress (Soyka and Heyer, 2000). More recently, Pérez- 

Amador et al. (2002) have shown that ADC2 gene 

expression is induced in response to mechanical 

wounding and methyl jasmonate treatment in 

Arabidopsis thaliana. All these observations appear to 

indicate that ADC2 is a key gene involved in the PA 

response to abiotic stress in Arabidopsis. We envisage 

that the extensive use of functional genomics and 

reverse genetic studies will facilitate the isolation of 

novel knock-out mutants affected in other PA 

biosynthetic genes. 

4.2. Transgenic plants 


With the availability of most of the genes involved in 

PA metabolism, it has become possible to manipulate 

this metabolic pathway using sense and antisense 

transgenic approaches. Thus, cellular PA content has


been modulated by overexpression or down regulation 

of the key genes ODC, ADC or SAMDC (Kumar et al., 

1997; Walden et al., 1997; Malmberg et al., 1998; 

Kumar and Minocha, 1998; Capell et al., 1998; Rajam 

et al.,1998; Roy and Wu, 2001; Bhatnagar et al., 2002). 

Most of the studies have used the constitutive 35S 

promoter, but only few of them were successful in 

using either inducible (Masgrau et al., 1997; Panicot et 

al., 2002a; Mehta et al., 2002) or tissue-specific 

promoters (Rafart-Pedros et al., 1999). Overexpression 

of heterologous ODC or ADC cDNAs generally causes 

the production of high levels of Put (DeScenzo and 

Minocha, 1993; Bastola and Minocha, 1995; Masgrau 

et al., 1997; Capell et al., 1998; Bhatnagar et al., 2002; 

Panicot et al., 2002a), but in most cases only a small 

increase or even no change in Spd and Spm has been 

observed. This indicates that elevated levels of Put 

resulting from genetic manipulation of a single step 

located upstream of the PA biosynthetic pathway (i.e. 

ODC or ADC) are not accompanied by an increase in 

subsequent biosynthetic reactions (i.e. Spd and Spm 

biosynthesis) (Bhatnagar et al., 2002). In contrast, 

overexpression of genes located downstream of the 

pathway (i.e. SAMDC or SPDS) generally lead to 

increased levels of Spd or Spm or both (Thu-Hang et 

al., 2002; Mehta et al., 2002). Taken together these 

results suggest that the levels of Spd and Spm in the 

cells are under a tight homeostatic regulation 

(Bhatnagar et al., 2002), which possibly could be 

related to a supramolecular organization of some of 

these enzymes (see Section 5). 

Discrepancies observed among different studies 

may have several causes. These include: transgene 

source, positional effects, recipient plant system, plant 

material analyzed and type of promoter used. A 

hierarchical accumulation of polyamines in different 

transgenic tissues/organs has been observed (Lepri et 

al., 2001). In general, less metabolically active tissues 

accumulate higher levels of polyamines (Lepri et al., 

2001). These results are in line with experiments in 

which metabolites such as vitamin A and 

pharmaceutical antibodies accumulate at high levels in 

seeds of different species. It is reasonable to assume 

that dormant or less metabolically active tissues 

provide a conducive environment for the accumulation 

of transgenic products (Thu-Hang et al., 2002). In this 

regard, it should be stressed that the most remarkable 

results have been obtained by controlled expression of 

transgenes using inducible or tissue-specific 

promoters. For example, tissue-specific expression of 

SAMDC gives rise to smaller potato tubers without 

affecting tuber yield (Rafart-Pedros et al., 1999). The 

distribution of tuber weights is of agronomic 

importance, and generally a reduction of tuber-size 

variation is economically advantageous, so that more 

tubers fall into a given size grade either for seed or 

ware (Rafart-Pedros et al., 1999). Similarly, fruitspecific 

expression of heterologous SAMDC in tomato 

resulted in ripening-specific accumulation of Spd and 

Spm which led to an increase in lycopene, prolonged 

vine life, and enhanced fruit juice quality (Mehta et al., 

2002). Besides the agronomic interest of this finding, 

this latter study constitutes one of the most striking 

evidence regarding the in vivo involvement of 

polyamines in a particular developmental process, i.e. 

fruit ripening (Mehta et al., 2002). 

5. Understanding the role of polyamines 

Phenotypic analyses of mutants and transgenic plants 

with altered PA levels gives further support to the 

previous physiological studies (see Section 3) with 

regard to the involvement of these compounds in 

several plant processes (reviewed by Tiburcio et al., 

2002). These include somatic embryogenesis (Bastola 

and Minocha, 1995), stem elongation and flowering 

(Gerats et al., 1988; Masgrau et al., 1997; Hanzawa et 

al., 2000; Panicot et al., 2002a), root growth (Watson 

et al., 1998; Cordeiro et al., unpublished), tuber 

development (Kumar et al., 1996; Rafart-Pedrós et al., 

1999), fruit ripening (Mehta et al., 1997; 2002), abiotic 

stresses (Minocha and Sun, 1997; Soyka and Heyer, 

1999; Roy and Nu, 2001). However, most of these 

mutants and transgenic plants have not been further 

exploited for the analysis of PA function. Application 

of advanced genomic and proteomic approaches will 

help to elucidate the role of PA in particular plant 

processes. 

5.1. Genomic approaches 

The availability of complete genome sequences 

permits the use of approaches to explore gene 

expression variations on a large genome scale. Either 

cDNAs or large oligonucleotide collections are 

attached on surfaces to create a microarray. The 

hybridisation of the microarray with fluorescent 

labelled RNA or cDNA yields an overall image of gene 

expression or ‘transcriptome’ (Lockhart and Winzeler,

2000). The global examination of gene expression 

should reveal the coincidence of spatial and temporal 

transcript expression profiles that may reflect a 

requirement of co-ordinated gene product expression 

in response to different type of signals. The technology 

developed for the Arabidopsis genome has been 

accelerated in the recent years both by public funding 

through the Arabidopsis Functional Genomics 

Consortium in the USA and the GARNet in the UK, 

and also by private initiatives like Monsanto, 

Affymetrix or Synteny/InCyte (Wisman and Ohlrogge, 

2000). 

Although there are already many examples in the 

literature showing the utility of this approach for 

unraveling complex plant responses and signal 

transduction processes (Schena et al., 1995; Schaffer et 

al., 2000), the use of this technology in our field is 

unfortunately in its infancy. So far, DNA microarray 

analysis has been used to reveal the induction of ADC 

genes during drought stress (Ozturk et al., 2002) or in 

response to wounding and methyl jasmonate treatment 

(Sasaki et al., 2001; Pérez-Amador et al., 2002). 

We envisage that global analysis of gene 

expression in well characterized mutant and transgenic 

plants with altered polyamine metabolism will provide 

novel clues in the near future for understanding the 

molecular mechanisms underlying polyamine effects 

on plant growth and development. 

5.2. Proteomic approaches 

Proteomics’ uses biochemical approaches aimed at 

systematically characterizing the ‘proteome’ or the 

‘protein complement of the genome’ (Wasinger et al., 

1995) in a given organism, tissue, cell or subcellular 

compartment. The means of proteome characterization 

include protein localization, expression and most 

importantly protein interaction maps. A plethora of 

innovative procedures has been employed in recent 

years for the large-scale analysis of protein signalling 

pathways, including the yeast two-hybrid system 

(Fields and Song, 1989), protein purification methods 

linked to detection by mass spectrometry (Neubauer et 

al., 1997; Verma et al., 2000); protein localization 

(Ferrando et al., 2000; 2001; Farràs et al., 2001), and 

protein microarray techniques (Zhu et al., 2001). 

The yeast two-hybrid system is a genetic tool to 

describe in vivo protein interactions using the yeast 

cell as a test tube. Each separated module of the GAL4 

transcription factor, either the DNA binding domain 


(DBD) or the transcriptional activation domain (AD), 

is translationally fused to proteins of interest X or Y, 

generating respectively the hybrid proteins X-DBD 

(bait) and Y-AD (prey). A powerful aspect of the yeast 

molecular genetics involves the facility to isolate the 

corresponding cDNAs coding for proteins X or Y, 

introduced in the form of plasmid DNA. This latter 

feature immediately favored the use of this system to 

identify interacting partners for a given bait protein X 

using cDNA libraries as a prey (reviewed by Walhout 

et al., 2000). The number of studies that have used 

proteomics in our field is still scanty. Here we will 

provide two examples that demonstrate the potential of 

these techniques to (i) unravel the role of PA in 

transcription; and (ii) to identify PA metabolons (see 

below). 

Although the potential role of PAs in affecting gene 

expression had already been reported, the molecular 

mechanisms underlying their effects were unknown 

(Wang et al., 2002). The identification of a polyamine 

responsive element and corresponding transacting 

protein factors that respond to polyamines has opened 

up an exciting new area to study the function of these 

compounds in transcription (Wang et al., 1999). By 

using the two-hybrid system, it was recently found that 

the human homologue of the Arabidopsis subunit 

COP9 signalosome complex binds to such transacting 

protein factors with the potential to directly affect gene 

expression (Wang et al., 2002). Remarkably, the COP9 

signalosome proteins were first identified in 

Arabidopsis and have been demonstrated to form a 

regulatory complex involved in light-activated 

development and playing a role in intracellular 

signalling (Deng et al., 2000). We envisage that similar 

type of experiments will be performed in the plant PA 

field that hopefully will provide new insights into the 

role of PAs in plant signal transduction. 

Increasing number of reports document that many 

metabolic reactions are catalysed by complexes of 

sequentially acting enzymes that show highly ordered 

structural organization (reviewed in Srere, 1987). In 

such multienzyme complexes the metabolites pass 

from one active enzyme site to the next through a 

process termed ‘substrate channeling’. The 

supramolecular arrangement of enzymes involved in 

such metabolic reactions is referred to as ‘metabolon’. 

Metabolons are multienzyme complexes in both 

prokaryotes and eukaryotes that represent highly 

organized assemblies of sequential enzymes in a 

metabolic pathway and are thought to provide


increased metabolic efficiency and higher substrate 

selectivity. Metabolons may also help to coordinate the 

activities of enzymes by sharing intermediates in a 

given pathway, as well as to ensure protection of labile 

substrates and sequestration of toxic intermediates 

(Sugumaran et al., 2000). In addition, the formation of 

multienzyme metabolon complexes may enhance 

enzyme stability, improve enzymatic performance and 

provide a means for adaptation to alterations of input 

of metabolic reactions, especially during demanding 

physiological conditions (Abadjieva et al., 2001). 

The relevant information about intrinsic properties 

of ‘metabolon’ formation can be acquired by studies of 

protein-protein interactions using modern proteomic 

approaches (Ferrando et al., 2002). In this regard, our 

laboratory has recently analyzed possible interactions 

between the SPDS and SPMS enzymes of polyamine 

biosynthetic pathway in the yeast two-hybrid system 

(Panicot et al., 2002b). Using the Arabidopsis 

spermidine synthase as bait, two similar proteins were 

identified to interact with SPDS2 that were named 

SPDS1 and SPMS. Yeast and bacterial mutant 

complementation tests revealed that SPDS1 encodes a 

novel spermidine synthase, whereas SPMS displays 

spermine synthase activity. The heterodimerization 

capabilities of enzymes catalyzing the two last steps of 

polyamine biosynthesis were also demonstrated in vivo 

by co-immunoprecipitation using epitope tagged 

SPDS1, SPDS2 and SPMS proteins (Ferrando et al., 

2000; Ferrando et al., 2001). Immunoaffinity 

purification and size fractionation of SPDS and SPMS 

enzymes labeled with different HA and c-Myc 

epitopes revealed that the SPDS and SPMS proteins 

co-purify with large multiprotein complexes of 650 to 

750 kDa. Further analysis of subunits of isolated 

SPDS-SPMS metabolon(s) by mass spectrometry is 

expected to yield important information about yet 

unknown regulatory subunits of SPDS-SPMS 

metabolon in the PA biosynthesis pathway. The 

available data support the conclusion that Spd 

synthesized by SPDS is effectively channeled to 

SPMS to control the formation of the end-product Spm 

thereby regulating the synthesis of high molecular 

weight polyamines (Panicot et al., 2002b). 

6. Conclusions 

Considerable evidence indicates that polyamines are 

involved in a wide array of plant processes, including 

DNA replication, transcription of genes, cell division, 

organ development, fruit development and ripening, 

leaf senescence and abiotic stresses. Despite ample 

evidence of their involvement in these processes, their 

precise role in these specific processes remains to be 

established. Recent developments of PA-deficient 

mutants and transgenic plants as well as of 

molecular genetic investigations should further our 

understanding of their role in plant growth and 

development. 

The polyamine pathway is now amenable to 

modulation by genetic approaches because it has been 

elucidated molecularly and biochemically in plants. 

Reverse genetics has identified an Arabidopsis 

knockout mutation of ADC2 gene which reveals 

inducibility by osmotic stress. Extensive use of 

functional genomics and reverse genetics studies will 

facilitate the isolation of novel knockout mutants 

affected in other polyamine metabolic genes. Sense 

and antisense transgenic approaches have revealed the 

feasibility of modulating cellular PA contents. 

Generally, genetic manipulation of single steps located 

upstream of the PA pathway (i.e. ODC or ADC) lead to 

elevated levels of Put, but no changes occur in the 

higher PAs, Spd and Spm. By contrast, overexpression 

of genes located downstream of the pathway (i.e. 

SAMDC or Spd synthase) generally leads to increased 

levels of Spd and Spm, indicating that the levels of Spd 

and Spm are under a tight homeostic cellular control. 

Phenotypic analyses of mutants and transgenic plants 

affected in polyamine metabolism further support 

previous physiological evidence, but the molecular 

mechanisms underlying PA effects on plant growth and 

development remain to be elucidated. Global analysis 

of gene expression by using the available DNA 

microarray genomic techniques will help to understand 

the role of these compounds. The potential of 

proteomics to unravel the role of polyamines in 

particular cellular processes is also examined. We 

envisage that the extensive use of the two-hybrid 

system and other proteomic approaches will provide 

new insights into the role of PAs on plant signal 

transduction. Furthermore, we provide evidence that 

proteomics is an excellent tool to unravel 

supramolecular organizations of PA metabolic 

enzymes which may help to understand homeostatic 

control of this metabolic pathway.

Acknowledgements 

AFT acknowledges the grants from Ministerio de 

Ciencia y Tecnología BIO-99-453 and BIO-2002- 

04459-C02-02. 

References 

Abadjieva A, Pauwels K, Hilven P and Crabeel M. A new 

yeast metabolon involving at least the two first enzymes 

of arginine biosynthesis. J Biol Chem. 276: 42869- 

42880, 2001. 

Apelbaum A, Burgoon A-C, Anderson JD, Lieberman M, 

Ben-Arie R and Mattoo AK. Polyamines inhibit 

synthesis of ethylene in higher plants. Plant physiol. 

68: 453-456, 1981. 

Apelbaum A, Goldlust A and Icekson I. Control by ethylene 

of arginine decarboxylase activity in pea seedlings and 

its implication for hormonal regulation of plant growth. 

Plant Physiol. 79: 635-640, 1985. 

Applewhite PB, Kaur-Sawhney R and Galston AW. A role of 

spermidine in the bolting and flowering of Arabidopsis. 

Physologia Plantarum. 108: 314-320, 2000. 

Bagni N, Torrigiani P and Barbieri P. Effect of various 

inhibitors of polyamine synthesis on the growth of 

Helianthus tuberosus. Med Biol. 59: 403-409, 1981. 

Bagni N and Pistocchi R. Uptake and transport of polyamine 

and inhibitors of polyamine metabolism in plants. In: 

Biochemistry and Physiology of Polyamines in Plants. 

Slocum RD and Flores HE (Ed). CRC Press Inc, Boca 

Raton, FL USA 105-120, 1991. 

Bais HP and Ravishankar GA. Role of polyamines in the 

ontogeny of plants and their biotechnological 

applications. Plant Cell, Tissue and Organ Culture. 

69: 1-34, 2002. 

Bastola DR and Minocha SC. Increased putrescine 

biosynthesis through transfer of mouse ornithine 

decarboxylase cDNA in carrot promotes somatic 

embryogenesis. Plant Physiol. 109: 63-71, 1995. 

Bey P, Danzin C and Jung M. Inhibition of basic amino acid 

decarboxylases involved in polyamine biosynthesis. In: 

Inhibition of Polyamine Metabolism. McCann PP, Pegg 

AE and Sjoerdsma A (Ed). Academic Press, Orlando, 

USA 1-32, 1987. 

Bhatnagar P, Minocha R and Minocha S. Genetic 

manipulation of the metabolism of polyamines in poplar 

cells. The regulation of putrescine catabolism. Plant 

Physiol. 128:1455-1469, 2002. 

Bitonti AJ, Carara PJ, McCann PP and Bey P. Catalytic 

irreversible inhibition of bacterial and plant arginine 

decarboxylase activities by novel substrate and product 

analogues. Biochem J. 242: 69-74, 1987. 

Borrel A, Culiañez-Marcià, Atabella T, Besford RT, Flores D 


and Tiburcio AF. Arginine decarboxylase is localized in 

chloroplasts. Plant Physiol. 109: 771-776, 1995. 

Cabanne F, Dalebroux MA, Martin-Tanguy J and Martin C. 

Hydroxycinnamic acid amides and ripening to flower of 

Nicotiana tabacum L. var. Xanthi n.c. Physiol Plant. 

53: 399-404, 1981. 

Capell T, Escobar C, Lui H, Burtin D, Lepri O and Christou P. 

Overexpression of the oat arginine decarboxylases 

cDNA in transgenic rice affects normal development 

patterns in vitro and results in putrescine accumulation in 

transgenic plants. Theor Appl Genet. 97:246-254, 1998. 

Cohen SS. A Guide to the Polyamines. Oxford University 

Press. New York, NY, 1998. 

Cohn M, Tabor CW and Tabor H. Regulatory mutations 

affecting ornithine decarboxylase activity in S. 

cereviseae. J Bacteriol. 142: 792-799, 1980. 

Deng XW, Dubiel W, Wei N, Hofmann K and Mundt K. 

Unified nomenclature for the COP9 signalosome and its 

subunits: An essential regulator of development. Trends 

Genet. 16: 289, 2000. 

DeScenzo RA and Minocha SC. Modulation of cellular 

polyamines in tobacco by transfer and expression of 

mouse ornithine decarboxylase cDNA. Plant Mol Biol. 

22: 113-127, 1993. 

Egea-Cortines M and Mizrahi Y. Polyamines in cell division, 

fruit set and development and seed germination. In: 


Slocum RD and Flores HE (Ed). CRC Press, Boca 

Raton, Florida, USA. 1991. 

Evans PT and Malmberg RL. Do polyamines have a role in 

plant development? Annu Rev Plant Physiol Plant Mol 

Biol. 40: 235-269, 1989. 

Even-Chen Z, Mattoo AK and Goren R. Inhibition of 

ethylene biosynthesis by aminoethoxyornylglycine and 

by polyamines shunt label from C14-methionine into 

spermidine in aged orange peel discs. Plant Physiol. 69: 

385-388, 1982. 

Farràs R, Ferrando A, Jásik J, Kleinow T, Ökresz L, 

Tiburcio AF, Salchert K, del Pozo C, Schell J and Koncz C. 

SKP1-SnRK protein kinase interactions mediate 

proteasomal binding of a plant SCF ubiquitin ligase. 

EMBO J. 20: 2742-2756, 2001. 

Feirer RP, Mignon G and Litvay JD. Arginine decarboxylase 

and polyamines required for embryogenesis in wild 

carrot. Science. 223: 1433-1434, 1984. 

Ferrando A, Farràs R, Jasik J, Schell J and Koncz C. Introntagged 

epitope: A tool for facile detection and 

purification of proteins expressed in Agrobacteriumtransformed 

plant cells. Plant J. 22: 553-560, 2000. 

Ferrando A, Koncz-Kálmán Z, Farràs R, Tiburcio AF, 

Schell J and Koncz C. Detection of in vivo protein 

interactions between Snf1-related kinase subunits with 

intron-tagged epitope-labelling in plants cells. Nucleic 

Acids Res. 29: 3685-3693, 2001. 

Ferrando A, Altabella T, Koncz C and Tiburcio AF.


Proteomics: Emerging tools to characterize plant 

metabolons. Curr Top Plant Biol. In press. 

Fields S and Song O. A novel genetic system to detect 

protein-protein interactions. Nature. 340: 245-246, 1989. 

Fuhrer J, Kaur-Sawhney R, Shih LM and Galston AW. 

Effects of exogenous 1,3-diaminopropane and 

spermidine on senescence of oat leaves. II. Effects of 

polyamines on ethylene biosynthesis. Plant Physiol. 

70: 1597-1600, 1982. 

Galston AW and Tiburcio AF (Ed). Lecture Course on 

Polyamines as Modulators of Plant Development 257: 

Fundacion Jaun Madrid, March, 1991. 

Galston AW and Kaur-Sawhney R. Polyamines as 

endogenous growth regulators. In: Plant Hormones, 

Physiology, Biochemistry and Molecular Biology 

(2 nd edn). Davies PJ (Ed). Kluwer Academic Publishers, 

Dordrecht, The Netherlands. 158-178, 1995. 

Galston AW, Kaur-Sawhney R, Altabella T and Tiburcio AF. 

Plant polyamines in reproductive activity and response 

to abiotic stress. Bot Acta. 110:197-207, 1997. 

Gerats AGM, Kaye C, Collins C and Malmberg ML. 

Polyamine levels in Petunia genotypes with normal and 

abnormal floral morphologies. Plant Physiol. 86: 390- 

393, 1988. 

Hafner EW, Tabor CW and Tabor H. Mutants of E.coli that 

do not contain 1,4-diaminobutane (putrescine or 

spermidine). J Biol Chem. 254: 12419-12426, 1979. 

Hamasaki-Katagiri N,Tabor CW and Tabor H. Spermidine 

biosynthesis in S. cereviseae: Polyamine requirement of 

a null mutant of the SPE3 gene (spermidine synthase). 

Gene 187:35-43. 1997. 

Hamasaki-Katagiri N, Katagiri Y, Tabor CW and Tabor H. 

Spermine is not essential for growth of S. cereviseae: 

Identification of the SPE4 gene (spermine synthase) and 

characterization of a spe4 deletion mutant. Gene. 

210: 195-201, 1998. 

Hanzawa Y, Takahashi T, Michael AJ, Burtin D, Long D, 

Pineiro M, Coupland G and Komeda Y. ACAULIS5, an 

Arabidopsis gene required for stem elongation, encodes 

a spermine synthase. EMBO J. 19: 4248-4256, 2000. 

Heimer YM and Mizrahi Y. Characterization of ornithine 

decarboxylase of tobacco cells and tomato ovaries. 

Biochem J. 201: 373-376, 1982. 

Hibasami H, Tanaka M, Nagai J and Ikeda T. 

Dicyclohexylamine, a potent inhibitor of spermidine 

synthase in mammalian cells. FEBS Letters. 116: 99- 

101, 1980. 

Icekson I, Goldlust A and Apelbaum A. Influence of ethylene 

on S-adenosylmethionine activity in etiolated pea 

seedlings. J Plant Physiol. 119: 335-345, 1985. 

Kakkar RJ and Rai VK. Plant polyamines in flowering and 

fruit ripening. Phytochemistry. 33: 1281-1288, 1993. 

Kallio A, McCann PP. and Bey P. DL-α (Difluoromethyl) 

arginine: A potent enzyme-activated irreversible 

inhibitor of bacterial arginine decarboxylase. 

Biochemistry. 20: 3163-3166, 1981. 

Kaur-Sawhney R, Flores HE and Galston AW. Polyamine 

oxidase in oat leaves: A cell wall-localized enzyme. 

Plant Physiol. 68: 494-498, 1981. 

Kaur-Sawhney R, Shih Flores HE. and Galston AW. 

Relation of polyamine synthesis and titer to aging and 

senescence in oat leaves. Plant Physiol. 69: 405-410, 

1982. 

Kaur-Sawhney R, Tiburcio AF and Galston AW. Spermidine 

and flower bud differentiation in thin-layer explants of 

tobacco. Planta. 173: 282-284, 1988. 

Kumar A, Taylor MA, Mad Arif SA and Davies HV. Potato 

plants expressing antisense and sense SAMDC 

transgenes show altered levels of polyamines and 

ethylene: Antisense plants display abnormal phenotypes. 

Plant J. 9: 147-158, 1996. 

Kumar A, Altabella T, Taylor MA and Tiburcio AF. Recent 

advances in polyamine research. Trends Plant Sci. 

2: 124-130, 1997. 

Kumar A and Minocha SC. Transgenic manipulation of 

polyamine metabolism. In: Transgenic Plant Research. 

Lindsey K (Ed). Academic Publishers. Harwood. 

187-199, 1998. 

Lepri O, Bassie L, Safwat G, Thu-Hang P, Trung-Nghia P, 

Hölttä E, Christou P and Capell T. Over-expression of 

the human ornithine decarboxylase cDNA in transgenic 

rice plants alters the polyamine pool in a tissue-specific 

manner. Mol Gen Genet. 266:303-312, 2001. 

Lockhart DJ and Winzeler EA. Genomics, gene expression 

and DNA arrays. Nature. 405: 827-836, 2000. 

Macrae M, Plasterk RHA and Coffino P. The ornithine 

decarboxylase gene of Caenorhabditis elegans-cloning, 

mapping and mutagenesis. Genetics. 140:517-525, 1995. 

Malmberg RL and Rose DJ. Biochemical genetics of 

resistance to MGBG in tobacco: Mutants that alter SAM 

decarboxylases or polyamine ratios and floral 

morphology. Mol Gen Genet. 207: 9-14, 1987. 

Malmberg RL, Watson MB, Galloway GL and Yu W. 

Molecular genetic analyses of plant polyamines. Critical 

Rev Plant Sci. 17: 199-224, 1998. 

Martin-Tanguy J. The occurrence and possible function of 

hydroxy-cinnamoyl acid amides in plants. Plant Growth 

Regul. 3: 383-399, 1985. 

Martin-Tanguy J. Metabolism and function of polyamines in 

plants: Recent development (new approaches). Plant 

Growth Regul. 34: 135-148, 2001. 

Masgrau C, Altabella T, Farrás R, Flores D, Thompson AJ, 

Besford RT and Tiburcio AF. Inducible overexpression 

of oat arginine decarboxylase in transgenic tobacco 

plants. Plant J. 11: 465-473, 1997. 

Mehta RA, Handa A, Li N and Mattoo AK. Ripeningactivated 

expression of S-adenosylmethionine 

decarboxylase increases polyamine levels and influences 

ripening in transgenic tomato fruits (abstract no. 134). 

Plant Physiol. 114: S-44, 1997.

Mehta RA, Cassol T, Li N, Ali N, Handa AK and Mattoo AK. 

Engineered polyamine accumulation in tomato enhances 

phytonutrient content, juice quality and vine life. Nat 

Biotech. 20: 613-618, 2002. 

Milton KW, Tabor H and Tabor CW. Paraquat toxicity is 

increased in E. coli defective in the synthesis of 

polyamines. Proc Natl Acad Sci USA. 87: 2851-2855, 

1990. 

Minocha SC and Sun D. Stress tolerance in plants through 

transgenic manipulation of polyamine biosynthesis 

(abstract no. 1552). Plant Physiol. 114: S-297, 1997. 

Mirza JI and Iqbal M. Spermine-resistant mutants of 

Arabidopsis thaliana with developmental abnormalities. 

Plant Growth Regul. 22:151-156, 1997. 

Montague MJ, Koppenbrink JW and Jaworski EG. 

Polyamine metabolism in embryogenic cells of Daucus 

carota. Plant Physiol. 62: 430-433, 1978. 

Muhitch MJ, Edwards LA and Fletcher JS. Influence of 

diamines and polyamines on the senescence of plant 

suspension cultures. Plant Cell Rep. 2: 82-84, 1983. 

Neubauer G, Gottschalk A, Fabrizio P, Seraphin B, 

Luhrmann R, and Mann M. Identification of the proteins 

of the yeast U1 small nuclear ribonucleoprotein complex 

by mass spectrometry. Proc Natl Acad Sci USA. 

94: 385-390, 1997. 

Ozturk ZN, Talame V, Deyholos M, Michalowski CB, 

Galbraith DW, Gomukirmizi N, Tuberosa R and 

Bohnert HJ. Monitoring large-scale changes in transcript 

abundance in drought- and salt-stresses barley. Plant Mol 

Biol. 48: 551-573, 2002. 

Panicot M, Masgrau C, Borrell A, Cordeiro A, Tiburcio AF 

and Altabella T. Effects of putrescine accumulation in 

tobacco transgenic plants with different expression of oat 

arginine decarboxylases. Physiol Plant. 114:281-287, 

2002a. 

Panicot M, Minguet E, Ferrando A, Alcázar R, Blázquez MA, 

Carbonell J, Altabella T, Koncz C and Tiburcio AF. 

A polyamine metabolon involving aminopropyl 

transferases complexes in Arabidopsis. Plant Cell. 

2002b. In press. 

Pérez-Amador MA, León J, Green PJ and Carbonell J. 

Induction of arginine decarboxylase ADC2 gene 

provides evidence for the involvement of polyamines in 

the wound response in Arabidopsis. Plant Physiol. 

In press. 

Rafart-Pedros A, Mac Leod MR, Ross HA, McRae D, 

Tiburcio AF, Davies HD and Taylor M. Manipulation of 

the S-adenosylmethionine decarboxylase transcript level 

in potato tubers. Over-expression leads to an increase in 

tuber number and a change in tuber size distribution. 

Planta. 209: 153-160, 1999. 

Rajam MV, Dagar S, Waie B, Yadav JS, Kumar PA, Shoeb F 

and Kumria R. Genetic engineering of polyamine and 

carbohydrate metabolism for osmotic stress tolerance in 

higher plants. J Biosci. 23:473-482, 1998. 


Roy M and Wu R. Arginine decarboxylase transgene 

expression and analysis of environmental stress 

tolerance in transgenic rice. Plant Sci. 160: 869-875, 2001. 

Sasaki Y, Asamizu E, Shibata D, Nakamura Y, Kaneko T, 

Awai K, Amagai M, Kuwata C, Tsugane T, Masuda T, 

Shimada H, Takamiya K, Ohta H and Tabata S. 

Monitoring of methyl jasmonate-responsive genes in 

Arabidopsis by cDNA macroarray: self-activation of 

jasmonic acid biosynthesis and crosstalk with other 

phytohormone signaling pathways. DNA Res. 8: 153- 

161, 2001. 

Schaffer R, Langraf J, Pérez-Amador MA and Wisman E. 

Monitoring genome-wide expression in plants. Curr 

Opin Biotechnol. 11: 162-167, 2000. 

Schena M, Shalon D, Davis RW and Brown PO. Quantitative 

monitoring of gene expression patterns with a 

complementary DNA microarray. Science. 270: 467-470, 

1995. 

Slocum RD, Kaur-Sawhney R and Galston AW. The 

physiology and biochemistry of polyamines in plants. 

Arch Biochem Biophys. 325: 283-303, 1984. 

Slocum RD and Galston AW. Changes in polyamine 

biosynthesis associated with post-fertilization growh and 

development in tobacco ovary tissue. Plant Physiol. 

79: 336-343, 1985. 

Slocum RD and Galston AW. Inhibition of polyamine 

biosynthesis in plants and plant pathogenic fungi. In: 

Inhibition of Polyamine Metabolism. Biological 

Significance and Basis for New Therapies. McCann PP, 

Pegg AE and Sjoerdsma A (Ed). Academic Press, 

New York. 305-316, 1987. 

Slocum RD. Polyamine biosynthesis in plants. In: 



Raton, FL, USA. 22-40, 1991a. 

Slocum RD. Tissue and subcellular localisation of 

polyamines and enzymes of polyamine metabolism. In: 



Raton, FL, USA. 93-103, 1991b. 

Smith TA and Marshall JHA. The di and polyamine oxidases 

of plants. In: Progress in Polyamine Research (Advances 

in Experimental Biology and Medicine, 250) Plenum 

Press, New York. 573-587, 1988. 

Soyka S and Heyer AG. Arabidopsis knockout mutation of 

ADC2 gene reveals inducibility by osmotic stress. FEBS 

Lett. 458: 219-223, 1999. 

Srere PA. Complexes of sequential metabolic enzymes. 

Annu Rev Biochem. 56: 89-124, 1987. 

Steglich C and Schefler IE. Selection of ornithine 

decarboxylase-deficient mutants of Chinese hamster 

ovary cells. Methods Enzymol. 94: 108-111, 1983. 

Sugumaran M, Nellaiappan K, Amaratunga C, Cardinale S 

and Scott T. Insect melanogenesis. III. Metabolon 

formation in the melanogenic pathway. Regulation of


phenoloxidase activity by endogenous dopachrome 

isomerase. Arch Biochem Biophys. 378: 393-403, 2000. 

Suttle JC. Effect of polyamines on ethylene production. 

Phytochemistry. 20: 1477-1480, 1981. 

Tabor CW and Tabor H. Polyamines. Annu Rev Biochem. 

5: 749-790, 1984. 

Thu-Hang P, Bassie L, Safwat G, Trung-Nghia P. Christou P 

and Capell T. Expression of a heterologous Sadenosylmethionine 

decarboxylase cDNA in plants 

demonstrates that changes in SAMDC activity determine 

levels of the higher polyamines spermidine and 

spermine. Plant Physiol. 129:1744-1754, 2002. 

Tiburcio AF, Kaur-Sawhney R and Galston AW. Polyamine 

metabolism. In: Intermedatory Nitrogen Metabolism. 

16, The Biochemistry of Plants. Miflin BJ. and Lea PJ 

(Ed). Academic Press. 283-325, 1990. 

Tiburcio AF, Campos JL, Figueras X and Besford RT. Recent 

advances in the understanding of polyamine functions 

during plant development. Plant Growth Regul. 12: 331- 

340, 1993. 

Tiburcio AF, Altabella T, Borrell A and Masgrau C. 

Polyamine metabolism and its regulation. Physiol Plant. 

100: 664-674, 1997. 

Tiburcio AF, Altabella T and Masgrau C. Polyamines. In: 

New Developments in Plant Hormone Research. 

Bisseling T and Schell J (Ed). Springer-Verlag, 

New York. 2002. In press. 

Torrigiani P, Serafini-Fracassini D, Biondi S and Bagni N. 

Evidence for the subcellular localization of polyamines 

and their biosynthetic enzymes in plant cells. J Plant 

Physiol. 124: 23-29, 1986. 

Verma R, Chen S, Feldman R, Schieltz D, Yates J, Dohmen J 

and Deshaies RJ. Proteasomal proteomics: Identification 

of nucleotide-sensitive proteasome-interacting proteins 

by mass spectrometric analysis of affinity-purified 

proteasomes. Mol Biol Cell. 11: 3425-3439, 2000. 

Walden R, Cordeiro A and Tiburcio AF. Polyamines: Small 

molecules triggering pathways in plant growth and 

development. Plant Physiol. 113: 1009-1013, 1997. 

Walhout AJM, Boulton SJ and Vidal M. Yeast two-hybrid 

systems and protein interaction mapping projects for 

yeast and worm. Yeast. 17: 88-94, 2000. 

Wang Y, Devereux W, Stewart TM and Casero RA. Cloning 

and characterization of human polyamine-modulated 

factor 1, a transcriptional cofactor that regulates 

the transcription of the spermidine/spermine N 1 - 

acetyltransferase gene. J Biol Chem. 274: 22095-22101, 

1999. 

Wang Y, Devereux W, Stewart TM and Casero RA. 

Polyamine-modulated factor-1 binds to the human 

homologue of the 7a subunit of the Arabidopsis COP9 

signalosome: implications in gene expression. Biochem 

J. 366: 79-86, 2002. 

Wasinger VC, Cordwell SJ, Cerpa-Poljak A, Yan JX, 

Gooley AA, Wilkins MR, Duncan MW, Harris R, 

Williams KL and Humphery-Smith I. Progress with 

gene-product mapping of the Mollicutes: Mycoplasma 

genitalium. Electrophoresis. 16: 1090-1094, 1995. 

Watson MB, Emory KK, Piatak RM and Malmberg RL. 

Arginine decarboxylase (polyamine synthesis) mutants 

of Arabidopsis thaliana exhibit altered root growth. 

Plant J. 13: 231-239, 1998. 

Whitney P and Morris D. Polyamine auxotrophs of 

S. cereviseae. J Bacteriol. 134: 214-220. 

Williams-Ashman HG and Schenone A. Methyl-glyoxyl-bis 

(guanylhydrazone) as a potent inhibitor of mammalian 

and yeast S-adenosylmethionine. Biochem Biophys Res 

Commun. 46: 288-295, 1972. 

Wisman E and Ohlrogge J. Arabidopsis microarray service 

facilities. Plant Physiol. 124: 1468-1471, 2000. 

Young ND and Galston AW. Are polyamines transported in 

etiolated peas? Plant Physiol. 73: 912-914, 1983. 

Zhu H, Bilgin M, Bangham R, Hall D, Casamayor A, 

Bertone P, Lan N, Jansen R, Bidlingmaier S, Houfek T, 

Mitchell T, Miller P, Dean RA, Gerstein M and 

Snyder M. Global analysis of protein activities using 

proteome chips. Science. 293: 2101-2105, 2001.




Valentine I. Kefeli 1 , Maria V. Kalevitch 2 * and Bruno Borsari 3 

1 Slippery Rock Watershed Coalition, 3016 Unionville Rd., Cranberry Twp., PA 16066, USA; 2 Robert 

Morris University, 881 Narrows Run Rd., Moon Township PA 15108, USA; 3 Slippery Rock University, 

101 Eisenberg Bldg., Slippery Rock PA 16057, USA (* author for correspondence) 

Received 30 October 2002; Accepted 15 November 2002 

Abstract 

Phenolic substances are synthesized in plants and in the soil. They exist in the form of polymers and monomers. The 

latter group of phenolics is assembled within the chloroplasts of plant cells, whereas soil phenolics are associated 

with the process of humus formation on the alumino-silicate matrix of the soil micelle. As plants grow, phenolics 

accumulate in cell vacuoles, or polymerize into lignin, which strengthens the secondary cell walls. In addition to this, 

phenolics possess also some physiological functions as they regulate cell elongation. When they are excreted from 

plant root systems they exert inhibitory growth function within adjacent rhizospheres. This work presents the latest 

experimental evidence of phenolic synthesis and transformation in the environment, while providing an 

understanding of their effect in plant-soil relations. 

KKeeyy wwoorrddss:: Allelopathy, chloroplasts, humus, phenolics, soil micelle 


Özet 

Fenolik maddeler bitkilerde ve toprakta sentezlenir. Bunlar polimerler ve monomerler fleklinde bulunurlar. 

Fenoliklerin monomer grubu bitki hücresinin kloroplastlar›nda biraraya gelirken, toprak fenolikleri toprak 

misellerinin alumino-silikat matriksi üzerinde humus oluflum olay› ile uyumluluk gösterir. Bitki büyürken hücre 

vakuollerinde fenolikler birikir veya sekonder hücre çeperlerine sa¤laml›k kazand›ran ligninlere polimerize olurlar. 

Bunlara ilave olarak fenolikler hücre uzamas›n› düzenleyerek baz› fizyolojik ifllevlere de sahiptirler. Bitki kök 

sistemlerinden sal›nd›klar› zaman hemen yak›n›ndaki rizosferlerde büyümeyi inhibe edici etki meydana getirirler. Bu 

çal›flma fenolik sentezlerinin en son deneysel verilerini ve çevredeki dönüflümlerini sunarken, bitki-toprak 

iliflkilerindeki etkilerini anlamam›za yard›m etmektedir. 

AAnnaahhttaarr ssöözzccüükklleerr:: Allelopati, kloroplastlar, humus, fenolikler, toprak miseli 

Introduction 

Phenolics are very stable products in plant organisms. 

Generally, they are characterized by a benzene ring 

and one hydroxyl group (-OH). They can be converted 

into lignin which is the main phenolic polymer in 

plants. Microorganisms break down these molecules 

and their fragments contribute to the mineralization of 

soil nitrogen and humus formation. Thus, humus 

participates actively in fulfilling plants nutritional 

needs and growth. Light enhances the biosynthesis of 

phenolic substances in plant chloroplasts and these 

constitute in addition to soil micelles (humus) a second 

formation site for this diverse group of organic 

13

14 Valentine I. Kefeli et al. 

molecules. It should be mentioned however, that 

phenolics tend to accumulate in plant vacuoles in 

relatively high amounts, or they deposit in the 

secondary cell wall as lignin. 

Chloroplasts as centers of phenolics biosynthesis 

Experiments with chloroplasts of willow (Salix spp.) 

leaves showed that the synthesis of phenol-carboxylic 

acids and flavonoids is strongly stimulated by light 

exposure. Metabolic inhibitors that depress 

photosynthetic activity (simazine, diurone, 

chloramphenicol), affect negatively the biosynthesis of 

flavonoids. Leaves chloroplasts have the capability to 

localize phenol compounds, some of which are 

specific to these organelles only. The chloroplasts of 

spring willow leaves contain more phenols than the 

chloroplasts of the same leaves in the autumn. Light is 

a mandatory condition to initiate phenolics synthesis 

and this is indicated also by the lack of such molecules 

in the protoplastids of etiolated willow shoots (Kefeli 

and Kalevitch, 2002). Light appears also to induce 

flavonols synthesis in the chloroplasts and cytoplasm. 

Chalcone and phenolcarbonic acid present in etiolated 

willow shoots can be considered metabolic precursors 

of light-synthesized flavonols. In certain cell 

compartments (vacuoles and cell wall) phenols are 

contained in significant amounts (Lewis and 

Yamamoto, 1990). However, it is not clear yet how 

phenols are translocated within plant cells and how 

they affect the function of cell organelles such as 

ribosomes and mitochondria. Phenolic substances that 

inhibit plant growth (hydroxy derivatives of cinnamic 

acid, coumarin and naringenin) are synthesized 

similarly to other phenolics. The synthesis of growth 

inhibitor derivatives of hydroxycinnamic acids follows 

the pathway: shikimic acid-chorismic acid-prephenic 

acid-cinnamic acid and p-coumaric acid. A theory of 

metabolism bifurcation among phenolic substances, 

some of which can inhibit growth and synthesis of 

indolic compounds has been proposed. According to 

this new approach, indolil-3-acetic acid (IAA) 

becomes the main natural auxin (Kefeli, 1978; Kefeli 

and Dashek, 1994; Kefeli and Kalevitch, 2002). 

Therefore, indole auxins (IAA, indoleacetonitrile) as 

well as phenolic inhibitors (p-coumaric acid, 

coumarin, naringenin and others) are derived from the 

common precursors, shikimic and chorismic acids 

(Figure 1). 

COOH 

CHO 

I 

C— O 

HCOH 

I 

CH2 Phosphoenolpyruvic 

acid 

HCOH 

CH2O Dehydroquinic acid 

5-Dehydroshikimic acid Erythrose-4-phosphate 

2-keto- 

3-desoxy- 

COOH 

7-phospho- 

D-Araboheptonic acid 

OH OH 

Shikimic acid 

OH 

HOOC CH2-CO-COOH Chorismic acid 

COOH 

OH 

Prephenic acid 

CH 2-CH-COOH 

Phenylalanine 

CH-CH-COOH 

OH 

p-Coumaric acid 

Anthranilic acid 

Muzafarov and collaborators (1992) investigated 

the functions of some phenolics in chloroplasts. They 

assumed that the essence of the relationship between 

photosynthesis and phenolics biosynthesis is that 

phenolics exert a direct and an indirect effect on the 

process of solar accumulation itself. From our point of 

view, flavonoids as polyfunctional compounds in 

green plastids fulfill three major functions as: 

• substrates (use polyphenols and their catabolic 

products for other kinds of biosynthesis); 

• energy sources (electron and proton transport, ion 

exchange and membrane potential, radicals 

formation); 

• regulators (involvement in enzyme reactions as 

inhibitors or activators). 

During photosynthesis under light, flavonoids 

change the rate of electron transport and 

photophosphorylation, bringing about the change of 

ATP/NADPH ratio. In the reactions of carbon 

metabolism they can shift the dynamic equilibrium of 

pentosephosphate reduction cycle to enhance the 

synthesis of certain metabolites both due to the change 

in energy substrate intake and to the interaction with 

enzymes of the cycle. Additionally, flavonoids 

N 

NH 2 

CHOH-CHOH-CH 2O 

Indolylglycerophosphate 

N 

I 

H 

CH 2-CO-COOH 

Indolyl-acetic acid 

IAA 

Figure 1: Phenol-propanoids in metabolic bifurcation.

exercise a feedback control over their own 

biosynthesis, although this phenomenon is not clearly 

understood. This questionable situation remains as the 

biosynthesis of the entire flavonoid structure within 

plastids has not been explained, nor the complete 

enzymatic package of their biosynthesis has been 

discovered yet. Lack of direct eveidence of flavonoids 

transport within the cell and through the whole plant 

constitues another challenge to a more accurate 

description of their functions. Noneless, a variety of 

phenolic compounds, present simultaneously within 

cells appear to be capable of influencing the rate and 

direction of plants metabolic activities. Thus, any 

change in the flavonoid structure, or qualitative 

composition of the phenol complex result in a change 

of the mechanism of its effect upon the processes of 

cell energy exchange. 

Chalcone and phenolcarbonic acids present in 

etiolated willow shoots can be viewed as the potential 

precursors of light synthesized flavonoids. However, 

the use of paper chromatography to investigate 

isosalidpurposide transformation products did not 

reveal the presence of any flavonols sensitive to 

conventional reagents. Therefore, the transformation 

of chalcone (isosalpurposide) in lightless vitro appears 

to terminate at a second stage. The synthesis of 

eriodyctiol and luteolin that occurred in willow leaves 

evidently took place in vivo and under light exposure. 

It should be pointed out however, that phloridzin and 

isosalipurposide were decomposed from aglycone and 

that phloridzin and phloretin produced yellow stains 

on the chromatogram as well as flavonoids. It is known 

that flavonoid glycosides are revealed as dark spots on 

chromatograms exposed to UV light. Therefore, our 

yellow stains were classified as flavanones, since they 

did not react with AlCl3, nor Na2CO3 like flavonols, 

that also form yellow spots. At the same time, similar 

to chalcones and aurones, these floridzin 

transformation products are yellow colored and they 

turn into orange-pink when exposed to Na2CO3 or 

NH4OH. Relatively easy transformations of 

isosalipurposide and phloridzin into compounds of 

other classes (flavanones, chalcones, or aurones) 

evidenced the role of these products in the general 

metabolism of flavonoids (Figure 2). 

OH 

Phenolic cycle 15 

HO 

6´ 

5´ 1´ 

4´ 

2´ 

3´ 

C 

O 

O 

CH = CH 

O6H10O5 1 4 ´OH 

6 5 

Isosalipurposide 

1 

2´, 4´, 6´, 4-tetroxychalcone- 

-2´-glucoside of chalconarin- 

HO 

genin (chalcone) 

O 

H 

2´ 3´ 

8 1 C 

7 

2 1´ 4´ OH 

6´ 5´ 

6 

3 

5 4 CH2 C 

O 

O 

C6H10O3 Salipurposide 

naringenin-5-glucoside 

2 

(Flavanone-glycoside) 

HO 7 

8 

O H 

1 C 2 

2´ 3´ 

1´ 4´ OH 

6 

5 

OH 

3 

4 CH2 C 

O 

6´ 5´ 

3 

Naringenin 

(Flavanone) 

Eriodictyol 

(Flavanone) 

4 

Luteolin 

(Flavone) 

Phenolic substances secreted by roots and leached 

from leaves 

Plants contain and secrete a diverse group of growth 

inhibiting substances that may affect other plants 

development, if grown in their vicinity (allelopathy). 

Leaf exudates of willow species such as Salix rubra or 

Salix viminalis, contain phenolic inhibitors like 

naringenin derivative isosalipurposide. Other species 

instead like apple trees (Malus spp.) contain 

phloridzin, which is a strong respiratory inhibitor. 

Roots and leaves of the wild plant Nanaphyton native 

to semi-desert regions of Mongolia contain also strong 

phenolic inhibitors. Seed as well may secrete 

allelochemicals. Tobacco seed (Nicotiana tabacum) 

for example suppress germination of its own seed 

when leachates come in contact with the seeds (Kefeli 

and Kalevitch, 2002). Although the inhibition of 

germination was observed at various levels of 

intensity, this phenomenon demonstrates the 

selectivity of these natural excreta, similar to the effect 

of synthetic herbicides. Therefore, increasing evidence 

indicates that phenolics and alkaloids play the role of 

selective agents. Secondary compounds can be 

modified in transgenic plants and genetic mutants. 

2 3 

Figure 2: Flavonoid biosynthesis.


Hence, molecular genetics becomes a tool, which may 

help to regulate the level of secondary metabolites in 

plants. Therefore, there is a need continue the search 

for botanical herbicides as a rise of ecological 

concerns has clearly identified the environmental 

impact of herbicides of synthesis. 

Root exudates affect the germination of seeds of 

different crops: monocots and dicots (Table 1 and 

Table 2). However, it must be pointed out that only 

some phenolics were studied in the exudates of willow 

roots (1) which have no analogues in the roots (2) and 

leaves found among the common allelopathogens. 

Although some of these substances could be retained 

by willow roots, others where excreted into an external 

medium. Chromatography of these water exudates and 

a subsequent investigation of their chromatograms 

with UV-B light showed that most of these substances 

are polyphenols such as coumarin, or phenolic acids. 

The phenolic substances retained by cells had different 

chemical properties than those located in the root 

exudates. Thus, the data confirm the hypothesis that 

Table 1: Effect of root exudation on germination of crop seeds (Non-concentrated exudates). 

excreted substances had an allelopathic nature and 

were involved in developing ecological relationships 

with adjacent plants of different species. 

During the composting process water extracts 

contain many inhibiting substances that might form 

toxic exudates (Kefeli et al., 2001). Paper 

chromatography reveals the presence of phenolic acids 

and coumarins in water extracts. The highest 

concentrations of these inhibitors was measured in 

abscised leaves of red maple (Acer rubrum L.). One 

gram of dry leaves was mixed in 29 ml of water to 

prepare the extracts. The pH of the solution was 

between 5.4 and 5.6 and the extracts were incubated 

for a week at room temperature while the pH raised to 

7.2. Further observations revealed that during 

composting the amount of phenolics was drastically 

reduced. Seed germination tests were performed with 

these water extracts and pure water (control) on lettuce 

and wheat seeds. Germination rate and seedling 

lengths were measured to demonstrate that phenolics 

decreased inhibiting properties after dilution, or after 

Variant % to tap water (control) 

wheat clover lettuce mustard 

Tap water 100 100 100 100 

Spider plants (Chlorophytum) exudates 54 93 75 100 

Willow (Salix vitaminalis) exudates 58 79 74 138 

Stem length (5 tallest plants, mm) 

Tap water 29 23 18 25 

Spider plants (Chlorophytum) exudates 15 21 14 2 

Willow (Salix vitaminalis) exudates 7 18 13.5 3.5 

Table 2: Biological activity of willow root exudates after paper chromatography (Biological activity in % to control (water)). 

Clover Lettuce 

Rf Colour in Germination Stem length Germination Stem length 

UV-B light 

0 Blue 91 76 90 64 

0.14 Blue 94 68 98 58 

0.3 Violet 86 80 93 76 

0.5 Blue 56 52 71 76 

0.67 Yellow 87 68 89 88 

0.88 Yellow 52 56 63 64

Secondary 

substances 

Active secretion 

from roots 

into the 

water and soil 

Microorganisms 

Composting process 

Transformed phenolics and 

other secondary substances 

Photosynthesis 

Plant biomass 

in living plants 

Biomass of 

dead plants 

Composting process 

(phenolics and N-sources) 

To alumo-silicate 

matrix 

Humus formation 

Figure 3: Secondary substances, plant biomass accumulation 

and humus formation during allelopathic effects. 

contact with fungi. Therefore, the whole process of 

allelopathogens formation in the environment could be 

tightly connected with the formation of secondary 

substances and plant biomass accumulation (Figure 3). 

Soil-microbial complex for phenolic decomposition 

Phenolic substances are the most resistant metabolites 

produced by plants. They undergo further 

transformation in the soil, forming humus molecules, 

strongly linked to the alumino-silicate matrix. Humus 

is more or less a stable fraction of soil organic matter; 

it adsorbs mineral elements that serve as important 

nutrients for plant growth and development (Kefeli, 

2002). The alumino-silicate matrix and humus form 

primary soil units. Humus is formed by carbonnitrogen 

interaction. Potential sources of carbon 

include cellulose and polyphenols from plant leaves, 

or transformed lignin polymers. 

In order to verify the efficacy of microbial activity 

during the humification process, four different soil 

horizons in a Grashem soil at the Macoskey Center of 

Slippery Rock University of Pennsylvania, USA were 

investigated. The presence and number of colonies of 

heterotrophic soil microflora were determined in each 

Phenolic cycle 17 

horizon (TSA (triple-soya-agar, 48 hours, room 

temperature). The topsoil (horizon A, 0-28 cm) was 

dark gray in color, sandy, high organic matter content 

(5.6%), with slightly alkaline pH=7.5. This horizon 

was also high in potassium, low in available nitrogen, 

and medium in phosphates content, while very high 

was the microbial activity. Horizon E (28-52 cm) was 

ochric in color, it contained more loam, less organic 

matter, lower microbial activity and pH=7.7. Horizon 

B (52-62 cm) had no organic matter, microbial activity 

was the lowest and pH=7.8. Water permeability was 

also measured for each horizon to evaluate penetration 

times. The fastest penetration rate was measured in 

horizon A (11 minutes), whereas it took 47 minutes for 

horizon B and longer (more than 6 hours) below 

horizon B. Soil fertility conditions were also assessed 

with a wheat/clover germination test. A sand substrate 

was used as control, which yielded 30-50% 

germination. Horizon A had a germination of 80-82%, 

horizon B 40-60%, horizon E/B (with lowest microbial 

activity) yielded 30-70% germination rate (Kalevitch 

et al., 2002). The results of these experiments appear to 

indicate that topsoil (for its highest microbial activity) 

Figure 4: Phenolic cycle.


is an effective medium usable to facilitate composting 

of maple and sumac leaves, contaning nature phenolic 

compounds. 

Conclusion 

Microorganisms have the capability to decompose 

phenolic compounds to their monomers, being 

deglicosidation of phenolic molecules, followed by 

lignin decomposition the biochemical pathways of the 

process. Leaves become a primary substrate for soil 

microorganisms, while woody materials and sawdust 

serve as secondary type of biomass and these 

substrates play a major role in humus formation 

(Figure 4). 

The biosynthesis of phenolic substances within 

chloroplasts and its further transformation on the 

alumino-silicate matrix of soil micelles led us to 

conclude about the existence of phenolics cycle in the 

plant-soil system. Although many aspects remain 

unknown, the ecological relevance of phenolic 

substances in the environment has been amply 

demonstrated as this cycle embrace lithosphere, 

microsphere and biosphere. 

These emerging concepts facilitate the 

understanding of complexity within our living systems 

and their physical habitat while reinforcing the idea of 

interconnectedness among living species and 

ecosystems. 

References 

Kalevitch MV, Kefeli VI, Borsari B and Liguory A. Soil 

microflora and fabricated soils. American Society for 

Microbiology. 103 rd General Meeting. Washington DC. 

2003 (In press). 

Kefeli VI. Fabricated soil for landscape restoration. SME 

Ann Meet. 02-142, 2002. 

Kefeli VI and Dashek WV. Non hormonal stimulators and 

inhibitors. Biol Rev Cambrige. 59: 273-288, 1984. 

Kefeli VI, Borsari B and Welton S. The isolation of 

inhibiting compounds from the leaves of the red maple 

(Acer rubrum L.) for the germination and growth of 

lettuce seeds (Lactuca sative L.) NE-Annual Meet of 

ASPP J Plant Phys Abstr. 433. 

Kefeli VI and Kalevitch MV. Natural Growth Inhibitors and 

Phytohormones in Plant and Environment. Kluwer Acad 

Publ. 1-310, 2002. In press. 

Muzafarov EN and Zolotareva EV. Uncoupling effect of 

hydrocinnamic acid derivatives in pea chloroplasts. 

Biochem Physiol Pflanzen. 184: 363-369, 1989. 

Yamamoto T, Yokotani-Tomita K, Kosemura S, Yamada K 

and Hasegava K. Allelopathic substances exuded from a 

serious weed. J Plant Growth Reg. 18: 65-67, 1999.




Tülin Aktaç 1 *, Ayflegül Kabo¤lu 1 , Elvan Bakar 1 and Hamiyet Karakafl 2 

1University of Trakya, Faculty of Arts and Sciences, Department of Biology, 22080, Edirne-Turkey; 

2University of Trakya, Faculty of Medicine, Department of Biochemistry, 22080, Edirne-Turkey 

(* author for correspondence) 

Received 12 April 2002; Accepted 03 July 2002 

Abstract 

The effects of LD25 (480 mg/kg.bw.) dose of citric acid, a food preservative, were investigated on body weight, organ 

weights (liver, kidney, spleen), creatin kinase (CK), lactate dehydrogenase (LDH), alanine aminotransferase (ALT) 

and aspartate aminotransferase (AST) enzymes in the blood serum, and the liver tissue of mice after 10 days. Citric 

acid (to experimental groups) and physiological saline (to control groups) were given intraperitoneally. The results 

of enzyme activities were evaluated using autoanalyzer as IU/L. Even though significant decreases in the body 

weights were noted when compared to those of the control group (p0.05, kidney: p>0.05, spleen: p>0.05) and serum enzyme levels 

(CK: p>0.05, LDH,: p>0.05, ALT: p>0.05, AST: p>0.05). Microscopical examination of the liver showed 

histopathological changes depending on the citric acid. These changes were tissue degeneration, cytoplasmic 

vacuolisations, nuclear membrane invaginations, picnotic nucleus and necrosis of the hepatocytes. 

KKeeyy wwoorrddss:: Citric acid, food preservative, enzymes, mouse, liver 


Özet 

Bir besin koruyucu olan sitrik asidin LD25 (480 mg/kg.va.) dozu farelere intraperitoneal yolla uyguland›. 10 gün 

sonra hayvanlar›n vücut a¤›rl›klar›, organ a¤›rl›klar› (karaci¤er, böbrek, dalak), kreatin kinaz (CK), laktat 

dehidrogenaz (LDH), alanin aminotransferaz (ALT) ve aspartat aminotransferaz (AST) enzimlerinin serum düzeyleri 

ile, karaci¤er dokusu üzerinde sitrik asidin etkileri araflt›r›ld›. Otoanalizörde tayin edilen enzim aktiviteleri U/L 

olarak de¤erlendirildi. Çal›flmada vücut a¤›rl›klar›nda kontrol grubuna k›yasla anlaml› bir azalma gözlenmesine 

ra¤men (p0.05 , böbrek: p>0.05, dalak: p>0.05) ve enzim aktivitelerinde 

(CK: p>0.05, LDH: p>0.05, ALT: p>0.05, AST: p>0.05) anlaml› olmayan bir art›fl gözlendi. Karaci¤erin mikroskopik 

incelenmesinde doku dejenerasyonu, sitoplazmik vakuolizasyon, nükleer zar çöküntüleri, piknotik nukleuslar ve 

hepatositlerde nekroz gibi histopatolojik de¤ifliklikler gözlendi. 

AAnnaahhttaarr ssöözzccüükklleerr:: Sitrik asit, besin koruyucu, enzimler, fare, karaci¤er 

Introduction 

Humans are exposed daily to complex mixtures of 

chemical compounds in their food. One of these 

substances are antioxidants which are used as food 

preservatives. However, peroxides of saturated fats 

and their secondary oxidation products, can be toxic 

and impair food quality (Würtzen, 1990). Thus despite 

19

20 Tülin Aktaç et al. 

their economic importance, they can have negative 

effects on living organisms. Xenobiotics entering the 

organism are held by intestine, kidney and liver cells 

for detoxification. These cells contain important 

detoxification enzymes. During the detoxification of 

xenobiotics, free radicals are produced in 

oxidation/reduction reactions, and these radicals can 

have destructive effects on tissues. 

The toxic effects of many food preservatives on 

living organisms have been studied by many 

researchers (Makoveç and Sindelar, 1984; Daniel, 

1986; Cabel et al., 1988; Kagan et al., 1990; Jung et 

al.,1992; Nijhoff and Peters, 1992; Fujitani, 1993; 

Weemaes et al., 1997; Mc Farlene et al., 1997; Safer 

and Nughamish, 1999; Kabo¤lu and Aktaç, 2002; 

Aktaç et al., 2002). Although the citric acid and metal 

salts (sodium or potassium citrat) are widely used in 

food industry, there is no report on more detailed 

effects of citric acid (or its salts) in liver. In addition, 

soft drinks, cosmetics and drugs, in which citric acid is 

approved for use, are consumed by most of humans 

every day. 

A way of analysing harmfull effects of foreign 

materials entered to organism is to determine the 

effects of the chemicals on the enzymes. Enzymes 

have a very important role in the metabolical process 

since they are biological catalysts. Thus, their abnormal 

serum levels indicates various diseases. Among these 

enzymes are, creatine kinase (CK), lactate 

dehydrogenase (LDH), alanine aminotransferase (ALT) 

and aspartate aminotransferase (AST) which are the 

most important. Therefore, we studied short term 

treatment of citric acid (10 days) in mice. In these 

experiments, firstly we tested total body weigths, 

organ weights (liver, kidney, spleen), and determined 

the serum levels of creatin kinase (CK), lactate 

dehydrogenase (LDH), alanin aminotransferase (ALT) 

and aspartat aminotransferase (AST), and secondly the 

liver tissue was investigated histopathologically. 

Material and Methods 

Male mice (Balb/C albino) weighing 25-30 g were 

used in our experiments. Five mice were used control 

group and ten mice were used the citric acid-treated 

group. Animals were fed by pellet baits and water. 

LD25 dose (480 mg/kg.bw.) of citric acid (Merck; in 

physiological saline) were injected intraperitoneally to 

experiment group mice, and the same amount of 

physiological saline to control group mice. 10 days 

after the injection, the mice were killed by cervical 

dislocation and then the necessary studies were 

commenced. The livers, kidneys and spleens dissected 

out, weighed, liver samples were seperated for 

microscopical examination. Blood samples were also 

taken for enzyme assays. The serum levels of enzymes 

were determined using a Merck Mega 600 

autoanalyser with the aid of Diasis Kits. Data were 

analyzed by M.Whitney U test for multiple 

comparisons for the differences between the control 

and treated groups. For histological examination, liver 

samples were fixed with 10% buffered formalin, 

processed and stained hematoxylin-eosin. 

Results 

The effects of citric acid injection on the body weight 

and liver, kidney, spleen weights was shown Table 1 

and 2. Although the liver, spleen and kidney weights 

were not changed significantly (p>0.05), the body 

weights were decreased significantly (p0.05) as shown in Table 2. The results 

of the microscopic investigation showed that liver 

of mice treated with citric acid has necrotic 

changes, compare to the control group (Figure 1-6). 

These changes were slightly degeneration of tissue 

(Figure 2), cytoplasmic vacuolisation, nuclear 

membran invaginations (Figure 3, 4) and picnotic nuclei 

(Figure 5). In addition, we observed degeneration of the 

blood vessel endothelium (Figure 6). 

Discussion 

The effects of xenobiotics in living organisms can 

investigate in various ways. Among these are, shortterm 

toxicity tests which are used very commonly. In 

these methods, many parameter are used to test the 

effects of xenobiotics. Some of these parameters are 

body weight, organ weights, blood profile, and 

histopathological examination. In this study, the shortterm 

effects of citric acid applied intraperitoneally 

were investigated. It was reported that the body weight 

decreases in mouse (Würtzen, 1990), and in rats 

(Nijhoff and Peters, 1992) by the effects of phenolic 

antioxidant butylated hydroxytoluene (BHT) and 

butylated hydroxyanisole (BHA) in chronic studies. In

Figure 1: The control group of the liver tissue, bar 

representes 20 µm. 

Figure 3: Citric acid group. Nuclear invaginations (arrows), 

vacuolisation (v), and damaged nucleus (n) in necrotic cells, 

bar representes 4 µm. 

Figure 5: Citric acid group. Picnotic nuclei (arrows) in 

hepatocytes, bar representes 10 µm. 

contrast, any significant change was seen in body 

weight in F344 rats (0.2, 2.5 and 3.0 % of sodium 

benzoate) and in B6C3F1 mice (1.81, 2.09 and 2.4 % 

Short-term effects of citric acid 21 

Figure 2: Citric acid group. Distortion of general 

histological structure of the liver, v: blood vessel, bar 

representes 10 µm. 

Figure 4: Citric acid group. Invaginations of hypertrophic 

cell nucleus (arrow), bar representes 4 µm. 

Figure 6: Citric acid group. Degenerated endothelium 

(arrows) of blood vessel (v), bar representes 10 µm. 

of sodium benzoate) for ten days by Fujitani (1993). 

Similarly, Kabo¤lu and Aktaç (2002) were determined 

that a significant decrease obtained at 3.0 and 4.0 % of

22 Tülin Aktaç et al. 

Table 1: Effect of citric acid on body weight in mice. 

sodium benzoate. Also, at the present study we 

determined a significant decrease of body weight in 

mice by the effect of citric acid (Table 1). 

Some autors have shown that food preservatives 

had increasing effects to organ weight. The effects of 

BHT and BHA on the increasing of the liver and 

thyroid weights were demonstrated in mice by 

Würtzen (1990). Similarly, the effects of BHT on 

increasing of the liver weight in rats was also shown 

by Mc Farlene et al. (1997) and Safer and Nughamish 

(1999). Fujitani (1993) was also obtained significant 

increasing of the liver and kidney by the effects of 

sodium benzoate in male rats. In our previous studies, 

increasing of the total liver weight were seen oral 

treatment of sodium benzoate (Kabo¤lu and Aktaç, 

2002) and citric acid (Aktaç et al., 2002) but it was not 

significant. Additionally, in the present study, we could 

not find any significant change the liver, kidney and 

spleen weights by the intraperitoneal injection of citric 

acid (Table 2). According to our results, serum CK, 

LDH, AST and ALT levels in the treated animals were 

Body weight (g) 

Before experiment Post experiment 

(1. day) (10. days) 

Citric acid (LD25 dose) 27.54 ± 0.813 24.76 ± 1.05 * 

Control 26.04 ± 1.18 26.58 ± 2.65 ** 

Values are mean ± SD for ten mice of experiment group and five mice of control group. 

(*) significant (p0.05). 

Table 2: Effects of citric acid on the organ weights and serum enzyme levels in mice. 

Control Citric acid treated 

Organ weight 

Liver (g) 1.278 ± 0.085 1.257 ± 0.043 * 

Kidney (g) 0.2260 ± 0.033 0.1910 ± 0.089 * 

Spleen (g) 0.1620 ± 0.036 0.1250 ± 0.014 * 

Serum enzyme levels 

CK (IU/L) 572 ± 122 1050 ± 255 * 

LDH (IU/L) 1296 ± 100 2245 ± 321 * 

ALT (IU/L) 695 ± 6.84 101.0 ± 19 * 

AST (IU/L) 177.8 ± 3.2 307.2 ± 46.6* 

Abbrevations : CK = creatin kinase; LDH = lactate dehydrogenase; ALT = alanine aminotransferase; AST = aspartate aminotransferase. 

Values are mean ± SD for ten mice of experiment group and five mice of control group. 

(*) not significant (p>0.05). 

not significant to compare with the control. These 

results were similar with findings obtained in F344 rats 

and B6C3F1 mice by Fujitani (1993). 

Although the organ weights and serum levels of 

enzymes were not changed significantly, the 

examination by light microscopy revealed 

pathological changes in liver of mice, such as 

vacuolisation and glassy cytoplasm in the hepatocyte, 

nuclear membrane invaginations, picnotic nuclei. 

Similarly, with the effect of sodium benzoate in the 

rats and mice, high vacuolisation and glassy 

appearance in hepatocyte cytoplasm was explained 

(Fujitani, 1993). Again, similar findings were obtained 

in the rats with oral treatment of BHT (Mc Farlene et 

al., 1997; Safer and Nughamish, 1999), and with 

sodium benzoate, benzoic acid and citric acid in mice 

(Kabo¤lu and Aktaç, 2002; Aktaç et al., 2002). The 

results of present study suggested that citric acid has 

hepatotoxic effects and long term exposure may 

induce severe damage in liver of mice. However, the 

mechanism of damaging effects of citric acid need to

e clarified by more detailed studies. 

Finally, we can conclude that consumption of the 

foodstuffs containing preservatives is important for the 

human health. 

References 

Aktaç T, Kabo¤lu A, Ertan F, Ekinci F, Hüseyinova G. The 

effects of citric acid (antioxidant) and benzoic acid 

(antimicrobial agent) on the mouse liver: Biochemical 

and histopathological study. Biologia Bratislava. 57(6): 

2002. In press. 

Cabel MC, Waldroup PW, Shermer WD, Calabotta DF 

Effects of ethoxyquin feed preservative and peroxide 

level on broiler performance. Poultry Science. 67: 1725- 

1730, 1988. 

Daniel JW. Metabolic aspects of antioxidants and food 

preservatives. Xenobiotica. 16: 10-11, 1986. 

Fujitani T. Short-term effect of sodium benzoate in F344 rats 

and B6C3F1 mice. Toxicol Lett. 69: 171-179, 1993. 

Jung R, Cojocel C, Müller W, Böttger D, Lück E. Evaluation 

of the genotoxic potential of sorbic acid and potassium 

sorbate. Food Chem Toxicol. 30: 1-7, 1992. 

Kabo¤lu A. and Aktaç T.A study of the effects of the sodium 

benzoate on the mouse liver. Biologia Bratislava. 57(3): 

373-380, 2002. 

Kagan VE, Serbinova EA, Packer L. Generation and 

recycling of radicals from phenolic antioxidants. Arc 

Biochem Biophysiol. 280: 33-39, 1990. 

Makoveç P. and Sindelar L. The effect of phenolic 

compounds on the activity of respiratory chain enzymes 

and on respiration and phosphorylation activities of 

potato tuber mitochondria. Biol Plant. 26: 415-422, 1984. 

McFarlane M, Price SC, Cottrel S, Grasso P, Bremme JN, 

Bomhard ME, Hinton HR. Hepatic and associated 

response of rats to pregnancy, lactation and simultaneous 

treatment with butylated hydroxytoluene. Food Chem 

Toxicol. 35: 753-767, 1997. 

Nijhoff WA and Peters WHM. Induction of rat hepatic and 

intestinal glutathion S-transferases by butylated 

hydroxyanisole. Biochem Pharmacol. 44: 596-600, 1992. 

Safer AM and Nughamish AJ. Hepatotoxicity induced by the 

antioxidant food additive butylated hydroxytoluene 

(BHT) in rats: An electron microscopical study. Histol 

Histopathol. 14: 391-406, 1999. 

Weemaes CA, De-Cordt SV, Ludikhuyze LR, Van Den 

Broeck I, Hendrickx ME, Tobback PP. Influenze of pH, 

benzoic acid, EDTA, and glutathione on the pressure 

and/or temperature inactivation kinetics of mashroom 

polyphenoloxidase. Biotechnol Prog. 13: 25-32, 1997. 

Würtzen G. Short comings of current strategy for toxicity 

testing of food chemicals: Antioxidants. Food Chem 

Toxicol. 28: 743-745, 1990. 

Short-term effects of citric acid 23



Characterisation of RRPPPP77 mutant lines of the col-5 ecotype of 

AArraabbiiddooppssiiss tthhaalliiaannaa 

Canan Can 1 , Mehmet Özaslan 1 *, Eric B. Holub 2 

1 University of Gaziantep, Faculty of Science & Arts, Department of Biology, 27310 Gaziantep; 2 Plant 

Genetics and Biotechnology Department, Horticulture Research International, Wellesbourne, Warwick, 

CV35 9EF, England (* author for correspondance) 

Received 21 May 2002; Accepted 20 November 2002 

Abstract 

In this study, phenotypic characterization of RPP7 that confers resistance to Hiks1 isolate of Peronospora parasitica, 

deficient mutant lines of Col-5 ecotype of Arabidopsis thaliana was investigated. The Col-5 plants that exposed to 

Fast Neutron (FN) were inoculated with 8 different P. Parasitica isolates and symptom development was 

investigated. A total of 4 mutant lines were analyzed. It was found that the RPP7 gene present in the Col-5 ecotype 

is a unique gene different from the other RPP genes present in Col-5. 

KKeeyy wwoorrddss:: Arabidopsis thaliana, Col-5, Hiks-1, Peronospora parasitica 

AArraabbiiddooppssiiss tthhaalliiaannaa’n›n Col-5 ekotipinden elde edilen mutant hatlardan RRPPPP77 geninin 

karakterizasyonu 

Özet 

Bu çal›flmada, Arabidopsis thaliana’n›n Col-5 ekotipinde bulunan ve Peronospora parasitica’n›n Hiks-1 izolat›na 

karfl› dayan›kl›l›¤› sa¤layan RPP7 geninde mutasyon içeren hatlar›n fenotipik olarak belirlenmesi üzerinde 

araflt›rmalar gerçeklefltirilmifltir. Fast Nötron (FN) uygulamalar› ile mutasyon meydana getirilmifl Col-5 

tohumlar›ndan geliflen bitkiler 8 farkl› P. parasitica izolat› ile inokule edilerek semptom geliflimleri incelenmifltir. 

Toplam olarak 4 mutant hatta gerçeklefltirilen analizlerde, RPP7 geninin Col-5 ekotipinde bulunan ve farkl› 

P. parasitica izolatlar›na karfl› dayan›kl›l›¤› sa¤layan genlerden ba¤›ms›z olarak fonksiyon gösteren bir gen oldu¤u 

belirlenmifltir. 

AAnnaahhttaarr ssöözzccüükklleerr:: Arabidopsis thaliana, Col-5, Hiks-1, Peronospora parasitica 

Introduction 

Following the isolation of the Pseudomonas syringae 

resistance genes (R-gene) from tomato, the research on 

isolation and characterization of R-genes against plant 

pathogens has been improved (Hammond-Kosack and 

Jones, 1997). Recently many R-genes conferring 

resistance to fungi, bacteria, nematode and viruses in 

rice wheat, tomato, pepper and some other important 

crops are isolated and the mechanism of resistance is 

determined (Richter and Ronald, 2000). The mutant 

lines with lack of R-genes have a potential importance 

in this type of work (Mc-Dowel et al., 1998). 

Arabidopsis thaliana is a member of cruciferae 

family and it is known to have a small genome size of 

120 Mb. It is a flowering plant and is a best model for 

25

26 Canan Can et al. 

the working on genome analyses, growth regulation, 

hormons, flowering, disease resistance and 

embryogenesis. Arabidopsis and tomato were used to 

determine the mechanisms of disease resistance 

(Thomas et al., 1997; Botella et al., 1998). It is also the 

host of many pests which attacks to crop plants. Many 

genes that provides resistance to bacteria and fungi 

disease have been isolated and characterized from A. 

thaliana (Dangl and Jones, 2001; Feys and Parker, 

2000). 

Peronospora parasitica is a causal agent of mildew 

disease in the genus cabbage, turnip etc. of cruciferae 

family. R-genes that determines resistance to P. 

parasitica (RPP) were isolated and characterized 

(Holub and Beynon, 1997; Parker et al., 1997; Botelli 

et al., 1998; McDowell et al., 1998; Bittner-Eddy et al., 

2000). The researches on RPP genes have shown that 

these genes are at the specific regions at certain places 

of each chromosome called as “Major recognition 

complexes-MRC” (Can, 1997; Holub and Beynon, 

1997). 

RPP7 gene is present in Col-5 ecotype and 

recognized by the Hiks-1 isolate of P. parasitica. This 

gene was placed onto the first chromosome between 

the markers M421 and M213 by using the hybrid lines 

of Col-5 and Nd1 ecotypes (Tor et al., 1994; Can et al., 

1995; Can, 1997). The Hiks1 isolate also recognizes 

the RPP1 gene which is present in Nd-1 ecotype, and 

has an epistatic effect on the RPP7 gene (Tor et al., 

1994). 

The mutant lines that lack the R-genes were 

studied in detail and has a wide area of interest such as 

molecular and classical genetics. However, in order to 

study the relationships between A. thaliana and the 

RPP genes and to investigate the genome 

organizations, some mutant lines were used (Parker et 

al., 1996). The mutant lines lacking the RPP genes 

were obtained from Ws-O that contain RPP14 gene 

exhibiting resistance to No-Co2 isolate by using Ethyl 

Methane Sulfate (EMS). The lines were then used to 

separate the RPP10 and RPP1 genes, which were 

allelic to RPP14 that is on the third chromosome. It 

was found that the WsEDS line was susceptible to all 

P. parasitica isolates tested and that the WsEDS locus 

was necessary for the function of the RPP genes 

(Parker et al., 1996; Bittner-Eddy and Beynon, 2001; 

Falk et al., 1999). The npr (Non expressor of PR 

protein) mutant lines of A. thaliana synthesize the 

proteins which are related with pathogenesis. So, 

systemic resistance is not seen following inoculation 

with many isolates (Century et al., 1995; Aarts et al., 

1998). Similarly, in (Ethylene Intensitive) mutant lines 

do have the ethylene synthesis. But it was found that 

the P. syringae f.s.p. tomato resistance continued in 

this mutant lines. This study showed that ethylene was 

not important for A. thaliana and bacteria relationships 

(Bent et al., 1994; Dong, 1998). Lsd (Lesions 

Simulating Disease resistance response) and acd 

(Accelerated Cell Death) mutant lines produce 

Hypersensitive Resistance (HR) like symptoms 

without a pathogen infection. These symptoms are 

formed by the influence of external factors like heat 

and light (Lam et al., 1999). So, it is accepted that lsd 

and acd loci are negative regulators for HR formation 

(Dietrich et al., 1994). In general, the presence of 

different resistance mechanisms in A. thaliana which 

are directed by RPP genes was found by the 

characterization of mutant lines (Glazebrook et al., 

1997; McDowell et al., 2000). 

In this study, the mutant lines of the Col-5 ecotype 

of A. thaliana were characterized, to understand the 

mechanisms of RPP7 gene that confers resistance to 

Hiks-1 isolate of P. parasitica. 

Material and methods 

Plant and fungus material 

In this study, Col-5 ecotype of A. thaliana lines having 

MRC-B, MRC-C and MRC-H regions (Can, 1997; 

Holub and Beynon, 1997) were used as wild type 

ecotype. The Fast Neutron (FN) applied mutant lines 

were obtained commercially and the selections of 

mutant lines were performed by using the Hiks-1 

isolate of P. parasitica. The Hiks-1 isolate recognizes 

the RPP7 gene which is in the MRC-B region of wild 

Col-5 ecotype, and 7 days after inoculation it induces 

a resistance which is defined with HR. Four mutant 

lines were used in this study denoted as FN3922, 

FN3928, FN3929 and FN3930. The HR does not occur 

in mutant lines, and the pathogen completes its life 

cycle by sexual and asexual sporulation. 

Regeneration of Hiks-1 isolate from oospore 

population 

The Hiks-1 isolate was regenerated by using oospore 

population. To do this, the seeds of A. thaliana that 

were susceptible to the Hiks-1 isolate were sown into

little plastic pots containing 4:1:1 (torf: perlit: sand) of 

mixture for 40-50 seeds each. The pots were irrigated 

to wet the seeds and 1-2 x 10 5 oospore/ ml were added 

to the pots. The containers were held at 4 °C for 1-2 

weeks to break the dormancy. Following this, the 

containers were placed into the climated room at 18-20 

°C, 10 h light and 14 hour dark period. Within 10-15 

days following seed germination, some seedlings 

having sporulation was collected and placed into 

eppendorf tubes containing 200 µl dH2O. The 

eppendorf tubes were shaked gently to allow the 

conidia to pass to water. The conidia suspension was 

used to inoculate 7 days old seedlings of EBH3529 and 

Ksk-1 ecotypes, and the plants were placed into the 

climate room. By this way, the regeneration of the 

Hiks-1 isolate was done by subculturing 3-4 times. The 

conidia were stored at –20 °C and were used when 

needed. 

The same procedure were applied for, Ahco-1, 

Ahco-2, Ahco-7, Wand-1, Cand-5, Hind-2 and Hind-4 

isolates using Nd-1 and Col-5 ecotypes (Can, 1997). 

Characterization of P. parasitica isolates by using 

different A. thaliana ecotypes 

Regenerated P. parasitica isolates were inoculated into 

Col-5, Ksk-1, Nd-1, Ws-3, Tsu-1, Ler-1, Oy-1 and 

Wei-1 ecotypes in order to do phenotypic 

characterization. The A. thaliana ecotypes were 

obtained from Dr. Eric Holub (HRI- UK) 

The conidia suspension was adjusted to 4-5 x 10 4 

conidia/ml concentration for plant inoculations. The 

cotyledons of 7-8 days of the A. thaliana ecotypes 

were inoculated in such a way that it would be one 

drop to each cotyledon. The plants were placed into 

climated room with 18-20 °C, 10 h light, 14 h darkness 

conditions after the inoculation and the plants were 

checked at the end of 3. and 7. days. The evaluation 

was done regarding the pathogen sporulation and 

hypersensitive reaction types (the interaction 

phenotypes). Phenotypic reactions were examined 

under the fine group as; pitting with no pathogen 

sporulation (PN), flecking with no pathogen 

sporulation (FN), flecks with delate and moderate 

pathogen sporulation, 1-20 sporangiophorus per each 

cotyledon (DM), flecking with delate pathogen 

sporulation, 5-10 sporangiophorus per each cotyledon 

(FDL), early and heavy pathogen sporulation, 20> 

sporangiophores per cotyledon (EH), (Holub et al., 

1994). 

Microscopic analysis 

Fungal development in plant tissue was examined 

under light and fluorescence microscope. The infected 

leaves were taken and put absolute methanol for 5-6 

hours followed by saturated chloral hydrate solution 

for 4-5 hours. Then, tissues were placed in 50 % 

glycerol solution for microscopic analyses. 

DNA analysis 

Total plant genomic DNA was isolated with some 

modifications by using the methods of Ausubel et al., 

(1994). Five to eight grams of plant material was 

grounded in N2 and transferred to the tubes containing 

15 ml buffers (100 mM Tris-HCl, 50 mM EDTA, 500 

mM NaCl, 10 mM Mercaptoethanol, %25 SDS) with 

100 mg/lt proteinase K. The solution was kept at 55 °C 

for 1 hour. At the end of this time period, 5 ml of 5 M 

potassium acetate was added and held in ice for 20 

minutes, and the solution was centrifuged at 17000 

rpm for 25 minutes. The supernatant was mixed with 

0,6 volume of isopropanol and held at -20 °C for 

minutes and the DNA was precipitated. Phenolchloroform 

was used to wash the DNA and a second 

precipitation was done. The DNA was dissolved in 

dH2O and stored at -20 °C. The isolated DNA was 

diluted in such to 50-100 ng/µl to use in polimerase 

chain reactions (PCR). For PCR reactions, the closest 

marker to the RPP7 gene was used (Can, 1997). To do 

this, the solution which contains 0.05 mm primer, 2 

mm dNTPs, 25 mm MgCl2, 1 x Taq buffer and IU Taq 

DNA polymerase was completed to 25 ml volume. The 

PCR reactions were performed at 94 °C for 5 min 

followed by 94 °C for 1 minute, 56 °C for 1 min, 72 °C 

for 13 minute (35 cycles) and 72 °C for 10 min. The 

samples were electrophoresed at 80 W for 4 hours. 

Results and discussions 

Characterisation of RPP7 gene 27 

Characterization of P. parasitica isolates 

In order to determine the changes at the RPP7 locus in 

the mutant lines, Ahco-1, Ahco-2, Ahco-7, Wand-1, 

Cand-5, Hind-2 and Hind-4 isolates were used. Ahco- 

1, Ahco-2, Ahco-7 recognize MRC-B region which is 

located at the first chromosome in the Nd-1 ecotype 

(Can, 1997), and these isolates were presumed to 

recognize RPP7 allele of Nd-1 ecotype. Wand-1,


Table 1: Interaction phenotypes of different P. parasitica isolates on the A. thaliana ecotypes. 

Cand-5, Hind-2 and Hind-4 isolates recognize MRC-B 

and MRC-C region which present at the second 

chromosome in the Col-5 ecotype. The isolates 

regenerated from the oospore populations were 

inoculated on different A. thaliana ecotypes (Col-5, 

Ksk-1, Nd-1, Ws-3, Ler-1, Oy-1 and Wei-1) to 

determine if they were original. The results are shown 

in Table1. 

As it could be seen in Table 1, the isolates 

generated from the oospore populations were found to 

be as original, and there was no variation (Can, 1997). 

Therefore these isolates were used to inoculate the 

mutant lines recovered through inoculation with the 

Hiks-1 isolate. 

Interaction phenotypes on different A. thaliana ecotypes* 

P. parasitica isolates Col-5 Ksk-1 Nd-1 Ws-3 Ler-1 Oy-1 Wei-1 

Hiks-1 FN EH PN PN FN DM FDL 

Ahco-1 DM FN FDL FN FDL FN FDL 

Ahco-2 DM FN FDL FN FN FN DM 

Ahco-7 DM FN FDL FN FDL FN EH 

Wand-1 FN FN EH FN FN FN DM 

Cand-5 FN EH EH FN CN FDL DL 

Hind-2 FN FN EH PN FN FN EH 

Hind-4 FR FN EH PN EH FN EH 

*Necrotic pits (PN), necrotic flecks (FN), cavities (CN), flecks with delate and moderate pathogen sporulation, 1-20 

sporangiophorus per each cotyledon (DM), flecking with delate pathogen sporulation, 5-10 sporangiophorus per each cotyledon 

(FDL), early and heavy pathogen sporulation, 20> sporangiophores per cotyledon (EH). 

Phenotypic characterization of mutant lines 

P. parasitica isolates were used to inoculate the Col-5 

lines. The results were shown in Table 2. 

As indicated in Table 1, DM and EH phenotypes 

developed, following inoculation of FN3922, FN3928, 

FN3929 and FN3930 mutant lines with the Hiks-1 

isolate. These results revealed that the RPP7 gene is 

not present in the mutant lines. However, Ahco-1, 

Ahco-2 and Ahco-7 isolates exhibited the EH 

phenotype compared to DM in the wild Col-5 ecotype. 

This result showed that absence of the RPP7 gene 

increased susceptibility. The important point in here 

was that mutation of one R-gene could effect the 

resistance in same plant to other isolate. The result 

Table 2: Interaction phenotypes exhibited by Col-5 mutant lines following inoculation with different P. parasitica isolates. 

The interaction phenotypes on wild type and mutant lines* 

P. parasitica isolates Col-5 Nd-1 Ksk-1 FN3922 FN3928 FN3929 FN3930 

Hiks-1 FN PN EH EH DM EH EH 

Ahco-1 DM FDL FN EH EH EH EH 



Wand-1 FN EH FN EH FN FN FDL 

Cand-5 FN EH EH EH FN FN FN 

Hind-2 FN EH FN FN FN FN FN 

Hind-4 FR EH FN EH FN FN FN 

*Necrotic pits (PN), necrotic flecks (FN), flecks with moderate and late pathogen sporulation, 1-20 sporangiophorus per each 

cotyledon (DM), flecking with delate pathogen sporulation, 5-10 sporangiophorus per each cotyledon (FDL), early and heavy 

pathogen sporulation, 20> sporangiophores per cotyledon (EH).

Figure 1: Microscopic reactions occurred in Col-5 ecotype 

following inoculation with the Hiks-1 isolate. (A) Six hours 

after inoculation. a. Haustorium formed in the mesofil cells. 

b. Cell death and (B) Twelve hours after inoculation. 

Hypersensitive reaction and cell death. 

750 bp → 

500 bp → 

Figure 3: Distribution of g2a markers in mutant lines. 

M indicates the 1 kb DNA marker. 

may also show that when the RPP genes are allelic 

they do function in coordination. 

Cand-5 isolate recognizes MRC-B region which is 

present in Col-5 and determined with FN interaction 

phenotype. FN3922 mutant line exhibited the EH 

phenotype with Cand-5 and FN with other isolates 

tested (Table 2). This result may show that FN3922 

may be a Col-5 contamination. On the other word it is 

possible that in this line, many RPP genes present in 

the MRC-B loci (Can, 1997) could be mutated. 

Therefore, FN3922 may not be a specific mutant of the 

RPP7 gene. As it is known the Fast Neutron 

application results point mutations, so that at MRC-B 

region in FN3922, many point mutations or deletions 

may have been occurred. 

Inoculations of the FN3928, FN3929 and FN3930 

mutant lines with Wand-1, Hind-2, Hind-4 resulted FN 

phenotype. This results shows that the RPP7 gene has 

no correlation with the RPP genes present in seconder 

chromosome at the MRC-C region (Table 2). 

Microscopic characterization of mutant lines 

The fungal development in plant tissue was examined. 

Following inoculations at the first, third and seven 

days the cotyledons from the mutant lines and the wild 

type Col-5 were taken and prepared as described 

before. At the first six hour of inoculation of Col-5 

with the Hiks-1 isolate cell death was observed. The 

susceptible genotypes and the mutant lines allowed the 

penetration and mycelial development without any cell 

(Figure 1, 2). Fungal development in the mutant lines 

supported the macroscopic results (Mc Dowell et al., 

2000). 

DNA analyses of mutant lines 

Characterisation of RPP7 gene 29 

Figure 2: Microscopic reactions occured the FN3929 

mutant line following inoculation with the Hiks-1 isolate. 

Formation of fungal haustorium in the mesophyl cell (a) and 

hif development (b), twelve hours after inoculation. 

nga280 and g2a markers present at the lower arm of 

the first chromosome of A. thaliana were detected to 

give less recombination with the RPP7 loci (Can, 

1997). Therefore the mutant lines were subjected to 

PCR analyses with these markers. Results are given in 

Figure 3. 

The distributions of the bands in mutant lines were 

similar to those in the wild type Col-5. This result may 

suggest that no deletions may occurred in the mutant 

lines and that the loss of function could be due to a 

point mutation.


References 

Aarts N, Metz M, Holub E, Staskawicz BJ, Daniels M and 

Parker JE. Differential requirements for EDS1 and 

NDR1 by disease resistance genes define at least two R 

gene-mediated pathways in Arabidopsis. Proc Natl Acad 

Sci USA. 95: 10306-10311, 1998. 

Ausubel F, Brent R, Kingston RE, Moore DA, Seiddman JG, 

Smith JA and Struhl K. Current Protocols in Molecular 

Biology. John Wiley and Sons. 1994. 

Bent AF, Kunkel BN, Dahlbeck D, Brown KL, Schmidt R, 

Giraudat J, Leung J and Staskawicz BJ. RPS2 of 

Arabidopsis thaliana, A leucine-rich repeat class of plant 

disease resistance genes. Science. 265 (5180): 1856- 

1860, 1994. 

Bittner-Eddy PD, Crute IR, Holub EB and Beynon JL. 

RPP13 is a simple locus in Arabidopsis thaliana for 

alleles that specify downy mildew resistance to different 

avirulence determinants in Peronospora parasitica. The 

Plant Journal. 21(2): 177-188, 2000. 

Bittner-Eddy PD and Beynon JL. The Arabidopsis downy 

mildew resistance gene, RPP13-Nd, functions 

independently of NDR1 and EDS1 and does not require 

the accumulation of salicylic acid. Mol Plant-Microbe 

Interactions. 14: 416-421, 2001. 

Botella MA, Parker JE, Frost LN, Bittner-Eddy PD, 

Beynon JL, Daniels MJ, Holub EB and Jones JDG. 

Three genes of the Arabidopsis RPP1 complex 

resistance locus recognize distinct Peronospora 

parasitica avirulence determinants. Plant Cell. 10: 1847- 

1860, 1998. 

Can C. Bittner-Eddy P, Tör M, Williams K, Gunn N, Bakht S, 

Atkinson L, Debener T, Chimot P, Crute I, Beynon J and 

Holub EB. Revealing the organization of RPP loci in the 

Arabidopsis thaliana genome, Poster abstract in 6 th 

International Conference on Arabidopsis Research, 

Medison, Winconsin. 7-11 June, 1995. 

Can C. Genomic organisation of pathogen recognition genes 

in Arabidopsis thaliana to Peronospora parasitica, 

Ph. D. Thesis, University of London, Wye Collage, UK. 

1997. 

Century KS, Holub EB and Staskawicz BJ. NDR1, a locus of 

Arabidopsis thaliana that is required for disease 

resistance both a bacterial and a fungal pathogen. Proc 

Natl Acad Sci USA. 92 (14): 6597-6601, 1995. 

Dangl JL and Jones JDG. Plant pathogens and integrated 

defense responses to infection. Nature. 411: 826-833, 

2001. 

Dietrich RA, Richberg MA, Schmidt R, Dean C and 

Dangl JL. A novel zinc finger protein is encoded by the 

Arabidopsis LSD1 gene and functions as a negative 

regulator of plant cell death. Cell. 88: 685-694, 1994. 

Dong X. SA, JA, ethylene, and disease resistance in 

plants. Curr Opin Plant Biol. 1: 316-323, 1998. 

Falk A, Feys BJ, Frost LN, Jones JDG, Daniels MJ and 

Parker JE. EDS1, an essential component of R genemediated 

disease resistance in Arabidopsis has 

homology to eukaryotic lipases. Proc Natl Acad Sci 

USA. 96: 3292-3297, 1999. 

Feys BJ and Parker JE. Interplay of signaling pathways in 

plant disease resistance. Trends Genet. 16: 449-455, 

2000. 

Glazebrook J, Rogers EE and Ausubel FM. Use of 

Arabidopsis for genetic dissection of plant defense 

responses. Annu Rev Genet. 31: 547-569, 1997. 

Hammond-Kosack KE and Jones JDG. Plant disease 

resistance genes. Annu Rev Plant Physiol Plant Mol 

Biol. 48: 575-607, 1997. 

Holub EB, Beynon J and Crute I. Phenotypic and genotypic 

characterisation of interactions between isolates of 

Peronospora parasitica and accessions of Arabidopsis 

thaliana. Mol Plant-Microbe Interacts. 7 (2): 223-239, 

1994. 

Holub EB. and Beynon J. Symbiology of mouse-ear cress 

(Arabidopsis thaliana) and oomycetes. Adv Bot Res. 

24: 227-273, 1997. 

Lam E, Pontier D and Pozo O. Die and let live-programmed 

cell death in plants. Curr Opin in Plant Biol. 2: 502-507, 

1999. 

Mc-Dowel JM, Dhandaydham M, Long TA, Aarts MGM, 

Goff S, Holub EB and Dangl JL. Intragenic 

recombination and diversifying selection contribute to 

the evolution of downy mildew resistance at the RPP8 

locus of Arabidopsis. Plant Cell. 10: 1861-1874, 1998. 

McDowel JM, Cuzick A, Can C, Beynon J, Dangl JL and 

Holub EB. Downy mildew (Peronospora parasitica) 

resistance genes in Arabidopsis vary in functional 

requirements for NDR1, EDS1, NPR1 and salicylic acid 

accumulation, The Plant Journal. 22 (6): 523-529, 2000. 

Parker JE, Holub EB, Frost LN, Falk A, Gunn ND and 

Daniels MJ. Characterization of eds1, a mutation in 

Arabidopsis supressing resistance to Peronospora 

parasitica specified by several different RPP genes. 

Plant Cell. 8 (11): 2033-2046, 1996. 

Parker JE, Coleman MJ, Szabo V, Frost LN, Schmidt R, Van 

der Biezen EA, Moores T, Dean C, Daniels MJ and 

Jones JDG. The Arabidopsis downy mildew resistance 

gene RPP5 shares similarity to the Toll and Interleukin-1 

receptors with N and L6. Plant Cell. 9: 879-894, 1997. 

Richter TE and Ronald PC. The evolution of disease 

resistance genes. Plant Molecular Biology. 42: 195-204, 

2000. 

Thomas CM, Jones DA, Parniske M, Harrison K, Balint- 

Kurti P, Hatzixanyhis K and Jones JDG. Characterization 

of the tomato Cf-4 gene for resistance to Cladosporium 

fulvum identifys sequences that determine recognitional 

specificity in Cf-4 and Cf-9. Plant Cell. 9: 2209-2224, 

1997. 

Tor M, Holub EB, Brose E, Mussker R, Gunn N, Can C, 

Crute I and Beynon JL. Map positions of three loci in 

Arabidopsis thaliana associated with isolate-specific 

recognition of Peronospora parasitica (downy mildew). 

Mol Plant-Microbe Interac. 7 (2): 214-222, 1994.

Introduction 



The effect of mmeettaa-topolin on protein profile in radish cotyledons 

Serap Ça¤ 1 and Narçin Palavan-Ünsal2 * 

1Istanbul University, Department of Biology, Botany Section, Süleymaniye 34460, Istanbul-Turkey; 

2Haliç University; Department of Molecular Biology and Genetics, F›nd›kzade 34280, Istanbul-Turkey 

(* author for correspondance) 

Received 27 September 2002; Accepted 30 November 2002 

Abstract 

Meta-topolin (mT) has been established as an active aromatic cytokinin recently. The present investigation assessed 

the effects of mT on radish cotyledon growth and protein content. 0.05 to 1 mM mT increased the cotyledon growth 

about 2 fold in fresh weight basis. mT at 0.1, 0.25 and 0.5 mM concentrations caused an increase in soluble protein 

levels compared to the control cotyledons almost in the same ratio by 3 %. Compared to control cotyledons analysis 

of the soluble proteins displayed different electrophoretic pattern in mT treated cotyledons. 

KKeeyy wwoorrddss:: Cotyledon growth, meta-topolin, protein 

MMeettaa-topolinin turp kotiledonlar›nda protein profiline etkisi 

Özet 

Son y›llarda meta-topolin (mT) aktif aromatik sitokinin olarak saptand›. Bu araflt›rma da mT’in turp kotiledonlar›n›n 

büyüme ve protein içeri¤ine etkisi araflt›r›ld›. 0.05-1 mM mT kotiledon büyümesini taze a¤›rl›k baz›nda yaklafl›k 2 

kat kadar teflvik etti. 0.05, 0.1 ve 0.25 mM mT çözünür protein düzeylerini kontrole oranla yaklafl›k % 3 oran›nda 

artt›rd›. Çözünür proteinlerin analizleri, mT uygulanan kotiledonlarda kontrole oranla farkl› bir elektroforetik dizilim 

gösterdi. 

AAnnaahhttaarr ssöözzccüükklleerr:: Kotiledon büyümesi, meta-topolin, protein 

Cytokinins, N 6 -substituted adenine derivatives are a 

class of plant hormones that were first identified as 

factors that promoted cell division (Miller et al., 1955; 

1956) and have been implicated in many other aspects 

of plant growth and development including shoot 

initiation and growth, apical dominance, senescence 

and photomorphogenetic development (Letham, 

1971; Thimann, 1980; Mok and Mok, 1994). 

Although the physiological effects of cytokinins have 

been well documented, the molecular mechanisms 

underlying cytokinin action remain obscure (Mok and 

Mok, 1994; Binns, 1994). 

Bioassays are used to establish the relative 

biological activity of plant hormones compared with 

others. The cytokinin bioassays used most frequently 

depend on growth of tissues in sterile culture (Letham 

1967). Such methods are extremely sensitive but it 

needs at least 3 weeks to get final results. Letham 

(1971) described a rapid bioassay for cytokinins based 

on the ability of these compounds to promote 

markedly the expansion of radish cotyledons excised 

soon after seed germination. 

To date the effects of common cytokinins i.e. 

kinetin, benzyladenine (BA) and its riboside have been 

31

32 Serap Ça¤ and Narçin Palavan-Ünsal 

documented in radish cotyledons. A new active 

aromatic cytokinin meta-topolin (mT) have been 

determined by Strnad et al. (1997) in poplar. The 

sensitivity of the radish cotyledon bioassay to mT has 

been established by us before (Palavan-Ünsal et al., 

2002). This study will focus on the effect of mT on 

soluble protein contents in radish cotyledons that has 

not been studied before. 


Plant material and bioassay 

Radish (Raphanus sativus L.) seeds were germinated in 

darkness for 4 days at 25 °C on moist filter paper in 5 

cm petri dishes. Cotyledons were excised excluding 

petiole tissues and four cotyledons were placed in each 

petri dish after measuring the fresh weight. The 

cotyledons were placed with their adaxial sides down 

on the paper. They were incubated in a growth chamber 

at 25°C ± 2°C and 12 h light-dark photoperiods. Three 

ml mT was applied per petri dish at 0.05, 0.1, 0.25, 0.5 

and 1.0 mM concentrations. Cotyledon growth was 

measured by determining fresh and dry weights 3 days 

after the application (Letham, 1971) and the data 

presented here representative of 15 experiments. 

Measurement of soluble protein content 

Soluble protein content was determined as in Bradford 

(1976) using bovine serum albumin as standard. Each 

experiment was repeated four times and each treatment 

included three replicates. 

Electrophoresis for proteins 

Sodium dodecylsulphate (SDS)-polyacrylamide slab 

gel electrophoresis was performed according to 

Laemmli (1970). Gel containing 3.0 % (stacking gel) 

and 10.0 % (separation gel) acrylamide were prepared 

from a stock solution of 30.0 % of acrylamide and 0.8 

% N, N’-bis methylene acrylamide. The gels were 

polymerized chemically by the addition of ammonium 

persulphate. The mixture was completely dissociated 

by immersing the samples for 3 min in boiling water. 

Electrophoresis was carried out with a current of 150 V 

per gel until the bromophenol blue marker reached the 

bottom of the gel. The proteins were stained in the gel 

with Coomassie brilliant blue solution for overnight at 

room temperature. The gels were diffusion-destained 

by repeated washing in the solution containing 7.5 % 

acetic acide, 5 % methanole and 87.5 % distilled water. 

Results and discussion 

The early observations revealed that cytokinins exert 

parallel effects in maintain protein or nucleic acid 

levels while inhibiting senescence. Cytokinins 

stimulate both structural and enzymatic protein 

synthesis. They are selectively increasing the levels 

of certain enzymes associated generally with 

photosynthetic process (Feierabend, 1969). It is not 

clear whether the enhanced activity is due to greater 

synthesis, inhibition of degradation or activation of the 

enzymes. 

We already observed that new aromatic cytokinin 

mT at 0.25 to 1 mM concentration range delayed 

the senescence in excised wheat leaf segments 

(Palavan et al., 2002). This concentration range was 

high for radish cotyledon growth therefore lower 

concentrations were examined (0.05 to 1 mM) in 

addition. 

Cotyledon growth increased with the treatments of 

mT significantly (Figure 1). Stimulation of cotyledon 

growth was closely related with increasing 

concentrations of mT; 0.05 to 1 mM mT increased the 

cotyledon growth about two fold in fresh weight basis 

(p

Figure 2: The effect of meta-topolin on soluble protein content 

during the growth of radish cotyledons. Values are average 

of 4 experiments. 

Figure 3: SDS-PAGE analysis of soluble proteins from 

meta-topolin treated radish cotyledons. Gel was stained with 

Coomassie blue. Lane 1: Control, Lane 2: 0.05 mM mT, 

Lane 3: 0.1 mM mT, Lane 4: 0.25 mM mT, Lane 5: 0.5 mM 

mT, Lane 6: 1 mM mT treatments. Molecular mass (kDa) of 

markers are indicated on left hand margin. 

from cell enlargement during cotyledon growth. 

Cytokinin treatment promotes additional cell 

expansion with no increase in the dry weight of the 

treated cotyledons (Huff and Ross, 1975). 

Letham (1971) reported the ability of cytokinins to 

promote markedly the expansion of radish cotyledons 

and explained this response by the promotion of cell 

enlargement. mT also as a most active aromatic 

meta-topolin effect on protein 33 

cytokinin as reported by Strnad et al. (1997) caused to 

cotyledon growth markedly as shown in Figure 1. 

mT was found to increase the soluble protein 

contents of radish cotyledons. Treatments with 0.1, 0.25 

and 0.5 mM mT resulted an increase in soluble protein 

content in the same ratio (by 3,4 and 3 % respectively) 

compared to the control cotyledons (Figure 2). 

These findings correlated with electrophoretic 

determinations (Figure 3). Soluble proteins of mT 

treated radish cotyledons were analyzed using SDS- 

PAGE technique in order to test whether and 

significant amount of difference in protein profile 

occurred with mT treatments. Analysis of the soluble 

proteins displayed different electrophoretic pattern in 

mT treated cotyledons compared to control. Protein 

bands were very sharp and dark in 0.05, 0.1 and 0.25 

mM mT treated samples and their molecular masses 

ranges between 66 to 45 kDa’s. Molecular mass of 45 

to 29 kDa’s were weak in 0.5 and 1 mM mT treated 

and in control cotyledons also. On the other hand 

protein bands were very sharp and dark in 0.05, 0.1 

and 0.25 mM mT treated cotyledons comparing with 

0.5 and 1 mM mT treated and control cotyledons. 

Obvious bands were also observed around 30 kDa in 

cotyledons treated with 0.05, 0.1 and 0.25 mM mT. 

Besides these there were additional bands in mT 

treated samples different from controls and these 

bands were weak in 0.5 and 1.0 mM mT treated 

samples compared to the other applications around 24 

kDa. 

There is good evidence that cytokinins play a role 

in regulating protein synthesis (Tepfer and Fosket, 

1978). Cytokinins can not only increase the rate of 

protein synthesis, but also change the spectrum of 

proteins produced by plant tissues. 

Results obtained in this study showed that, total 

soluble protein content in radish cotyledons not 

effected from exogenously applied mT. On the other 

hand, when protein profile was examined 

electrophoretically additional bands were observed in 

mT treated samples. These can be explained by the fact 

that mT stimulate new protein synthesis without 

effecting total protein content. 

In conclusion, natural aromatic cytokinin mT has 

an important role in the control of cotyledon growth 

and this response closely associated with protein 

profile. The results of this research are exhibited mTas 

a promising plant growth regulators in physiological 

studies.

34 Serap Ça¤ and Narçin Palavan-Ünsal 

Acknowledgement 

We thank to Dr. M. Strnad and his colleagues for the 

generous gift of aromatic cytokinins and to Damla 

Büyüktunçer for technical assistance. This study was 

supported by Istanbul University Research Fund 

(Project number: B-430/13042000). 

References 

Binns AN. Cytokinin accumulation and action: biochemical, 

genetic and molecular approaches. Ann Rev Plant 

Physiol Plant Mol Biol. 45: 173-196,1994. 

Bradford AM. A rapid and sensitive method for the 

quantification of microgram quantities of protein 

utilizing the principle of protein-dye binding. Anal 

Biochem. 72: 248-254, 1976. 

Feierabend J. Der Einfluss von Cytokinin auf die Bildung 

von Photosyntheseenzyme im Roggenkeimlingen. 

Planta. 84: 11-29, 1969. 

Huff AK, Ross CW. Promotion of radish cotyledon 

enlargement and reducing sugar content by zeatin and 

red light. Plant Physiol. 56: 429-433, 1975. 

Laemmli UK. Cleavage of structural proteins during the 

assembly of the head of bacteriophage T4. Nature. 

227: 680-685, 1970. 

Letham DS. Chemistry and physiology of kinetin-like 

compounds. Ann Rev Plant Physiol. 18: 349-364, 1967. 

Letham DS. Regulators of cell division in plant tissues. XII. 

A cytokinin bioassay using excised radish cotyledons. 

Physiol Plant. 25: 391-396, 1971. 

Miller CO, Skoog F, Von Saltza, MH, Strong F. Kinetin a cell 

division factor from deoxyribonucleic acid. J Am Chem 

Soc. 77: 1392-1293, 1955. 

Miller CO, Skoog F, Okomura FS, von Saltza MH, 

Strong FM. Isolation, structure and synthesis of kinetin a 

substance promoting cell division. J Am Chem Soc. 

78: 1345-1350, 1956. 

Mok DWS, Mok MC. Cytokinins: Chemistry, Activity and 

Function. CRC Press, Boca Raton. 1994. 

Palavan-Ünsal N, Ça¤ S, Çetin E. Growth responses of 

excised radish cotyledons to meta-topolin. Canadian J 

Plant Sci. 82: 191-194, 2002. 

Strnad M, Hanus J, Vanek T, Kaminek M, Ballantine JA, 

Fussell B, Hanke DE. Meta-topolin, a highly active 

aromatic cytokinin from poplar leaves (Populus x 

canadensis Moench., cv. Robusta). Phytochemistry. 

45: 213-218, 1997. 

Tepfer DA, Fosket DE. Hormone-mediated translational 

control of protein synthesis in cultured cells of Glycine 

max. Dev Biol. 62: 486-497, 1978. 

Thimann KV. Senescence in Plants. 85-115. CRC Press, 

Boca Raton. 1980.




Serkan ‹fller 1 and Günhan Erdem 2 * 

1 Department of Biology, Institute of Applied Sciences, Çanakkale Onsekiz Mart University, Çanakkale, 

Turkey; 2 College of Health, Çanakkale Onsekiz Mart University, Çanakkale, Turkey (*author for 

correspondence) 

Received 30 September 2002; Accepted 26 December 2002 

Abstract 

Many recent studies have focused on the investigation of the biological effects of electromagnetic field. Although 

the several types of biological effects of electromagnetic fields have been shown, the molecular mechanisms of these 

effects have not been explained yet. Some epidemiological studies have suggested that exposure to ambient, lowlevel 

50-60 Hz electromagnetic fields increase risk of disease including cancer such as leukemia among children who 

live close to power lines or among men whose jobs expose them to electromagnetic field, while others have 

suggested that electromagnetic fields exposure could increase both the concentration of free radicals and oscillating 

free radicals. Electromagnetic fields are known to affect radical pair recombination and they may increase the 

concentration of oxygen free radicals in living cells. In this study, oxidative stress was formed by the oxidation of 

ascorbic acid and the effect of 50 Hz, 0.3 mT electromagnetic fields on the oxidative DNA damage has been 

investigated. The results of the study showed that extremely low-frequency electromagnetic fields enhanced the 

effect of oxidative stress on DNA damage and supported the idea obtained from the previous studies on an increasing 

effect of electromagnetic fields on the concentration and the life-time of free radicals. 

KKeeyy wwoorrddss:: Electromagnetic fields, DNA damage, ascorbic acid, vitamin C, oxidative stress 


Özet 

Günümüzdeki birçok çal›flma, elektromanyetik alan›n biyolojik etkilerinin araflt›r›lmas› üzerinde odaklanm›flt›r. 

Elektromanyetik alan›n biyolojik etkilerinin baz› türlerinin gösterilmifl olmas›na ra¤men, bu etkilerin moleküler 

mekanizmalar› henüz aç›klanamam›flt›r. Baz› epidemiyolojik çal›flmalar, 50-60 Hz dolay›ndaki düflük düzeyli 

elektromanyetik alana maruz kalman›n yüksek gerilim hatlar›na yak›n yaflamakta olan çocuklarda veya 

elektromanyetik alana maruz kalarak çal›flanlarda görülen lösemi gibi kanser vakalar›n› kapsayan hastal›klara iliflkin 

riski art›rd›¤›n› öne sürerken, baz› çal›flmalar ise elektromanyetik alan maruziyetinin serbest radikal 

konsantrasyonunu ve serbest radikallerin izlenebilirli¤ini art›rabilece¤ini ileri sürmüfltür. Elektromanyetik alan›n 

radikal çifti rekombinasyonunu etkiledi¤i bilinmektedir ve bu da, hücrelerdeki oksijene dayal› serbest radikal 

konsantrasyonunu art›rabilir. Bu çal›flmada, askorbik asit oksidasyonu ile oksidatif stres oluflturulmufl ve 50 Hz, 0.3 

mT düzeyindeki elektromanyetik alan›n, oksidatif DNA hasar› üzerindeki etkisi araflt›r›lm›flt›r. Bu çal›flman›n 

sonuçlar›, oldukça düflük frekansl› elektromanyetik alan›n, oksidatif stresin DNA hasar› üzerindeki etkisini art›rd›¤›n› 

göstermifl ve önceki araflt›rmalardan elde edilen, elektromanyetik alan›n serbest radikal konsantrasyonu ve yar› ömrü 

üzerindeki art›r›c› etkisine dair düflünceleri desteklemifltir. 

AAnnaahhttaarr ssöözzccüükklleerr:: Elektromanyetik alan, DNA hasar›, askorbik asit, C vitamini, oksidatif stres 

35

36 Serkan ‹fller and Günhan Erdem 

Introduction 

There are many reports on the biological effects of 

electromagnetic fields (EMF) and there have been 

many attempts to develop a theoretical explanation of 

this phenomenon. Some epidemiological studies have 

suggested that exposure to ambient, low-level 50/60 

Hz EMF increases risk of disease including cancer 

such as leukemia among children who live close to 

power lines or among men whose jobs expose them to 

EMF (Wertheimer and Leeper, 1979; Tomenius, 1986; 

Savitz et al., 1988; London et al., 1991). EMF firstly 

affects the cell membrane. Some ion channels such as 

Na-K ATPase have been affected according the level 

of EMF. The alteration in the activity of these proteins 

causes an increasing or decreasing intracellular 

concentration of many ions such as Na + , K + , Mg 2+ and 

Ca 2+ which plays very important roles in cell signaling. 

Therefore, the biological effects of EMF expand 

among the cellular systems (Goodman et al., 1995). 

Although the several types of biological effects of 

EMF have been shown, the molecular mechanisms of 

these effects have not been explained yet. Some 

studies have suggested that EMF exposure could be 

due to both the increase in the concentration (Jajte, 

2000) and oscillating of free radicals (Scaiano et al., 

1995). EMF is known to affect radical pair 

recombination and they may increase the 

concentration of oxygen free radicals in living cells 

(Jajte, 2000). Increasing the concentration of free 

radicals creates oxidative stress and some biological 

reactions such as DNA damage occur under this 

condition. Metabolic energy production or effects of 

chemicals and radiation can form oxidative stress. 

In this study, oxidative stress was formed by the 

oxidation of ascorbic acid with Cu 2+ ions and the effect 

of 50 Hz, 0.3 mT EMF on the oxidative DNA damage 

was investigated. 

Materials and methods 

DNA isolation 

High molecular weight (app. 10 kb) human genomic 

DNA was isolated from the white blood cells with the 

modified method of Poncz et al. (1982) by using MBI 

Fermentas genomic DNA isolation system. Molecular 

weight and purity of DNA samples were controlled by 

agarose gel electrophoresis. The concentration of 

DNA samples was spectrophotometrically determined. 

All DNA samples were free from proteins, RNAs and 

solvents used for extraction. 

Oxidative DNA cleavage reactions 

Cleavage reactions were carried out in a medium 

containing 0.5 µg DNA, 20 mM Tris-HCl (Sigma) pH 

7.8, 0.25 mM ultra-pure ascorbic acid (Merck) and 

CuCl2 (Sigma) in the final concentrations of 2.5, 5, 7.5 

and 10 µM, in a final volume 10 µl. Other antioxidants 

(glutathion, cystein and dithiothreitol, Sigma) and 

metal chelator (EDTA, Merck) were added to the 

reaction mixtures at a final concentration of 0.5 mM. 

The mixtures were incubated at room temperature for 

10, 20 and 30 minutes. Adding EDTA at a final 

concentration of 25 mM stopped reactions. DNA 

cleavages in the reaction mixtures were analyzed on 

the 1% agarose gel (Promega) electrophoresis. 

EMF exposure system 

Electromagnetic fields were applied by using the 

Helmholtz coil. The coil system was constructed by 

using the polyester sphere that was surrounded by 

copper wire with 0.75 cm diameter (Galt et al., 1995). 

The diameter and height of the sphere were 16 and 26 

cm, respectively. 50 Hz, 4.5 V electricity was applied 

to coil system. As a result, 0.3 mT EMF was generated 

at the center of the coil system which includes handles 

for the sample tubes. 

Results and discussion 

The oxidative DNA damage was induced by the 

concentration of cupric ions (Figure 1). In the constant 

ascorbate concentration (0.25 mM), oxidative DNA 

breakage was started in the presence of 2.5 µM 

copper(II) ions and EMF also induced the DNA 

breakage at this condition (Figure 1, lanes 4 and 10). 

Therefore, main DNA band in the lane 10 of Figure 1 

is thinner than the lane 4. In the presence of high 

cupric ions concentration, excess scission of DNA 

molecules occurred at the EMF when compared to 

normal conditions (Figure 1, lanes 6 and 12). 

Electromagnetic fields did not have an effect on the 

oxidation of ascorbic acid in the absence of cupric ions 

(specific data was not shown, but the sample in Figure 

3, lane 12 had reflected this result, because EDTA was

1 2 3 4 5 6 7 8 9 10 11 12 

Figure 1: The effect of EMF and Cu(II) concentrations on 

oxidative DNA damage. All lanes include 0.5 µg DNA. The 

DNA samples in the lanes from 1 to 6 were incubated under 

normal condition, 7 to 12 were incubated in EMF at room 

temperature in the presence of 0.25 mM ascorbic acid 

except the 1 and 7 which were control lanes. Cu(II) 

concentrations were 1.25 µM in 2 and 8, 2.5 µM in 3 and 9, 

5 µM in 4 and 10, 7.5 µM in 5 and 11, 10 µM in 6 and 12 

lanes. Incubation time was 30 min for all samples. 

chelating cupric ions and eliminated their oxidative 

effects). 

The results of this study showed that the oxidative 

DNA damage depends on the incubation time (Figure 2). 

DNA breakages could be observed at the 20 th minute of 

incubation time (Figure 2, lanes 6 and 8). EMF 

exposure enhances the oxidative DNA damage after 

the 20 th minute (Figure 2, lanes 2 and 4). 

1 2 3 4 5 6 7 8 9 10 11 12 

Figure 3: The effect of some antioxidants (glutathione, 

cystein, dithiothreitol) and metal chelator (EDTA) on 

excessive oxidative DNA damage in EMF. All lanes include 

0.5 µg DNA. Ascorbic acid and Cu(II) concentrations were 

0.25 mM and 7.5 µM in all lanes except the control DNA 

lanes 1 and 7, respectively. Lanes 2 and 8 were scission 

controls. Glutathione (3 and 9), cystein (4 and 10), 

dithiothreitol (5 and 11) and EDTA (6 and 12) 

concentrations were 0.5 mM. The samples in 1 to 6 were 

incubated at normal condition. The others were incubated in 

EMF. Incubation time was 30 min for all samples. 

EMF and oxidative DNA damage 37 

1 2 3 4 5 6 7 8 9 10 11 12 

Figure 2: The effect of incubation time on oxidative DNA 

damage depends in electromagnetic fields. All lanes include 

0.5 µg DNA. Incubation times were 30 min from 1 to 4, 20 

min from 5 to 8 and 10 min from 9 to 12. Ascorbic acid 

concentration were 0.25 mM in all lanes. Cu(II) 

concentrations were 2.5 µM in 1, 3, 5, 7, 9 and 11 while 

5 µM in 2, 4, 6, 8, 10 and 12. The samples in 1, 2, 5, 6, 9 and 

10 were incubated in EMF. Other samples were incubated at 

normal conditions. 

In the presence of EDTA as a cationic metal 

chelator, oxidative DNA damage was not observed. 

This result showed that ascorbate oxidation and 

oxidative DNA damage depend on cupric ions as an 

oxidizing agent (Figure 3, lanes 6 and 12). As an 

antioxidant, cystein did not block the oxidative DNA 

damage (Figure 3, lanes 4 and 10). Glutathione 

reduced the oxidative stress. Therefore, the DNA 

damage was formed as aggregation rather than 

fragmentation in the presence of glutathione (Figure 3, 

lanes 3 and 9). Dithiotreitol (DTT) was the most 

effective antioxidant of all investigated but EMF 

exposure inhibited the effectiveness of DTT (Figure 3, 

lanes 5 and 11). 

The oxidative species produced by ascorbate 

oxidation in the presence of copper(II) ions damage 

the DNA molecules (Figure 1). Previously DNA 

damage depending on ascorbate oxidation had been 

studied (Erdem et al., 1994; Zareie et al., 1996). 

Oxidative DNA damage was observed as 

fragmentation or aggregation. The degree of oxidative 

DNA damage varies in the levels and reactivity of free 

radicals produced in the reaction medium. In the 

presence of oxygen, the hydroxyl and peroxyl radicals 

such as superoxide anion and hydroperoxyl radical are 

produced by the reaction between radical form of 

ascorbic acid (ascorbyl radical) and molecular oxygen 

(Fuchs et al., 1990). 

These radicals attack to electrophilic nuclei on the 

targets and create secondary carbon radicals. At the

38 Serkan ‹fller and Günhan Erdem 

high level or high reactivity of these radicals, excess 

formation of secondary carbon radicals on the same 

DNA molecule causes a reaction between each other 

and then the DNA damage occurs as fragmentation. 

Therefore, DNA size became smaller and gave the 

smeared patterns on gel electrophoresis (lanes 5 and 6 

in Figure 1) However, at the low level or low reactivity 

of oxygen species, the oxidative DNA damage results 

in aggregation of the DNA molecules with the 

intermolecular reaction of the secondary carbon 

radicals. Thus, the DNA samples became heavier and 

were retarded on the gel electrophoresis (lanes 3 and 9 

in Figure 3). 

Our results showed that extremely low-frequency 

EMF enhanced the effect of oxidative stress on DNA 

damage and supported the idea obtained from previous 

studies on an increasing effect of EMF on the 

concentration and the life-time of free radicals (Jajte, 

2000; Scaiano et al., 1995; Jajte and ZmySlony, 2000). 

Especially the comparisons of lane 2 to lane 4 in 

Figure 2 and lane 5 to lane 11 in Figure 3, indicate that 

the degree of the oxidative stress under the EMF is 

greater than the normal condition. 

In the brain cells of rats, an increase in DNA 

single- and double-strand breaks had been found after 

acute exposure to a sinusoidal 60 Hz magnetic field. 

When the experiment was carried out in the presence 

of melatonin or a radical scavenger compound N-tertbutyl-alpha-phenylnitrone 

(PBN), the effect of 

magnetic fields on brain cell DNA was not observed 

(Lai and Singh, 1997). Melatonin is a neurohormone 

and it is also an antioxidant and a free radical 

scavenger. Therefore, this hormone could protect 

biological systems against oxidative damage. The 

increasing effect of EMF on the concentration of free 

radicals has been suggested that melatonin suppression 

in humans may increase the probability of mutagenic 

and carcinogenic risk (Jajte and ZmySlony, 2000). 

EMF (≥1 mT) increases the concentration of free 

radicals that escape from the alkyl sulphate and 

sulphonate micelles. The effect of extremely lowfrequency 

EMF on the radicals formed from singlet 

precursors is larger than triplet precursors. Some 

radicals such as hydroxyl and peroxyl radicals 

generated in the biological reactions are formed from 

singlet precursors (Eveson et al., 2000). 

In conclusion, the results obtained from our study 

suggest that the effects of extra low frequency EMF on 

the concentration of free radicals and the 

recombination of radical pairs might trigger the 

carcinogenesis in the populations living close to the 

overhead electric power distribution lines. 

References 

Erdem G, Öner C, Önal AM, K›sakürek D and Ö¤üfl A. Free 

radical mediated interaction of ascorbic acid and 

ascorbate/Cu(II) with viral and plasmid DNAs. J Biosci. 

19: 9-17, 1994. 

Eveson RW, Timmel CR, Brocklehurst B, Hore PJ and 

McLauchland KA. The effects of weak magnetic fields 

on radical recombination reactions in micelles. Int J 

Radiation Biol. 76: 1509-1522, 2000. 

Fuchs J, Mehlhron RJ and Packer L. Assay for free radical 

reductase activity in biological tissue by electron spin 

resonance spectroscopy. Methods in Enzymology. 

186: 670-674, 1990. 

Galt S, Whalstrom J, Hamnerius Y, Holmqvist D and 

Johannesson T. Study of effects of 50 Hz magnetic fields 

on chromosome aberration and growth-related enzyme 

ODC in human amniotic cells. Bioelectrochemistry and 

Bioenergetics. 36: 1-8, 1995. 

Goodman EM, Greenebaum B and Marron MT. Effects of 

electromagnetic fields on molecules and cells. In: Int 

Rew Cytology, A Survey of Cell Biology. Jean KW and 

Jarvik J (Ed). Academic Press. 158: 279-338, 1995. 

Jajte J and ZmySlony M. The role of melatonin in the 

molecular mechanism of weak, static and extremely low 

frequency (50 Hz) magnetic fields (ELF). Medycyna 

Pracy. 51: 51-57, 2000. 

Jajte JM. Programmed cell death as a biological function of 

electromagnetic fields at a frequency of (50/60 Hz). 

Medycyna Pracy. 51: 383-389, 2000. 

Lai H and Singh NP. Melatonin and N-tert-butyl-alphaphenylnitrone 

block 60-Hz magnetic field-induced DNA 

single and double strand breaks in rat brain cells. 

J Pineal Res. 22: 152-62, 1997. 

London SJ, Thomas DC, Bowman JD, Sobel E, Cheng TC 

and Peters JM. Exposure to residential electric and 

magnetic fields and risk of childhood leukemia. Am J 

Epidemiol. 134: 923-37, 1991. 

Poncz M, Solowiejczyk D, Harpel B, Mory Y, Schwartz E 

and Surrey S. Construction of human gene libraries from 

small amounts of peripheral blood: Analysis of ß-like 

globin genes. Hemoglobin. 6: 27-36, 1982. 

Savitz DA, Wachtel H, Barnes FA, John EM and Tvrdik JG. 

Case-control study of childhood cancer and exposure to 

60-Hz magnetic fields. Am J Epidemiol. 128: 21-38, 1988. 

Scaiano JC, Cozens FL and Mohtat N. Development of a 

model and application of the radical pair mechanism to 

radicals in micelles. Photochemistry and Photobiology. 

62: 818-829, 1995. 

Tomenius L. 50-Hz electromagnetic environment and the 

incidence of childhood tumors in Stockholm County. 

Bioelectromagnetics. 7: 191-207, 1986. 

Wertheimer N and Leeper E. Electrical wiring configurations 

and childhood cancer. Am J Epidemiol. 109: 273-284, 

1979. 

Zareie MH, Erdem G, Öner C, Öner R, Ö¤üfl A and Piflkin E. 

Investigation of ascorbate-Cu (II) induced cleavage of 

DNA by scanning tunneling microscopy. Int J Biol 

Macromol. 19: 69-73, 1996.



Chromosomes of a balanced translocation case evaluated with 

atomic force microscopy 

Zerrin Y›lmaz 1 *, Mehmet Ali Ergun 2 , Erdal Tan 3 

1 Department of Medical Biology and Genetics, Baskent University, Faculty of Medicine, 06570, 

Maltepe, Ankara, Turkey; 2 Department of Medical Biology and Genetics, Gazi University, Faculty of 

Medicine, 06510, Besevler, Ankara, Turkey; 3 Materials Research Department, Ankara Nuclear Research 

and Training Center, 06100, Besevler, Ankara, Turkey (*author for correspondence) 

Received 2 December 2002; Accepted 30 December 2002 

Abstract 

A couple was referred to our genetics department for cytogenetic analysis because of two previous abortions. The 

cytogenetic analysis of the male was found as 46, XY and the female revealed a balanced translocation; 46, XX,t 

(7;12) (p21;q14) and also she had 14 cenh+ as her mother. Atomic force microscopy (AFM) is a useful method for 

detecting detailed structures of chromosomes. With the help of this new technique the surface topography of human 

chromosomes can be examined. We used AFM in order to analyse the surface topography of derivative chromosomes 

of the patients, and found a 0.6 µm gap region. In this study, we aimed to examine the differences between the images 

of the derivative chromosomes detected by light and atomic force microscopy analyses. 

KKeeyy wwoorrddss:: Balanced translocation, chromosome polymorphism, atomic force microscopy 



Özet 

Ardarda iki gebelik kayb› nedeniyle departman›m›za yönlendirilen çiftin sitogenetik analizleri yap›lm›flt›r. Erkekte 

normal kromozom kuruluflu 46, XY saptanm›fl ancak kad›nda dengeli translokasyonla birlikte 

14. kromozoma ait sentromer art›fl› saptanm›flt›r; 46, XX, t (7;12) (p21;q14), 14 cenh+. Proband›n ailesinde yap›lan 

sitogenetik çal›flma ile ayn› kromozom kuruluflunun proband›n annesinden kal›t›ld›¤› saptanm›flt›r. Atomik güç 

mikroskobu kromozomlar›n yap›sal olarak detayl› incelenmesinde kullan›lmaktad›r. Bu yeni tekni¤in yard›m›yla 

insan kromozomlar›n›n yüzey topografisi incelenebilmektedir. Biz de atomik güç mikroskobunu kullanarak derivatif 

kromozomun yüzey topografisini araflt›rd›k ve 0.6 µm’lik bir gap bölgesi saptad›k. Bu çal›flmada probanda ait 

derivatif kromozom yap›s›n› hem ›fl›k mikroskobu hem de atomik güç mikroskobu ile ayr› ayr› de¤erlendirerek 

sonuçlar›m›z› karfl›laflt›rd›k. 

AAnnaahhttaarr ssöözzccüükklleerr:: Dengeli translokasyon, kromozom polimorfizmi, atomik güç mikroskobu 

Introduction 

Cytogenetics is the study of genetic material at the 

cellular level. Human cytogenetics is almost always 

concerned with light microscope studies of 

chromosomes. Staining procedures which provide a 

39

40 Zerrin Y›lmaz et al. 

uniform unbanded appearance to chromosomes are 

referred to solid or covential staining. They can, 

however, be useful for studies on chromosome 

breakage as scoring gaps and breaks can be difficult in 

lightly stained chromosome bands. Giemsa banding 

(G-banding) has become the most widely used 

technique for the routine staining of human 

chromosomes. The chromosome banding patterns 

obtained reflect both the structural and functional 

composition of chromosomes. Consititutive 

heterochromatin is the structural chromosomal 

material seen as dark staining material in interphase as 

well as during mitosis. It includes both repetetive 

DNA, satellit DNA and some non-repetetive DNA. 

C-banding can be used to demonstrate the repetetive 

DNA (Benn and Perle, 1992). 

Atomic force microscopy (AFM) is a diagnostic 

tool for detecting detailed structures of the 

chromosomes and the surface topography of human 

chromosomes can be examined using this new 

technique (Binning et al., 1986; Musio et al., 1997). 

AFM could be considered as a tool for further 

chromosomal studies. 

In our previous studies using AFM, we showed 

that, unbanded human metaphase chromosomes 

displayed a banding pattern similar to G-bands, and for 

the first time we have provided an AFM imaging of 

chromosomes in trisomy 13, 21 and Klinefelter 

Syndrome patients (Ergun et al., 1999). Besides, G and 

C-banding patterns of chromosomes were also 

investigated (Sahin et al., 2000; Tan et al., 2001). 

In this study, we used AFM in order to analyse the 

surface topography of derivative chromosomes of a 

female patient whose daughter was referred to our 

Genetics department with the chief complaints of 

abortions. 

Materials and methods 

Case presentation 

In this study we evaluated the chromosomes of a 

family. This family was referred to our genetics 

department for cytogenetic analysis because of two 

previous abortions during the first trimester. They had 

no live-born children after a marriage of 5 years. The 

male was 37 years old and healthy, and his nonconsanguineous 

wife was 36 years old. Her physical 

examination revealed no abnormalities in 

genitourinary, endocrinological and other organ 

systems; also laboratory findings were normal. 

Light microscopy analysis 

Metaphase chromosome preparation was obtained 

from peripheral blood lymphocytes using standard 

techniques (Verma and Babu, 1995). Conventional 

cytogenetic analysis was carried out using GTGbanding 

and C-banding techniques (Benn and Perle, 

1992). The chromosome images were captured by 

computer imaging (Cytovision system, Image 

analysis, Applied Imaging, Saunderland, UK). 

For each patient we analysed 20 metaphases, and 

C-banding procedure was performed while 

investigating 14 cenh+. 

Atomic force microscopy and analysis 

The AFM used in this study was TopoMetrix 

TMX2000 Explorer, operating in contact mode and air. 

Throughout the surface analysis, we have used 

standard pyramidal tip (1520-00) with the radius of 

curvature of approximately 1000 A°. During the 

surface analysis, the metaphase region was primarily 

determined and addressed by light microscopy. Later, 

the region under consideration was scanned via AFM 

at various scan ranges changing from 150 µm down to 

10 µm or less to image the chromosomes in a good 

manner. The applied force and the image resolution 

were between 1 and 3 nN and 400x400 pixels (or 

higher) respectively for each image acquisition. The 

raw data gathered were analysed by using the software 

of the microscopy system in two or three-dimensional 

patterns. 

In our study, the chromosomes of the patient were 

spread on glass surface. Then, the metaphase spreads 

were analysed by AFM. Line measure analysis was 

performed on derivative chromosomes. 

Results 

The karyotype of the male revealed 46, XY, while the 

cytogenetic analysis of his wife was karyotyped as 

46, XX, t (7;12) (p 21;q 14); a balanced translocation. 

She also had 14cenh+. In order to understand the origin 

of this translocation chromosome, her mother was 

karyotyped and she was also found to be a translocation 

carrier; 46, XX, t (7;12) (p 21; q14) and 14 cenh+.

Figure 1: The partial karyotype of the daughter (a) and the 

mother (b); derivative chromosomes 7 and 12 and 

chromosomes 14 with C-banding images are shown. 

Figure 2: AFM image of derivative chromosome 7 and line 

measure analysis of the gap region. 

The karyotypes of the mother and the daughter 

were similar; 46, XX, t (7;12) (p 21; q 14). The partial 

karyotype of the daughter (Figure 1a) and the mother 

(Figure 1b) with the derivative chromosomes 7, 12 and 

chromosomes 14 with C-banding images are shown. 

We performed detailed measurements on the 

derivative chromosomes of the mother and detected 

the attached chromosome fragment from derivative 

chromosome 12 to the derivative chromosome 7. Our 

measurements revealed a 0.6 µm gap region (Figure 2). 

Besides we also analysed the satellite region of 

chromosome 14 (Figure 3) of the mother. 

Discussion 

AFM in chromosome evaluation 41 

Figure 3: AFM image of chromosome 14 indicated by an 

arrow. 

In this study we evaluated the detailed structures of 

translocated chromosomes of the mother with AFM. 

Line measure analysis revealed a gap region on the 

derivative chromosome 7, which was measured as 0.6 

µm. It was equivalent to a mid-sized G-band region 

(Figure 2). 

In the previous studies, it was reported that these 

gap regions correspond to narrowing and grooving 

regions on chromosomes, and are considered as 

negative G- band regions (Uehara et al., 1996). The 

gap regions were also observed in fragile X (Harrison 

et al., 1983) and in radiation exposed chromosomes 

(Mullinger and Johnson, 1987). It was thought that, 

these regions correspond to high-order structural 

aberrations resulting from an incomplete or irregular 

composition of chromatid fibres induced by a 

translocation of a chromosomal fragment. The gap 

regions were the results of chromosomal 

rearrangements (Uehara et al., 1996). 

Our second analysis was on the satellite region of 

the chromosome 14 of the mother. First of all, GTGbanding 

revealed an increase in the heterochromatin 

region of the short arm of chromosome 14. Then, we 

performed C-banding procedure to understand if this 

region was belonging to a constitutive heterochromatin 

region, or to an extra banding region. The results of 

C-banding confirmed that these regions were 

belonging to heterochromatin region. Our 3dimensional 

AFM analysis for the satellite region 

showed an augmentation on the short arm of this 

chromosome (Figure 3). These heterochromatin 

regions are polymorphic regions, and they are highly

42 Zerrin Y›lmaz et al. 

repetitive regions that are located on the centromeres 

of chromosomes 1, 9, and 16 and on the distal arm of 

Y chromosome (Burkholder and Duczek, 1980; Cook, 

1995). Our AFM image also helps us to understand 

that these regions were not belonging to G- banding 

regions, as there was not a banding pattern (Tan et al., 

2001). 

AFM can be considered as a novel technique for 

analysing detailed structures of chromosomes for its 

line measure analysis and 3-D image capture 

capabilities. Reflecting these capabilities, AFM helped 

us to investigate the gap region on the derivative 

chromosome and this study is also novel by making 

new implementations on the mechanism of 

translocation. As a conclusion, the capability of AFM 

for detecting chromosomal abnormalities will reflect 

light into further studies. 

References 

Benn PA and Perle MA. Chromosome staining and banding. 

In: Human Cyotgenetics. A Practical Approach. 

Rooney DE and Czepulowski BH (Ed). New York. 

Oxford University Press. 1: 91-118, 1992. 

Binning G, Rohrer H and Gerber C. Atomic force 

microscopy. Phys Rev Lett. 56: 930- 933, 1986. 

Burkholder GD and Duczek LL. Proteins in chromosome 

banding. II. Effect of R- and C-banding treatments on the 

proteins of isolated nuclei. Chromosoma. 79: 43-51, 1980. 

Cook PR. A chromomeric model for nuclear and 

chromosome structure. Journal of Cell Science. 

108: 2927-2935, 1995. 

Ergun MA, Tan E, Sahin FI and Menevse A. Numerical 

chromosome abnormalities detected by atomic force 

microscopy. Scanning. 21: 182-186, 1999. 

Harrison CJ, Jack EM, Allen TD and Harris R. The fragile 

X: A scanning electron microscope study. J Med Genet. 

20: 280-5, 1983. 

Mullinger AM and Johnson RT. Scanning electron 

microscope analysis of structural changes and 

aberrations in human chromosomes associated with the 

inhibition and reversal of inhibition of ultraviolet light 

induced DNA repair. Chromosoma. 96: 39-44, 1987. 

Musio A, Mariani T, Frediani C, Ascoli C and Sbrana I. 

Atomic force microscopy imaging of chromosome 

structure during G-banding treatments. Genome. 

40: 127-131, 1997. 

Sahin FI, Ergun MA, Tan E and Menevse A. The mechanism 

of G- banding detected by atomic force microscopy. 

Scanning. 22: 24-27, 2000. 

Tan E, Sahin FI, Ergun MA, Ercan I and Menevse A. 

C-banding visualised by AFM. Scanning. 23: 32-35, 

2001. 

Uehara S, Sasaki H, Takabayashi T and Yajima A. Structural 

aberrations of metaphase derivative chromosomes from 

reciprocal translocations as revealed by scanning 

electron microscopy. Cytogenet Cell Genet. 74: 76-79, 

1996. 

Verma RS and Babu A Banding techniques. In: Human 

Chromosomes Principles and Techniques. Verma RS and 

Babu A (Ed). McGraw-Hill Inc. New York. 72-133,1995.




Gül Özcan Ar›can* and Mehmet Topçul 

‹stanbul University, Faculty of Science, Department of Biology, 34459 Vezneciler, ‹stanbul, Turkey 

(*author for correspondence) 

Received 9 December 2002; Accepted 30 December 2002 

Abstract 

Cancer chemotherapy is an additional application to surgical operations and radiotherapy in the treatment of 

widespread tumors. An anthracycline-derived antibiotic, epirubicin (EPI) is one of the clinically used antineoplastic 

drugs. In this study the cytotoxic effects of EPI in transformed mouse fibroblasts (L-cell) were examined. EPI 

concentrations of 0.001 µg/ml, 0.01 µg/ml and 0.1 µg/ml were applied to the cells for 2, 4, 8, 16 and 32 hours. The 

results showed that EPI diminished mitotic index of L-cells depends upon time and applied concentrations. This 

decrease was found statistically significant in each treatment group when compared to control (p

44 Gül Özcan Ar›can and Mehmet Topçul 

Skladonowski and Konopa, 1994). In addition, 

topoisomerase-II has also been shown to be inactivated 

by EPI (Robert and Gianni, 1993; Haldane et al., 

1993). 

In vitro studies showed that EPI possesses 

cytotoxicity at least equivalent to that of doxorubicin 

against a variety of animal and human tumor cell lines 

including those derived from breast, liver, lung, 

gastric, colorectal, squamous cell, cervical, bladder, 

ovarian carcinomas, neuroblastoma and leukaemia 

(Bagnara et al., 1987; Zhang et al., 1992; Bartkowiak 

et al., 1992). 

EPI is a cell cycle phase non-specific 

anthracycline, with maximal cytotoxic effects in the S 

and G2 phases. Preliminary in vitro studies were 

carried out on HeLa cells. The first tests demonstrated 

that EPI and doxorubicin gave essentially the same 

inhibition of HeLa cell colony formation (Di marco et 

al., 1976). Similarly, EPI was as active as doxorubicin 

on mouse embryo fibroblast proliferation (Di marco et 

al., 1977), but was taken up in greater amount than 

doxorubicin by L1210 leukemia cells in vitro (Wilson 

et al., 1981). 

There have been few studies about the effect of EPI 

on mitotic index of rapidly proliferating cells. In this 

study, we have therefore studied the effect at EPI, 

employed in the concentrations of 0.001 µg/ml, 

0.01 µg/ml and 0.1 µg/ml for a period of 2 to 32 hours, 

on proliferation of transformed L-cells in culture 

which was investigated by measuring mitotic index in 

order to investigate the effectiveness of this drug in 

chemotherapy. 


Chemical 

EPI (4’-epidoxorubicin), an anthracycline antibiotic, is 

a doxorubicin stereoisomer, possessing the L-arabino 

instead of the L-lyxo configuration of the sugar moiety 

(Figure 1). In EPI therefore the hydroxyl group on the 

sugar moiety, possessing the stable 1 C4 conformation, 

has an equatorial orientation (Plosker and Faulds, 

1993). 

Cell line 

The cells used in this study were derived from mouse 

fibroblast by in vitro malign transformation (Earle, 

1943). Transformed L-cells obtained from mouse 

subcutaneous connective tissue in 1943. They were 

supplied by Dr. P.P. Dendy of Department of 

Radiotherapeutics, Cambridge University, in 1975. 

The cells were grown in Medium-199 (M-199, Gibco 

lab.) containing 10% foetal bovine serum (FBS, Gibco 

lab.), 100 µg/ml streptomycin and 100 IU/ml 

penicillin, and were passaged twice a week in 

appropriate number of 25 cm 2 flasks and the volume of 

the complete medium in each flask was completely to 

12 ml. Cells were removed from the surface of culture 

flasks by addition of 0.25% trypsine (Gibco lab.) and 

centrifuged for 3 minutes at 1500 cycle/min. 

Following the addition of M-199 on the cell 

precipitate, the cells became ready for the experiment. 

Cell doubling time (Tc) of L-cells was 22.8 hours 

(Özcan and R›dvano¤ullar›, 1996). L-cells were 

cultured on the cover-slips as 3.10 4 cells/ml in petri 

dishes and incubated for 24 hours with 95% air and 5% 

CO2 containing medium at 37°C with pH 7.2 in a 

dessicator. At the end of this incubation medium was 

removed, replaced with medium containing EPI 

concentrations. 

Drug application 

Figure 1: Structural formulae of EPI. 

Epirubicin (Farmorubicin, Carlo Erba) was dissolved 

immediatedly before use in sterile medium (M-199) to 

give the required concentration. We used 0.001 µg/ml, 

0.01 µg/ml and 0.1 µg/ml concentrations of EPI. Cells 

were treated with these doses for 2, 4, 8, 16 and 32 

hours.

Mitotic index analysis 

Mitotic index were studied by the methods of Feulgen. 

Before the cells were treated with Feulgen, they were 

prepared with 1 N HCl at room temperature for 1 

minute and then hydrolized with 1 N HCl for 10.5 

minutes at 60°C. After slides were treated with 

Feulgen, they were rinsed for few minutes in distilled 

water and stained with 10% Giemsa stain solution pH 

6.8, for 3 minutes and washed twice in phosphate 

Figure 2: Mitosis in L-cells under the light microscope 

(3.3x100). 

buffer. After staining, the slides were rinsed in distilled 

water. And then the slides were air dried. At last 

mitotic index were calculated by counting metaphases, 

anaphases and telophases for each tested drug 

concentration and control (Figure 2). At least three 

thousands cells were examined from each slide for 

mitotic index. 

Statistical analysis 

Mitotic index values which obtained from experiments 

were calculated to evaluate the statistical analysis. The 

differences between the percentage distrubition of M 

phase of the various treatment groups and control were 

compared by the Student-t test (n=25). 

Results 

Epirubicin effect on mitotic index 45 

The effect of EPI on mitotic index of L-cells 

in culture was investigated. EPI concentrations of 

0.001 µg/ml, 0.01 µg/ml and 0.1 µg/ml were applied to 

the cells for time periods of 2, 4, 8, 16 and 32 hours. 

In this study, EPI diminished the mitotic index of Lcells 

with increasing both treatment time and drug 

concentration compared to controls (untreated group). 

From the value of 2 hours treatment, we saw that all 

EPI concentrations had a rapid effect. In subsequent 

hours, this effect seemed to continue. The values of 

mitotic index reached a minimum at EPI concentration 

of 0.1 µg/ml with increasing drug concentration. Table 

1 reveals that treatments of EPI decreased the 

percentage of the cells at M phase. With increasing 

time the differences among the effects of various drug 

concentrations tended to be lower being very small at 

2 to 8 hours applications. The inhibition of mitosis was 

higher in 16 and 32 hours applications than those in 2, 

4 and 8 hours EPI applications in Table 1 especially 

with EPI concentration of 0.1 µg/ml. However, in the 

treatment of 0.1 µg/ml concentration, mitotic 

inhibition reached a maximum at 32 hours application. 

The values of mitotic index of the cells treated with 

EPI for 32 hours showed that mitotic index decreased 

as drug concentrations were increased. 

Table 1: Mitotic index values in cultures of L-cells treated with various concentrations of EPI, given in mean ± Standard deviation 

(SD). 

Mitotic index (%) 

EPI 2 hours 4 hours 8 hours 16 hours 32 hours 

concentrations 

Control 1.44 ± 0.12 SD 

0.001 µg/ml 1.35 ± 0.09 a 

0.01 µg/ml 1.29 ± 0.11 a 

0.1 µg/ml 0.94 ± 0.02 c 

a : p < 0.05, b : p < 0.01, c : p < 0.001 

1.93 ± 0.14 3.04 ± 0.08 3.39 ± 0.15 3.84 ± 0.21 

1.80 ± 0.10 a 

2.72 ± 0.07 a 

2.96 ± 0.04 b 

3.10 ± 0.30 b 

1.79 ± 0.05 b 

2.61 ± 0.06 b 

2.70 ± 0.13 b 

2.99 ± 0.16 b 

1.04 ± 0.01 c 

1.77 ± 0.09 c 

1.85 ± 0.08 c 

1.02 ± 0.22 c


EPI significantly decreased the mitotic index in 

cultures of L-cells. The results show that EPI 

decreased the mitotic index at significant level p

seem to be concordant with the above mentioned 

studies suggesting that cytotoxic effects of EPI might 

occur in the G1 and M phases at higher drug 

concentration. 

In our study, decreases in the mitotic index of cells 

with increasing both treatment time and EPI 

concentration have confirmed that EPI is an effective 

inhibitor of mitosis. 

In conclusion, the results of this study declared the 

cell kinetics and cytotoxic effects of the anticancer 

drug, EPI, in treated cultures of L-cell line. Although 

EPI has less systemic and cardiac toxicity than 

doxorubicin and other anthracyclines with an 

equivalent spectrum of antitumor action, it still has 

cytotoxic effects. 

Acknowledgment 

We would like to thank Prof. Dr. Atilla Özalpan for his 

kind help and critics. 

References 

Bagnara GP, Rocchi P, Bonsi L. The in vitro effects of 

epirubicin on human normal and leukemic hemopoietic 

cells. Anticancer Research. 7: 1197-1200, 1987. 

Bartkowiak D, Hemmer J, Rottinger E. Dose dependence of 

the cytokinetic and cytotoxic effects of epirubicin in 

vitro. Cancer Chemother Pharmacol. 30 (3):189-192, 

1992. 

Cantoni O, Sestili P, Cattabeni F. Comparative effects of 

doxorubicin and 4’-epidoxorubicin on nucleic acid 

metabolism and cytotoxicity in a human tumor cell line. 

Cancer Chemother and Pharmacol. 27: 47-51, 1990. 

Casazza AM and Giuliani FC. Preclinical properties of 

epirubicin. In: Advances in Anthracycline Chemotherapy: 

Epirubicin. Bonadonna G (Ed). Masson, Milano-Italy. 

31-40, 1984. 

Chabner BA and Myers CE. Antitumor antibiotics, 

In: Cancer: Principles and Practice of Oncology. De vita 

VT (Ed). AJF Lippincott, Philadelphia. 374-385, 1993. 

Di marco A, Casazza AM, Gambetta R, Supino R, Zunino F. 

Relationship between activity and aminosugar 

stereochemistry of daunorubicin and adriamycin 

derivates. Cancer Res. 36: 1962-1966, 1976 

Di marco A, Casazza AM, Dasdia T, Necco A, Pratesi G, 

Rivolta P, Velcich A, Zaccara A, Zunino F. Changes of 

activity of daunorubicin, adriamycinand stereoisomers 

following the introduction or removal of hydroxyl 

groups in the amino sugar moiety. Chem Biol Interac. 

19: 291-302, 1977. 

Epirubicin effect on mitotic index 47 

Di marco A. Epirubicin: Mechanism of action at the cellular 

level. In: Advances in Anthracycline Chemotherapy: 

Epirubicin. Bonadonna G (Ed). Masson, Milano-Italy. 

41-47, 1984. 

Earle WR. Production of malignancy in vitro. IV. The mouse 

fibroblast cultures and changes seen in the living cells. 

J Nat Cancer Inst. 4: 165-212, 1943. 

El-Mahdy Sayed Othman O. Cytogenetic effect of the 

anticancer drug epirubicin on Chinese hamster cell line 

in vitro. Mutation Res. 468: 109-115, 2000. 

Haldane A, Finlay GJ, Baguley BC. A comparison of the 

effects of aphidicolin and other inhibitors on 

topoisomerase II-directed cytotoxic drugs. Oncol Res. 

5 (3): 133-138, 1993. 

Hill BT and Whelan RDH. A comparison of the lethal and 

kinetic effects of doxorubicin and 4’-epidoxorubicin 

in vitro. Tumori. 68: 29-37, 1982. 

Lollini PL, De Giovanni C, Del Re B. Myogenic 

differentiation of human rhabdomyosarcoma cells 

induced in vitro by antineoplastic drugs. Cancer 

Research. 49: 3631-3636, 1989. 

Nistico C, Garufic C, Barni S, Frontini L, Galla DA, 

Giannaarelli D, Vaccaro A, Dottovio AM, Terzoli E. 

Phase II study of epirubicin and vinorelbine eith 

granulocyte colony-stimulating factor: A high-activity, 

dose-dense weekly regimen for advanced breast cancer. 

Ann Oncol. 10 (8): 937-942, 1999. 

Özcan G and R›dvano¤ullar› M. The effect of epirubicin on 

the cell cycle of L-cells. 13 th National Congress of 

Biology. Istanbul, Turkey. 3: 267-276, 1996. 

Özcan FG, Topçul MR, Y›lmazer N, R›dvano¤ullar› M. 

Effect of epirubicin on 3 H-thymidine labelling index in 

cultured L-strain cells. J Exp Clin Cancer Res. 16(1): 23- 

27, 1997. 

Plosker GL and Faulds D. Epirubicin. A review of its 

pharmacodynamic and pharmacokinetic properties, and 

therapeutic use in cancer chemotherapy. In: Drugs. 

Chrisps P (Ed). 0012-6667. 45 (5): 788-856, 1993. 

Robert J and Gianni L. Pharmacokinetics and metabolism of 

anthracyclines. Cancer Surv. 17: 219-252, 1993. 

Rocchi P, Ferreri AM, Simone G. Epirubicin-induced 

differentiation of human neuroblastoma cells in vitro. 

Anticancer Research.7: 247-250, 1987. 

Sinha BK and Politi PM. Anthracyclines. Cancer chemother 

apy. Biol Response Modif. 11: 45-57, 1990. 

Skladanowski A and Konopa J. Interstrand DNA 

crosslinking induced by anthracyclines in tumour cells. 

Biochem Pharmacol. 47 (12): 2269-2278, 1994. 

Stewart DJ, Cripps MC, Dahrouge RGS, Yau J, Tomiak E, 

Huan S, Soltys K, Prosser A, Davies RA. Pilot study of 

multiple chemotherapy resistance modulators plus 

epirubicin in the treatment of resistant malignancies. 

Cancer Chemother Pharmacol. 41: 1-8, 1997. 

Topçul MR, Ar›can Özcan G, Erensoy N, Özalpan A. Effect 

of epirubicin and tamoxifen on labelling index in FM3A 

cells. J Cell and Mol Biol. 2: 81-85, 2002


Wilson RG, Kalonaros V, King M. Comparative inhibition of 

nuclear RNA synthesis in cultured mouse leukemia 

L1210 cells by adriamycin and 4’-epi-adriamycin. 

Chemico-Biological Interaction. 37: 351-363, 1981. 

Young CW. Clinical toxicity of epirubicin. Update on 

epirubicin. In: Advances in Clinical Oncology. 

Robustelli della cuna G and Bonadonna G (Ed). Edimes- 

Pavia, Italy. 29-38, 1989. 

Zhang W, Zalcberg JR, Cosolo W. Interaction of epirubicin 

with other cytotoxic and anti-emetic drugs. Anticancer 

Drugs. 3 (6): 593-597, 1992. 

Zuckerman KS, Case-Dc JR, Gams RA. Chemotherapy of 

intermediate- and high-grade Non-Hodgkins’s 

lymphomas with an intensive epirubicin-containing 

regimen. Blood. 82 (12): 3564-3573, 1993.

Letter to editor 

P rostate cancer and importance of 

tumor marker studies 

P rostat kanseri ve tümor mark›rlar› ile 

ilgili çal›flmalar›n önemi 

Prostate cancer is the most commonly new diagnosed 

noncutaneous malignancy in men in USA. In the year 

2002, according to the health statistics 189,000 men in 

the United States are expected to be diagnosed with the 

disease and 30,200 men are expected to die of it. 

Incidence varies greatly, with African Americans 

having the highest incidence in the world (224 cases 

per 100,000 population). The incidence of prostate 

cancer in African Americans stands in stark contrast to 

the incidence in white Americans (150 per 100,000) 

and that in men in Western Europe (39.6 per 100,000), 

Japan (8.5 per 100,000), and China (1.1 per 100,000). 

Tumor markers are biological molecules that 

indicate the presence of malignancy. They are 

potentially useful in cancer screening, aiding 

diagnosis, assessing prognosis, predicting in advance a 

likely response to therapy, and monitoring patients 

before and after diagnosis. Because of low prevalance 

of most cancers in the general population and the 

limited sensivity and spesificity of avaible markers, 

these tests alone are generally of little value in 

screening for cancer in healthy subjects. Currently, 

however, prostate spesific antigen (PSA) in 

combination with digital rectal examination (DRE) are 

undergoing evaluation as screening modalities for 

prostate cancer. Because of a lack of sensitivity and 

spesificity markers are rarely of use in early diagnosis 

of cancer. Also they can be used as monitoring disease 

evaluation with therapy. The goal of future research 

should be that development of the most specific, cheap 

and easy markers for common cancer types as prostate 

cancer. 

P rostate spesific antigen: Screening prostate cancer 

provides a dilemma unique among cancer sites. The 

best strategy is determination of the ratio of the 

prostate serum antigen (PSA) to the volume of the 

prostate gland in prostate cancer diagnosis. 

Determination of the free PSA (i.e., the percentage of 

PSA that is unbound to serum proteins) has also been 

suggested as a means of distinguishing malignancy 

from benign hyperplasia. PSA has revolutionized the 

management of prostate cancer since its development 

in the 1980s. For unclear reasons, PSA derived from 

malignant epithelial cells tends to bind more avidly to 

serum proteins. Thus, in men with an elevated serum 

PSA level, cancer is more likely to be present when the 

percentage of free PSA is low. Because the relative 

sensitivity versus specificity varies, depending on the 

free PSA cutoff, the optimal cutoff value for free PSA 

is still under debate. Prostate specific antigen (PSA) 

represents the best serum marker for prostatic 

carcinoma and is considered as most perfect tumor 

marker available today. Nevertheless, the use of PSA 

to detect prostate cancer is clinically imprecise since 

benign and malignant prostate disease can cause 

elevations in PSA. It is sensitive but spesificity is not 

good to show tumor agressiveness, and so does not 

benign prostatic hypertropy from invasive cancer. 

Age-spesific cut-offs have been suggested to improve 

spesificity, but there is still substantial overlap 

between normals and those with cancer. Further 

markers of tumor agressiviness, either measured in 

serum or needle biopsy specimens are needed to 

determine which patients are in need of curative 

treatment. 

Serum acid phosphatase: Serum acid phosphatase 

(ACP) served as the only serum tumor marker for 

prostate cancer between the 1930s and 1980s. more 

sensitive serum tumor marker in detection of localized 

disease and in monitoring response to therapy. In the 

past two decades, the use of ACP has diminished 

because of problems with lack of sensitivity and 

specificity and because of the discovery of prostatespecific 

antigen (PSA), a is an independent predictor 

of biochemical recurrence in men who undergo 

surgery. ACP level is independently predictive of 

biochemical recurrence following radical retropubic 

prostatectomy (RRP), when adjusted for other 

predictive variables. 

Granins: The nomenclature for chromogranin-A 

continues to evolve; for simplicity,it is referred as 

49

50 

granin-A (GRN-A). GRN-A is a 49-kilodalton protein 

that is produced exclusively by endocrine and 

neurondocrine (NE) cells. It is costored and cosecreted 

with the resident hormones of these cells, such as 

catecholamines and calcitonin (CT). Although the 

function of GRN-A is not known, it can serve as a 

tissue and serum marker for a variety of endocrine 

cells and tumors. There are several major cancer types 

are characterized by NE differentiation. Recently, the 

importance of NE differentiation and the attendant 

expression of chromogranin-A has become 

appreciated for prostate cancer. Clinical and basic 

roles of chromogranin-A in human prostate cancer are 

still studied. Although the function of GRN-A is not 

known, several theories have emerged about its role: 

(1) that it participates and perhaps regulates the storage 

and secretion of its coresident hormones in secretory 

vesicles; (2) that it inhibits proteolytic cleavage 

enzymes; (3) that it binds calcium and thus regulates 

the biologic effects of this ion; and (4) that it is a 

precursor for peptides that have unique biologic effects 

on the function and growth of its resident cells. 

Function notwithstanding, the production of GRN-A in 

NE prostate cancers has resulted in the availability of 

a new serum and tissue marker for the tumors. The 

clinical potential of GRN-A as a serum and tumor 

marker in prostate cancer. It is now wellestablished 

that GRN-A can be a marker for advanced disease. 

More importantly, GRN-A may be a marker for early 

and recurrent disease, even in the absence of abnormal 

PSA. GRN-A serum levels may also have prognostic 

significance, especially for androgen-independent 

prostate cancer. 

E-cadherin: In attempts to determine which cancers 

of patients with clinically localized disease who 

undergo radical prostatectomy will recur, the most 

well-characterized and accepted predictors are model 

equations that take into account preoperative serum 

prostate-specific antigen (PSA), final Gleason score, 

and final pathologic stage. Prediction of regression for 

the individual patient using these statistical models, 

however, is still not precise, and these models could 

still be improved on. Thus, additional markers are 

needed to more accurately target high-risk patients for 

inclusion in clinical trials involving investigational 

therapies for locally advanced prostate carcinoma. 

Several other approaches show promise in this regard, 

including nuclear morphometry, where the results have 

been quite consistent. Other more controversial 

markers include DNA ploidy and other biomarkers, 

such as the amount of tumor angiogenesis, and 

immunohistochemical levels of various markers, 

including Ki-67, Bcl-2, p53, and E-cadherin. Ecadherin 

as a biomarker to predict prognosis in 

patients at risk of disease recurrence after radical 

prostatectomy is warranted. 

Serum total homocystein: Homocystein (Hcy) as a 

tumor marker targets to reveal chemotherapy effects 

on patients. It is largely derived from cellular 

methionine, an essential amino acid drawn from 

dietary intake. Intracellular homocysteine is normally 

secreted extracellularly, at rapid rates. In the 

circulating blood, the majority of the homocysteine 

binds to albumin, forming a disulfide linkage. 

Approximately 10% to 20% of the Hcy also exists as a 

mixed disulfide with cysteine or with homocysteine 

itself . Very little Hcy is present in the circulating 

blood in a free reduced form (approximately 

1%).Elevated serum tHcy (total homocysteine, free 

and protein-bound) are detectable in patients with 

malignant diseases. Finding increased circulating tHcy 

in tumor cells may also be related to the so-called 

‘‘methionine dependency’’ of many, but not all, tumor 

cells. Many tumor cells are methionine dependent 

because of their inability to convert homocysteine 

(Hcy) to methionine by way of the remethylation 

reaction. On the other hand, normal cells have no 

problem obtaining methionine from homocysteine. 

Folate is critical to the remethylation reaction. Any 

folate deficiency will result in the impairment of 

function of the remethylation reaction, causing 

accumulation of Hcy. Therefore, it was generally 

believed that the rapid proliferation rate of tumor cells, 

such as in prostate cancer and in the so-called 

methionine dependency of tumor cells, was due to the 

depletion of folate by the rapid growing tumor cells 

and changing levels of fLV (a form of folate) in 24 h 

after therapy. In other words, with a better 

understanding of the effects of various drugs, the rise 

and fall of circulating tHcy could be used as a new 

tumor marker to monitor cancer patients during 

therapy, complementing commonly used tumor 

markers. The general impression that elevated tHcy is 

detectable in cancer patients derives from the fact that 

many cancer patients take anti-folate drugs such as 

methotrexate. It is important to know that the level of 

tHcy reflects the tumor cell proliferation rate. 

Regardless of the folate status, it is very likely given 

our results and others that the rapid proliferation of 

tumor cells is one of the major reasons that elevated

circulating tHcy can be detected in cancer patients. 

Conceivably, circulating tHcy could very well be used 

as a marker to monitor cancer patients during therapy, 

complementing the currently used tumor markers. 

Choline kinase: Choline kinase (ChoK) is the first 

enzyme in the Kennedy pathway, responsible for the 

de novo synthesis of phosphatidylcholine (PC), one of 

the basic lipid components of membranes. ChoK is 

responsible of the generation of phosphorylcholine 

(PCho) from its precursor, choline. Both ChoK and its 

product, PCho, have been recently reported as 

essential molecules in cell proliferation and 

transformation. Generation of Pcho from ChoK 

activity has been described as an essential event in 

growth factor-induced mitogenesis in fibroblasts and 

has been found to cooperate with several mitogens. 

Furthermore, overexpression of several oncogenes 

induces increased levels of ChoK and the intracellular 

levels of PCho. A strong correlation can be established 

between ChoK activity and cancer onset at least in 

some human tumors. Additional evidence gives 

support for a role of ChoK in the generation of human 

tumors, since studies using nuclear magnetic 

resonance (NMR) techniques have demonstrated 

elevated levels of PCho in human tumoral tissues with 

respect to the normal ones, including breast, prostate 

carcinomas. ChoK is overexpressed with high 

incidence in both, tumor- derived cell lines and 

tumoral tissues, these results indicate the putative use 

of ChoK as a tumor marker, potentially useful in 

diagnosis and screening of the progression of tumors. 

The recent findings show that overexpression of 

the polycomb group transcriptional represor enhancer 

zeste Gene (EZH2) in prostate cancer raises the 

possibility that transcriptional regulation at the 

chromatin level play a role in the development of the 

metabolic phenotype and suggest new exploration 

prespective on patient stratification, therapeutics and a 

tumor marker identity. 

Also proliferation markers in biopsies such as K67, 

expression levels of mRNA and/or proteins for bcl2, 

p53, p27 etc. and molecular changes in tumor 

supressor genes such as PTEN or mutations in genes or 

mutations in genes can be candidate markers. This is 

urgently needed since radical surgery carries a high 

morbidity leading to impotence and/or incontinence. 

Serdar Ar›san 

fiiflli Etfal State Hospital 

1. Urology Clinics, fiiflli, ‹stanbul 

51

Book reviews 

Çetin ALGÜNEfi, Radyasyon Biyofizi¤i, Trakya 

Üniversitesi Yay›nlar›, Edirne, 135 sayfa, ISBN: 975- 

374-051-4, 2002. 

Kitapta atom ve çekirde¤inin yap›s›, karars›z 

çekirdekler, iyonizan radyasyon tipleri ve özellikleri, 

radyasyonun madde ile etkileflmesi ve radyasyon 

birimleri, iyonizasyona sebep olmayan radyasyonlar, 

iyonizan radyasyonlar›n biyolojik etkileri ve 

radyasyondan korunma konular› tart›fl›lm›flt›r. 

Bölümlerin ayr›nt›l› incelenmesinde, kitab›n 

diziliminin geleneksel tarzda, flekil ve tablolar›n 

geçtikleri yerlerde metin aras›nda verildi¤i 

görülmektedir. Bütün konular, aç›klay›c› flekil, tablo ve 

örneklerle desteklenmifltir. 

Bu kitab›n radyasyon biyofizi¤i konusunda önemli 

bilgiler kazand›rmas› aç›s›ndan çok yararl› bir rehber 

olaca¤› kan›s›nday›m. Ayr›ca Türkiye’de bu alanlarda 

kaynak oluflturacak Türkçe eserlerin say›lar› da son 

derece s›n›rl› oldu¤u için, bu kitab› biyologlara, 

radyobiyologlara, fizikçilere, radyasyon etkileri ile 

ilgilenen ziraatç› ve veterinerlere öneririm. 


Haliç Üniversitesi, 

Moleküler Biyoloji ve Genetik Bölümü 

Çetin ALGÜNEfi, Radiation Biophysics, Published 

by Trakya University, Edirne, 135 pp, ISBN: 975-374- 

051-4, 2002. 

In the book, atomic and nuclear structure, unstable 

nuclei, types and properties of ionizing radiations, 

interaction of radiation with matter, radiation units, 

non-ionizing radiations, biological effects of ionizing 

radiations and radiation protection are discussed. 

In detail, the layout of the book has a traditional 

format in that figures and tables have been integrated 

into the text at appropriate places. All statement are 

supported with a plenty of explenatory figures, tables 

and examples. 

53 

The book is a valuable guide of radiation 

biophysics. On the other hand, this book is a good 

document because there are very limited Turkish 

publication in this area. For this reason, I 

recommended this book for the biologists, physisists, 

agriculturists and veterinarians who apply radiation on 

living organisms for several purposes. 


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Redford IR. Evidence for a general relationship 

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Taccioli CE, Cottlieb TM and Blund T. Ku 80: Product 

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Weaver RF. Molecular Biology. WCB/Mc 

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CONTENTS Volume 2, No. 1, 2003 

Dedication 

Review articles 



R. Kaur-Sawhney, A.F. Tiburcio, T. Altabella, A.W. Galston 1-12 



V. I. Kefeli, M. V. Kalevitch, B. Borsari 13-18 

Research papers 



T. Aktaç, A. Kabo¤lu, E. Bakar, H. Karakafl 19-23 

Characterisation of RPP7 mutant lines of the col-5 ecotype of Arabidopsis thaliana 

Arabidopsis thaliana’n›n col-5 ekotipinden elde edilen mutant hatlardan RPP7 

geninin karakterizasyonu 

C. Can, M. Özaslan, E. B. Holub 25-30 

The effect of meta-topolin on protein profile in radish cotyledons 

Meta-topolinin turp kotiledonlar›nda protein profiline etkisi 

S. Ça¤, N. Palavan-Ünsal 31-34 



S. ‹fller, G. Erdem 35-38 

Chromosomes of a balanced translocation case evaluated with atomic force microscopy 



Z. Y›lmaz, M. A. Ergun, E. Tan 39-42 


Epirubisinin kültürdeki L-hücrelerinde mitotik indekse etkisi 

G. Özcan Ar›can, M. Topçul 43-48 

Letter to editor 49-51 

Book reviews 53 

Instructions to authors 55-56

(Converted)-4 - Journal of Cell and Molecular Biology - Haliç ...

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