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VOLUME 2 • NO.

1 • 2003 • ISSN 1303-3646

HAL‹Ç UNIVERSITY
‹STANBUL
FACULTY OF ARTS AND SCIENCES
1998

Journal of Cell and


Molecular Biology
Journal of Cell and
M o l e c u l a r Biology
Haliç University
Published by Haliç University
Faculty of Arts and Sciences Faculty of Arts and Sciences
J o u r n a l o f C e l l a n d M o l e c u l a r Biology
Founder Editor
Prof. Dr. Gündüz GED‹KO⁄LU
Atilla ÖZALPAN
President of Board of Trustee
Rights held by
Prof. Dr. Ahmet YÜKSEL Associate Editor
Rector Narç›n PALAVAN ÜNSAL

Correspondence Address:
The Editorial Office Editorial Board
J o u r n a l o f C e l l a n d M o l e c u l a r Biology
Çimen ATAK
Haliç Üniversitesi, Fen-Edebiyat Fakültesi,
Ahmet Vefik Pafla Cad., No: 1, 34280, Atok OLGUN
F›nd›kzade, ‹stanbul-Turkey
P›nar ÖZKAN
Phone: 90 212 530 50 24
Fax: 90 212 530 35 35 Nihal BÜYÜKUSLU
E-mail: jcmb@halic.edu.tr Kürflat ÖZD‹LL‹
J o u r n a l o f C e l l a n d M o l e c u l a r Biology is Damla BÜYÜKTUNÇER
indexed in EBSCO database.
Özge EM‹RO⁄LU
Summaries of all articles in this journal are
available free of charge from www.halic.edu.tr Mehmet Ali TÜFEKÇ‹
ISSN 1303-3646 Merve ALO⁄LU
printed at yaflar printing house Asl› BAfiAR

Advisory Board
Igor ALEXANDROV, Dubna, Russia Ünal EGEL‹, Bursa, Turkey
Çetin ALGÜNEfi, Edirne, Turkey Candan JOHANSEN, ‹stanbul, Turkey
Aglaia ATHANASSIADOU, Patros, Greece As›m KADIO⁄LU, Trabzon, Turkey
fiehnaz BOLKENT, ‹stanbul, Turkey Valentine KEFEL‹, Pennsylvania, USA
Nihat BOZCUK, Ankara, Turkey Göksel OLGUN, Edirne, Turkey
‹smail ÇAKMAK, ‹stanbul, Turkey U¤ur ÖZBEK, ‹stanbul, Turkey
Adile ÇEV‹KBAfi, ‹stanbul, Turkey Zekiye SULUDERE, Ankara, Turkey
Beyaz›t ÇIRAKO⁄LU, ‹stanbul, Turkey ‹smail TÜRKAN, ‹zmir, Turkey
Ayfl›n ÇOTUK, ‹stanbul, Turkey Mehmet TOPAKTAfi, Adana, Turkey
Zihni DEM‹RBA⁄, Trabzon, Turkey Meral ÜNAL, ‹stanbul, Turkey
Mustafa DJAMGOZ, London, UK Mustafa YAT‹N, Boston, USA
Aglika EDREVA, Sofia, Bulgaria Ziya Z‹YLAN, ‹stanbul, Turkey
Journal of Cell and
M o l e c u l a r Biology

Volume 2/2003

Haliç University
Faculty of Arts and Sciences
‹stanbul-TURKEY
Journal of Cell and
M o l e c u l a r Biology
Haliç University
Published by Haliç University
Faculty of Arts and Sciences Faculty of Arts and Sciences
J o u r n a l o f C e l l a n d M o l e c u l a r Biology
Founder Editor
Prof. Dr. Gündüz GED‹KO⁄LU
Atilla ÖZALPAN
President of Board of Trustee
Rights held by
Prof. Dr. Ahmet YÜKSEL Associate Editor
Rector Narç›n PALAVAN ÜNSAL

Correspondence Address:
The Editorial Office Editorial Board
J o u r n a l o f C e l l a n d M o l e c u l a r Biology
Çimen ATAK
Haliç Üniversitesi, Fen-Edebiyat Fakültesi,
Ahmet Vefik Pafla Cad., No: 1, 34280, Atok OLGUN
F›nd›kzade, ‹stanbul-Turkey
P›nar ÖZKAN
Phone: 90 212 530 50 24
Fax: 90 212 530 35 35 Nihal BÜYÜKUSLU
E-mail: jcmb@halic.edu.tr Kürflat ÖZD‹LL‹
J o u r n a l o f C e l l a n d M o l e c u l a r Biology is Damla BÜYÜKTUNÇER
indexed in EBSCO database.
Özge EM‹RO⁄LU
Summaries of all articles in this journal are
available free of charge from www.halic.edu.tr Mehmet Ali TÜFEKÇ‹
ISSN 1303-3646 Merve ALO⁄LU
printed at yaflar printing house Asl› BAfiAR

Advisory Board
Igor ALEXANDROV, Dubna, Russia Ünal EGEL‹, Bursa, Turkey
Çetin ALGÜNEfi, Edirne, Turkey Candan JOHANSEN, ‹stanbul, Turkey
Aglaia ATHANASSIADOU, Patros, Greece As›m KADIO⁄LU, Trabzon, Turkey
fiehnaz BOLKENT, ‹stanbul, Turkey Valentine KEFEL‹, Pennsylvania, USA
Nihat BOZCUK, Ankara, Turkey Göksel OLGUN, Edirne, Turkey
‹smail ÇAKMAK, ‹stanbul, Turkey U¤ur ÖZBEK, ‹stanbul, Turkey
Adile ÇEV‹KBAfi, ‹stanbul, Turkey Zekiye SULUDERE, Ankara, Turkey
Beyaz›t ÇIRAKO⁄LU, ‹stanbul, Turkey ‹smail TÜRKAN, ‹zmir, Turkey
Ayfl›n ÇOTUK, ‹stanbul, Turkey Mehmet TOPAKTAfi, Adana, Turkey
Zihni DEM‹RBA⁄, Trabzon, Turkey Meral ÜNAL, ‹stanbul, Turkey
Mustafa DJAMGOZ, London, UK Mustafa YAT‹N, Boston, USA
Aglika EDREVA, Sofia, Bulgaria Ziya Z‹YLAN, ‹stanbul, Turkey
J o u r n a l o f C e l l a n d M o l e c u l a r Biology

C O N T E N T S Volume 2, No.1, 2003

Dedication
Review articles
P o l y a m i n e s i n p l a n t s : An overview
Bitkilerde poliaminler: Genel bir bak›fl
R. Kaur-Sawhney, A.F. Tiburcio, T. Altabella, A.W. Galston 1-12
P h e n o l i c c y c l e i n p l a n t s a n d e n v i ro n m e n t
Bitkilerde fenolik döngü ve çevre
V. I. Kefeli, M. V. Kalevitch, B. Borsari 13-18
R e s e a rc h p a p e r s
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
C h a r a c t e r i s a t i o n o f R P P 7 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 - t o p o l i n o n p r o t e i n p r o f i l e i n r a d i s h c o t y l e d o n s
Meta-topolinin turp kotiledonlar›nda protein profiline etkisi
S. Ça¤, N. Palavan-Ünsal 31-34

T h e e f f e c t o f e l e c t romagnetic fields on oxidative DNA d a m a g e


Elektromanyetik alan›n oksidatif DNA hasar› üzerindeki etkisi
S. ‹fller, G. Erdem 35-38
C h romosomes of a balanced translocation case evaluated with atomic force
m i c r oscopy
Dengeli translokasyon vakas›nda kromozomlar›n atomik güç mikroskobu ile
de¤erlendirilmesi
Z. Y›lmaz, M. A. Ergun, E. Tan 39-42
E f f e c t o f e p i r u b i c i n o n m i t o t i c i n d e x i n c u l t u r ed L-cells
Epirubisinin kültürdeki L-hücrelerinde mitotik indekse etkisi
G. Özcan Ar›can, M. Topçul 43-48
L e t t e r to editor 49-51
Book reviews 53
Instructions to authors 55-56
This issue is dedicated to

P rof. Dr. A r t h u r W. Galston


for his invaluable contribution to plant biology
A r t h u r W. G a l s t o n , C u r r i c u l u m Vitae

B o r n : April 21, 1920 Eaton Professor of Botany, Emeritus, Department of


Molecular, Cellular and Developmental Biology,
E d u c a t i o n : 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

H o n o r s : 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.

E x p e r i e n c e : 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 re s e n t o f p a s t E d i t o r i a l B o a r d M e m b e r : 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.

F o r m e r M e m b e r : 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, 2nd Ed., 1964, 3rd 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. 1
Haliç University, Printed in Turkey.

P o l y a m i n e s i n p l a n t s : An overview
Ravindar Kaur-Sawhney1*, Antonio F. Tiburcio2, Teresa Altabella2, and Arthur W. Galston1
1
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT,
06520-8103, USA; 2Laboratori 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.

Key words: Polyamines, mutants, transgenic plants, genomics, proteomics

Bitkilerde poliaminler: Genel bir bak›fl

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.

Anahtar sözcükler: Poliaminler, mutantlar, transgenik bitkiler, genomik, proteomik


2 Ravindar Kaur-Sawhney et al.

1. I n t roduction This article presents an overview of the role of PAs


in plants with particular emphasis on recent
Polyamines (PAs) are low molecular weight investigations using molecular and genetic
polycations found in all living organisms (Cohen, approaches.
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 2. Polyamine biosynthesis
diamine putrescine (Put), triamine spermidine (Spd)
and tetramine spermine (Spm) constitute the major The PA biosynthetic pathway in plants has been
PAs. They occur in the free form or as conjugates thoroughly investigated and reviewed in detail (Evans
bound to phenolic acids and other low molecular and Malmberg, 1989; Tiburcio et al., 1990; Slocum,
weight compounds or to macromolecules such as 1991a; Martin-Tanguy, 2001). Briefly, PAs are
proteins and nucleic acids. As such, they stimulate synthesized from arginine and ornithine by arginine
DNA replication, transcription and translation. They decarboxylase (ADC) and ornithine decarboxylase
have been implicated in a wide range of biological (ODC) as illustrated in Figure 1. The intermediate
processes in plant growth and development, including agmatine, synthesized from arginine, is converted to
senescence, environmental stress and infection by Put, which is further transformed to Spd and Spm by
fungi and viruses. Their biological activity is attributed successive transfers of aminopropyl groups from
to their cationic nature. These findings have been decarboxylated S-adenosylmethionine (dSAM)
discussed in several recent review articles (Tiburcio et catalysed by specific Spd and Spm synthases. The
al., 1993; Galston et al.,1997; Bais and Ravishankar, aminopropyl groups are derived from methionine,
2002). which is first converted to S-adenosylmethionine
The use of PA biosynthesis inhibitors has shown a (SAM), and then decarboxylated in a reaction
causal relationship between changes in endogenous PA catalyzed by SAM decarboxylase (SAMDC). The
levels and growth responses in plants. These resulting decarboxylated SAM is utilized as an
observations led to further studies into undestanding aminopropyl donor. SAM is a common precursor for
the mode of PA action. Some of the important both PAs and ethylene, and SAMDC regulates both
observations suggest that PAs can act by stabilizing biosynthetic pathways as illustrated in Figure 1.
membranes, scavenging free radicals, affecting nucleic A number of investigators have used PA inhibitors
acids and protein synthesis, RNAse, protease and other to modulate the cellular PA titer in order to determine
enzyme activities, and interacting with hormones, their role in various plant processes. Four commonly
phytochrome, and ethylene biosynthesis (reviewed in used inhibitors of PA synthesis are: 1.
Slocum et al., 1984; Galston and Tiburcio, 1991). Difluoromethylornithine (DFMO), an irreversible
Because of these numerous biological interactions of inhibitor of ODC (reviewed in Bey et al., 1987); 2.
PAs in plant systems, it has been difficult to determine Difluoromethylarginine (DFMA), an irreversible
their precise role in plant growth and development. inhibitor of ADC (Bitonti et al., 1987); 3. Methyl-
In recent years, however, investigations into glyoxyl-bis guanylhydrazone (MGBG), a competitive
molecular genetics of plant PAs have led to isolation of inhibitor of S-adenosyl-methionine decarboxylase
a number of genes encoding PA biosynthetic enzymes (SAMDC) (Williams-Ashman and Schenone,1972);
and development of antibodies to some of the genes. and 4. Cyclohexylamine (CHA), a competitive
Furthermore mutants and transgenic plants with altered inhibitor of spermidine synthase (Hibasami et al.,
PA metabolism have also been developed. Genomic 1980). Common oxidases are diamine oxidase and
and proteomic approaches are being used to further polyamine oxidase (PAO), as reviewed by Smith and
gain an understanding into the role of PAs in plant Marshall (1988). Each PA has been found to be
developmental processes. These findings will hopefully catabolized by a specific oxidase.
lead to a better understanding of their specific functions Several investigations have dealt with localization
in plants. Several useful reviews on these aspects have of PAs and their biosynthetic enzymes in plants
been published (Galston et al., 1997; Walden et al., (reviewed by Slocum, 1991b). However, paucity of
1997; Malmberg et al., 1998; Martin-Tanguy, 2001; information regarding the exact cellular and
Bais and Ravishankar, 2002). subcellular localization of these entities remains one of
Polyamines in plants 3

Ornithine Arginine
ODC ADC
Methionine DFMA
DFMO
Agmatine

MGBG Putrescine
S - adenosylmethionine dSAM Spdsynthase
Spermidine
SAMDC
ACC synthase Spmsynthase
AVG
ACC Spermine

ACC oxidase
Ethylene

F i g u re 1: Polyamine biosynthetic pathway and its linkage to ethylene biosynthetis. Biosynthetic enzymes are ADC, ODC and
SAMDC and the inhibitor DFMA, DFMO and MGBG.

the obstacles in understanding their biological role. 3. Polyamines in plant growth and development
Recent studies have shown that PAs are present in the
cell wall fractions, vacuole, mitochondria and The availability of specific inhibitors of PA
chloroplasts (Torrigiani et al., 1986; Slocum, 1991b; biosynthesis has helped in investigating the
Tiburcio et al., 1997). The biosynthetic enzymes, mechanisms involved in PA interactions to some extent,
ODC, SAMDC, and Spd synthase have been reported providing a partial understanding of their physiological
to be localized in the cytoplasm, whereas ADC is role in plant growth and development. Clearly, PAs are
localized in the thylakoid membrane of chloroplast involved in many plant developmental processes,
(Borrell et al., 1996; Tiburcio et al., 1997) and PAO in including cell division, embryogenesis, reproductive
the cell wall (Kaur-Sawhney et al., 1981). ODC organ development, root growth, tuberization, floral
activity has also been observed in the nucleus initiation and development, fruit development and
(Slocum, 1991b). However, these findings have to be ripening as well as leaf senescence and abiotic stresses
interpreted with caution because various procedural (reviewed by Evans and Malmberg, 1989; Galston et
problems can mask the results. Despite these advances al., 1997; Bais and Ravishankar, 2002; Tiburcio et al.,
in understanding the metabolic processes involving 2002). Changes in free and conjugated PAs and their
PAs and their localization in plant cells, the precise biosynthetic enzymes, namely ADC, ODC, and
role of PAs in plant morphogenesis remains elusive. 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
4 Ravindar Kaur-Sawhney et al.

and Koppenbrink, 1978; Feirer et al., 1984), tomato Sawhney,1995). Thus, PAs which may or may not be
ovaries (Heimer and Mizrahi, 1982), tobacco ovaries mobile in plants (Young and Galston, 1983; Bagni and
(Slocum and Galston, 1985), and fruit development Pistocchi, 1991) can serve as intracellular mediators of
(reviewed in Kakkar and Rai, 1993). Similar results hormone actions (Galston and Kaur-Sawhney, 1995).
have been reported for many other plant species Supporting evidence for this hypothesis has been
(reviewed in Bais and Ravishankar, 2002). In contrast, obtained in experiments using specific inhibitors of PA
several other studies have suggested that correlations biosynthesis (Bagni et al., 1981; Egea-Cortines and
between PAs and their biosynthetic enzymes and plant Mizrahi, 1991; reviewed in Galston et al., 1997; Bais
growth processes, especially somatic embryogenesis, and Ravishankar, 2002).
are not universal and may be species specific (reviewed Of the major plant hormones, ethylene has been
in Evans and Malmberg, 1989; Galston et al., 1997; most intensively investigated with respect to PA
Bais and Ravishankar, 2002). metabolism. The two metabolites, PAs and ethylene,
In general, cells undergoing division contain high play antagonistic roles in plant processes. While PAs
levels of free PAs synthesized via ODC, and cells inhibit senescence of leaves (Kaur-Sawhney et al.,
undergoing expansion and elongation contain low 1982), cell cultures of many monocot and dicot species
levels of free PAs synthesized via ADC (see review by (Muhitch et al., 1983) and fruit ripening (Kakkar and
Galston and Kaur-Sawhney, 1995). High levels of Rai, 1993), ethylene promotes these processes. The
endogenous PAs and their conjugates have also been most commonly held view is that PAs and ethylene
found in apical shoots and meristems prior to regulate each other’s synthesis, either directly or
flowering (Cabbane et al., 1981) and flower parts of through metabolic competition for SAM, a common
many plants (Martin-Tanguy, 1985). Our experiments precursor for their biosynthesis (Figure 1). PAs inhibit
using callus cultures derived from thin layer explants ethylene biosynthesis, perhaps by blocking the
of pedicels from tobacco inflorescence show that conversion of SAM to ACC and of ACC to ethylene
endogenous Spd increased more rapidly than other (Apelbaum et al., 1981; Suttle, 1981; Even-Chen et al.,
PAs in floral buds than in vegetative buds. Addition of 1982; Furer et al., 1982). Ethylene, on the other hand,
CHA, an inhibitor of Spd synthesis, to the culture is an effective inhibitor of ADC and SAMDC, key
medium reduced flower formation in a dose dependent enzymes in PA biosynthetic pathway (Apelbaum et al.,
manner and such inhibition was correlated with a 1985; Icekson et al., 1985). Thus, PAs may affect
switch to initiation of vegetative instead of flower senescence and fruit ripening by modulating PA and
buds. This inhibition was reversed by the addition of ethylene biosynthesis.
exogenous Spd (Kaur-Sawhney et al., 1988). More Apparently, PAs are essential members of an array
recently, we have found that higher levels of of internal metabolites required in many plant
endogenous PAs occur in flowers and siliques when developmental processes, but their precise role in these
compared with their levels in leaves and bolts of processes has yet to be established. Whereas, specific
certain strains of Arabidopsis. Addition of the PA PAs at specific concentrations may be required at
biosynthetic inhibitors, DFMA and CHA to the culture critical stages of growth and morphogenetic events, no
medium, at time of seed germination, inhibited bolting definitive data are available to establish their role as
and flower formation and this was partially reversed plant hormones.
by addition of exogenous Spd (Applewhite et al.,
2000). These results clearly show that Spd is involved
in flower initiation and development. Similar results 4. Manipulation of the polyamine pathway
have been reported in other plants also (reviewed by
Galston et al.,1997; Bais and Ravishankar, 2002). The PA pathway is ubiquitous in living organisms and
Many plant growth and development processes is relatively short (see Section 2) in terms of the
known to be regulated by plant hormones, such as number of enzymes involved. Most of the genes
auxins, 2,4-D, GA and ethylene, have also been coding for enzymes involved in the pathway have been
correlated with changes in PA metabolism. These cloned from different sources (Kumar et al., 1997;
changes occur in both endogenous levels of PAs and Walden et al., 1997; Galston et al., 1997; Tiburcio et
their biosynthetic enzymes and appear to be tissue al., 1997; Malmberg et al., 1998; Kumar and Minocha,
specific (reviewed by Galston and Kaur- 1998; Panicot et al., 2002b). Thus, the PA pathway
Polyamines in plants 5

represents an excellent model to test various which may correspond to the two gene copies
hypotheses and to answer fundamental biological encoding ADC, ADC1 and ADC2 (Watson et al.,
questions derived from pathway manipulation (Thu- 1998). The mutations have not been mapped and
Hang et al., 2002; Bhatnagar et al., 2002). therefore it cannot be excluded that other functions,
Initially, approaches to manipulate the PA pathway i.e. regulatory elements, are affected (Soyka and
made use of suicide inhibitors, but the effects of Heyer, 1999). More recently, Hanzawa et al. (2000)
DFMO and DFMA on ODC and ADC respectively, are reported that the inactivation of the Arabidopsis
variable in different plant systems, ranging from ACAULIS5 (ACL5) gene causes a defect in the
inhibition to stimulation or no effect and depending on elongation of stem internodes by reducing cell
the concentration, plant system tested and the expansion. It was suggested that ACL5 encodes a Spm
existence of compensatory mechanisms (Slocum and synthase, but the possibility that ACL5 may exhibit
Galston, 1987). Therefore, alternative approaches to broad amine substrate specificities and be involved in
manipulate polyamine metabolism have been the synthesis of other polyamines could not be
developed during the recent years. excluded (Hanzawa et al., 2000).
Thus far the only well characterized plant
4.1. Mutants polyamine biosynthetic mutant has been generated by
using reverse genetics. The availability of mutant
Mutants deficient in PA biosynthesis have been collections generated either by transposon or T-DNA
isolated from several biological systems. Hafner et al. tagging now facilitates the identification of knockouts
(1979) isolated PA mutants in Escherichia coli in any gene of interest using PCR-based mutant
showing decreased growth and increased sensitivity to screening techniques (Ferrando et al., 2002). By using
paraquat (Milton et al., 1990). Yeast mutants these techniques, Soyka and Heyer (2000) isolated an
presenting ODC as the sole pathway, show reduced Arabidopsis thaliana mutant line carrying an insertion
growth and altered sporulation on PA deficient of the En-1 transposable element at the ADC2 locus
medium (Cohn et al., 1980; Whitney and Morris, which should be regarded as a complete loss-of-
1978). Chinese hamster ovary cells lacking ODC function or knockout mutation. The ADC2 knockout
activity do not grow in medium lacking PA (Steglich mutant shows no obvious phenotype change under
and Scheffler, 1983) and a moderately reduced brood normal growth conditions, but is completely devoid of
size was observed in a Caenorhabditis elegans ODC ADC induction by osmotic stress. As ADC1 gene
deletion mutant (Macrae et al., 1995). Mutations in expression was not affected in the mutant, it was
genes affecting Spd and Spm biosynthesis have also concluded that ADC2 is the gene responsible for
been isolated in yeast. The spe3 Spd synthase mutation induction of ADC and PA biosynthesis under osmotic
causes a growth arrest, which can be complemented stress (Soyka and Heyer, 2000). More recently, Pérez-
with externally added Spd (Hamasaki-Katagiri et al., Amador et al. (2002) have shown that ADC2 gene
1997), while the yeast spe4 mutant is defective in Spm expression is induced in response to mechanical
biosynthesis (Hamasaki-Katagiri et al., 1998). wounding and methyl jasmonate treatment in
Less is known about mutants affecting PA Arabidopsis thaliana. All these observations appear to
metabolism in plants. Mutants with high levels of indicate that ADC2 is a key gene involved in the PA
ADC activity have been identified in petunia because response to abiotic stress in Arabidopsis. We envisage
of their abnormal morphology (Geerats et al., 1988), that the extensive use of functional genomics and
but the basis of the mutation is still not known. reverse genetic studies will facilitate the isolation of
Screening for resistance to the SAMDC inhibitor novel knock-out mutants affected in other PA
MGBG (Malmberg and Rose, 1987) or to inhibitory biosynthetic genes.
concentrations of Spm (Mirza et al., 1997), yielded
mutants that showed reduced sensitivity to the 4.2. Transgenic plants
respective agent, but these mutants have not been
further exploited for the analysis of PA function. With the availability of most of the genes involved in
Watson et al. (1998) isolated EMS mutants of A. PA metabolism, it has become possible to manipulate
thaliana that are reduced in ADC activity. The mutants this metabolic pathway using sense and antisense
fall into two complementation groups, spe1 and spe2, transgenic approaches. Thus, cellular PA content has
6 Ravindar Kaur-Sawhney et al.

been modulated by overexpression or down regulation SAMDC gives rise to smaller potato tubers without
of the key genes ODC, ADC or SAMDC (Kumar et al., affecting tuber yield (Rafart-Pedros et al., 1999). The
1997; Walden et al., 1997; Malmberg et al., 1998; distribution of tuber weights is of agronomic
Kumar and Minocha, 1998; Capell et al., 1998; Rajam importance, and generally a reduction of tuber-size
et al.,1998; Roy and Wu, 2001; Bhatnagar et al., 2002). variation is economically advantageous, so that more
Most of the studies have used the constitutive 35S tubers fall into a given size grade either for seed or
promoter, but only few of them were successful in ware (Rafart-Pedros et al., 1999). Similarly, fruit-
using either inducible (Masgrau et al., 1997; Panicot et specific expression of heterologous SAMDC in tomato
al., 2002a; Mehta et al., 2002) or tissue-specific resulted in ripening-specific accumulation of Spd and
promoters (Rafart-Pedros et al., 1999). Overexpression Spm which led to an increase in lycopene, prolonged
of heterologous ODC or ADC cDNAs generally causes vine life, and enhanced fruit juice quality (Mehta et al.,
the production of high levels of Put (DeScenzo and 2002). Besides the agronomic interest of this finding,
Minocha, 1993; Bastola and Minocha, 1995; Masgrau this latter study constitutes one of the most striking
et al., 1997; Capell et al., 1998; Bhatnagar et al., 2002; evidence regarding the in vivo involvement of
Panicot et al., 2002a), but in most cases only a small polyamines in a particular developmental process, i.e.
increase or even no change in Spd and Spm has been fruit ripening (Mehta et al., 2002).
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. 5. U n d e r s t a n d i n g t h e role of polyamines
ODC or ADC) are not accompanied by an increase in
subsequent biosynthetic reactions (i.e. Spd and Spm Phenotypic analyses of mutants and transgenic plants
biosynthesis) (Bhatnagar et al., 2002). In contrast, with altered PA levels gives further support to the
overexpression of genes located downstream of the previous physiological studies (see Section 3) with
pathway (i.e. SAMDC or SPDS) generally lead to regard to the involvement of these compounds in
increased levels of Spd or Spm or both (Thu-Hang et several plant processes (reviewed by Tiburcio et al.,
al., 2002; Mehta et al., 2002). Taken together these 2002). These include somatic embryogenesis (Bastola
results suggest that the levels of Spd and Spm in the and Minocha, 1995), stem elongation and flowering
cells are under a tight homeostatic regulation (Gerats et al., 1988; Masgrau et al., 1997; Hanzawa et
(Bhatnagar et al., 2002), which possibly could be al., 2000; Panicot et al., 2002a), root growth (Watson
related to a supramolecular organization of some of et al., 1998; Cordeiro et al., unpublished), tuber
these enzymes (see Section 5). development (Kumar et al., 1996; Rafart-Pedrós et al.,
Discrepancies observed among different studies 1999), fruit ripening (Mehta et al., 1997; 2002), abiotic
may have several causes. These include: transgene stresses (Minocha and Sun, 1997; Soyka and Heyer,
source, positional effects, recipient plant system, plant 1999; Roy and Nu, 2001). However, most of these
material analyzed and type of promoter used. A mutants and transgenic plants have not been further
hierarchical accumulation of polyamines in different exploited for the analysis of PA function. Application
transgenic tissues/organs has been observed (Lepri et of advanced genomic and proteomic approaches will
al., 2001). In general, less metabolically active tissues help to elucidate the role of PA in particular plant
accumulate higher levels of polyamines (Lepri et al., processes.
2001). These results are in line with experiments in
which metabolites such as vitamin A and 5.1. Genomic approaches
pharmaceutical antibodies accumulate at high levels in
seeds of different species. It is reasonable to assume The availability of complete genome sequences
that dormant or less metabolically active tissues permits the use of approaches to explore gene
provide a conducive environment for the accumulation expression variations on a large genome scale. Either
of transgenic products (Thu-Hang et al., 2002). In this cDNAs or large oligonucleotide collections are
regard, it should be stressed that the most remarkable attached on surfaces to create a microarray. The
results have been obtained by controlled expression of hybridisation of the microarray with fluorescent
transgenes using inducible or tissue-specific labelled RNA or cDNA yields an overall image of gene
promoters. For example, tissue-specific expression of expression or ‘transcriptome’ (Lockhart and Winzeler,
Polyamines in plants 7

2000). The global examination of gene expression (DBD) or the transcriptional activation domain (AD),
should reveal the coincidence of spatial and temporal is translationally fused to proteins of interest X or Y,
transcript expression profiles that may reflect a generating respectively the hybrid proteins X-DBD
requirement of co-ordinated gene product expression (bait) and Y-AD (prey). A powerful aspect of the yeast
in response to different type of signals. The technology molecular genetics involves the facility to isolate the
developed for the Arabidopsis genome has been corresponding cDNAs coding for proteins X or Y,
accelerated in the recent years both by public funding introduced in the form of plasmid DNA. This latter
through the Arabidopsis Functional Genomics feature immediately favored the use of this system to
Consortium in the USA and the GARNet in the UK, identify interacting partners for a given bait protein X
and also by private initiatives like Monsanto, using cDNA libraries as a prey (reviewed by Walhout
Affymetrix or Synteny/InCyte (Wisman and Ohlrogge, et al., 2000). The number of studies that have used
2000). proteomics in our field is still scanty. Here we will
Although there are already many examples in the provide two examples that demonstrate the potential of
literature showing the utility of this approach for these techniques to (i) unravel the role of PA in
unraveling complex plant responses and signal transcription; and (ii) to identify PA metabolons (see
transduction processes (Schena et al., 1995; Schaffer et below).
al., 2000), the use of this technology in our field is Although the potential role of PAs in affecting gene
unfortunately in its infancy. So far, DNA microarray expression had already been reported, the molecular
analysis has been used to reveal the induction of ADC mechanisms underlying their effects were unknown
genes during drought stress (Ozturk et al., 2002) or in (Wang et al., 2002). The identification of a polyamine
response to wounding and methyl jasmonate treatment responsive element and corresponding transacting
(Sasaki et al., 2001; Pérez-Amador et al., 2002). protein factors that respond to polyamines has opened
We envisage that global analysis of gene up an exciting new area to study the function of these
expression in well characterized mutant and transgenic compounds in transcription (Wang et al., 1999). By
plants with altered polyamine metabolism will provide using the two-hybrid system, it was recently found that
novel clues in the near future for understanding the the human homologue of the Arabidopsis subunit
molecular mechanisms underlying polyamine effects COP9 signalosome complex binds to such transacting
on plant growth and development. protein factors with the potential to directly affect gene
expression (Wang et al., 2002). Remarkably, the COP9
5.2. Proteomic approaches signalosome proteins were first identified in
Arabidopsis and have been demonstrated to form a
Proteomics’ uses biochemical approaches aimed at regulatory complex involved in light-activated
systematically characterizing the ‘proteome’ or the development and playing a role in intracellular
‘protein complement of the genome’ (Wasinger et al., signalling (Deng et al., 2000). We envisage that similar
1995) in a given organism, tissue, cell or subcellular type of experiments will be performed in the plant PA
compartment. The means of proteome characterization field that hopefully will provide new insights into the
include protein localization, expression and most role of PAs in plant signal transduction.
importantly protein interaction maps. A plethora of Increasing number of reports document that many
innovative procedures has been employed in recent metabolic reactions are catalysed by complexes of
years for the large-scale analysis of protein signalling sequentially acting enzymes that show highly ordered
pathways, including the yeast two-hybrid system structural organization (reviewed in Srere, 1987). In
(Fields and Song, 1989), protein purification methods such multienzyme complexes the metabolites pass
linked to detection by mass spectrometry (Neubauer et from one active enzyme site to the next through a
al., 1997; Verma et al., 2000); protein localization process termed ‘substrate channeling’. The
(Ferrando et al., 2000; 2001; Farràs et al., 2001), and supramolecular arrangement of enzymes involved in
protein microarray techniques (Zhu et al., 2001). such metabolic reactions is referred to as ‘metabolon’.
The yeast two-hybrid system is a genetic tool to Metabolons are multienzyme complexes in both
describe in vivo protein interactions using the yeast prokaryotes and eukaryotes that represent highly
cell as a test tube. Each separated module of the GAL4 organized assemblies of sequential enzymes in a
transcription factor, either the DNA binding domain metabolic pathway and are thought to provide
8 Ravindar Kaur-Sawhney et al.

increased metabolic efficiency and higher substrate DNA replication, transcription of genes, cell division,
selectivity. Metabolons may also help to coordinate the organ development, fruit development and ripening,
activities of enzymes by sharing intermediates in a leaf senescence and abiotic stresses. Despite ample
given pathway, as well as to ensure protection of labile evidence of their involvement in these processes, their
substrates and sequestration of toxic intermediates precise role in these specific processes remains to be
(Sugumaran et al., 2000). In addition, the formation of established. Recent developments of PA-deficient
multienzyme metabolon complexes may enhance mutants and transgenic plants as well as of
enzyme stability, improve enzymatic performance and molecular genetic investigations should further our
provide a means for adaptation to alterations of input understanding of their role in plant growth and
of metabolic reactions, especially during demanding development.
physiological conditions (Abadjieva et al., 2001). The polyamine pathway is now amenable to
The relevant information about intrinsic properties modulation by genetic approaches because it has been
of ‘metabolon’ formation can be acquired by studies of elucidated molecularly and biochemically in plants.
protein-protein interactions using modern proteomic Reverse genetics has identified an Arabidopsis
approaches (Ferrando et al., 2002). In this regard, our knockout mutation of ADC2 gene which reveals
laboratory has recently analyzed possible interactions inducibility by osmotic stress. Extensive use of
between the SPDS and SPMS enzymes of polyamine functional genomics and reverse genetics studies will
biosynthetic pathway in the yeast two-hybrid system facilitate the isolation of novel knockout mutants
(Panicot et al., 2002b). Using the Arabidopsis affected in other polyamine metabolic genes. Sense
spermidine synthase as bait, two similar proteins were and antisense transgenic approaches have revealed the
identified to interact with SPDS2 that were named feasibility of modulating cellular PA contents.
SPDS1 and SPMS. Yeast and bacterial mutant Generally, genetic manipulation of single steps located
complementation tests revealed that SPDS1 encodes a upstream of the PA pathway (i.e. ODC or ADC) lead to
novel spermidine synthase, whereas SPMS displays elevated levels of Put, but no changes occur in the
spermine synthase activity. The heterodimerization higher PAs, Spd and Spm. By contrast, overexpression
capabilities of enzymes catalyzing the two last steps of of genes located downstream of the pathway (i.e.
polyamine biosynthesis were also demonstrated in vivo SAMDC or Spd synthase) generally leads to increased
by co-immunoprecipitation using epitope tagged levels of Spd and Spm, indicating that the levels of Spd
SPDS1, SPDS2 and SPMS proteins (Ferrando et al., and Spm are under a tight homeostic cellular control.
2000; Ferrando et al., 2001). Immunoaffinity Phenotypic analyses of mutants and transgenic plants
purification and size fractionation of SPDS and SPMS affected in polyamine metabolism further support
enzymes labeled with different HA and c-Myc previous physiological evidence, but the molecular
epitopes revealed that the SPDS and SPMS proteins mechanisms underlying PA effects on plant growth and
co-purify with large multiprotein complexes of 650 to development remain to be elucidated. Global analysis
750 kDa. Further analysis of subunits of isolated of gene expression by using the available DNA
SPDS-SPMS metabolon(s) by mass spectrometry is microarray genomic techniques will help to understand
expected to yield important information about yet the role of these compounds. The potential of
unknown regulatory subunits of SPDS-SPMS proteomics to unravel the role of polyamines in
metabolon in the PA biosynthesis pathway. The particular cellular processes is also examined. We
available data support the conclusion that Spd envisage that the extensive use of the two-hybrid
synthesized by SPDS is effectively channeled to system and other proteomic approaches will provide
SPMS to control the formation of the end-product Spm new insights into the role of PAs on plant signal
thereby regulating the synthesis of high molecular transduction. Furthermore, we provide evidence that
weight polyamines (Panicot et al., 2002b). proteomics is an excellent tool to unravel
supramolecular organizations of PA metabolic
enzymes which may help to understand homeostatic
6. Conclusions control of this metabolic pathway.

Considerable evidence indicates that polyamines are


involved in a wide array of plant processes, including
Polyamines in plants 9

Acknowledgements and Tiburcio AF. Arginine decarboxylase is localized in


chloroplasts. Plant Physiol. 109: 771-776, 1995.
AFT acknowledges the grants from Ministerio de Cabanne F, Dalebroux MA, Martin-Tanguy J and Martin C.
Ciencia y Tecnología BIO-99-453 and BIO-2002- Hydroxycinnamic acid amides and ripening to flower of
Nicotiana tabacum L. var. Xanthi n.c. Physiol Plant.
04459-C02-02.
53: 399-404, 1981.
Capell T, Escobar C, Lui H, Burtin D, Lepri O and Christou P.
Overexpression of the oat arginine decarboxylases
R e f e r ences cDNA in transgenic rice affects normal development
patterns in vitro and results in putrescine accumulation in
Abadjieva A, Pauwels K, Hilven P and Crabeel M. A new transgenic plants. Theor Appl Genet. 97:246-254, 1998.
yeast metabolon involving at least the two first enzymes Cohen SS. A Guide to the Polyamines. Oxford University
of arginine biosynthesis. J Biol Chem. 276: 42869- Press. New York, NY, 1998.
42880, 2001. Cohn M, Tabor CW and Tabor H. Regulatory mutations
Apelbaum A, Burgoon A-C, Anderson JD, Lieberman M, affecting ornithine decarboxylase activity in S.
Ben-Arie R and Mattoo AK. Polyamines inhibit cereviseae. J Bacteriol. 142: 792-799, 1980.
synthesis of ethylene in higher plants. Plant physiol. Deng XW, Dubiel W, Wei N, Hofmann K and Mundt K.
68: 453-456, 1981. Unified nomenclature for the COP9 signalosome and its
Apelbaum A, Goldlust A and Icekson I. Control by ethylene subunits: An essential regulator of development. Trends
of arginine decarboxylase activity in pea seedlings and Genet. 16: 289, 2000.
its implication for hormonal regulation of plant growth. DeScenzo RA and Minocha SC. Modulation of cellular
Plant Physiol. 79: 635-640, 1985. polyamines in tobacco by transfer and expression of
Applewhite PB, Kaur-Sawhney R and Galston AW. A role of mouse ornithine decarboxylase cDNA. Plant Mol Biol.
spermidine in the bolting and flowering of Arabidopsis. 22: 113-127, 1993.
Physologia Plantarum. 108: 314-320, 2000. Egea-Cortines M and Mizrahi Y. Polyamines in cell division,
Bagni N, Torrigiani P and Barbieri P. Effect of various fruit set and development and seed germination. In:
inhibitors of polyamine synthesis on the growth of Biochemistry and Physiology of Polyamines in Plants.
Helianthus tuberosus. Med Biol. 59: 403-409, 1981. Slocum RD and Flores HE (Ed). CRC Press, Boca
Bagni N and Pistocchi R. Uptake and transport of polyamine Raton, Florida, USA. 1991.
and inhibitors of polyamine metabolism in plants. In: Evans PT and Malmberg RL. Do polyamines have a role in
Biochemistry and Physiology of Polyamines in Plants. plant development? Annu Rev Plant Physiol Plant Mol
Slocum RD and Flores HE (Ed). CRC Press Inc, Boca Biol. 40: 235-269, 1989.
Raton, FL USA 105-120, 1991. Even-Chen Z, Mattoo AK and Goren R. Inhibition of
Bais HP and Ravishankar GA. Role of polyamines in the ethylene biosynthesis by aminoethoxyornylglycine and
ontogeny of plants and their biotechnological by polyamines shunt label from C14-methionine into
applications. Plant Cell, Tissue and Organ Culture. spermidine in aged orange peel discs. Plant Physiol. 69:
69: 1-34, 2002. 385-388, 1982.
Bastola DR and Minocha SC. Increased putrescine Farràs R, Ferrando A, Jásik J, Kleinow T, Ökresz L,
biosynthesis through transfer of mouse ornithine Tiburcio AF, Salchert K, del Pozo C, Schell J and Koncz C.
decarboxylase cDNA in carrot promotes somatic SKP1-SnRK protein kinase interactions mediate
embryogenesis. Plant Physiol. 109: 63-71, 1995. proteasomal binding of a plant SCF ubiquitin ligase.
Bey P, Danzin C and Jung M. Inhibition of basic amino acid EMBO J. 20: 2742-2756, 2001.
decarboxylases involved in polyamine biosynthesis. In: Feirer RP, Mignon G and Litvay JD. Arginine decarboxylase
Inhibition of Polyamine Metabolism. McCann PP, Pegg and polyamines required for embryogenesis in wild
AE and Sjoerdsma A (Ed). Academic Press, Orlando, carrot. Science. 223: 1433-1434, 1984.
USA 1-32, 1987. Ferrando A, Farràs R, Jasik J, Schell J and Koncz C. Intron-
Bhatnagar P, Minocha R and Minocha S. Genetic tagged epitope: A tool for facile detection and
manipulation of the metabolism of polyamines in poplar purification of proteins expressed in Agrobacterium-
cells. The regulation of putrescine catabolism. Plant transformed plant cells. Plant J. 22: 553-560, 2000.
Physiol. 128:1455-1469, 2002. Ferrando A, Koncz-Kálmán Z, Farràs R, Tiburcio AF,
Bitonti AJ, Carara PJ, McCann PP and Bey P. Catalytic Schell J and Koncz C. Detection of in vivo protein
irreversible inhibition of bacterial and plant arginine interactions between Snf1-related kinase subunits with
decarboxylase activities by novel substrate and product intron-tagged epitope-labelling in plants cells. Nucleic
analogues. Biochem J. 242: 69-74, 1987. Acids Res. 29: 3685-3693, 2001.
Borrel A, Culiañez-Marcià, Atabella T, Besford RT, Flores D Ferrando A, Altabella T, Koncz C and Tiburcio AF.
10 Ravindar Kaur-Sawhney et al.

Proteomics: Emerging tools to characterize plant Biochemistry. 20: 3163-3166, 1981.


metabolons. Curr Top Plant Biol. In press. Kaur-Sawhney R, Flores HE and Galston AW. Polyamine
Fields S and Song O. A novel genetic system to detect oxidase in oat leaves: A cell wall-localized enzyme.
protein-protein interactions. Nature. 340: 245-246, 1989. Plant Physiol. 68: 494-498, 1981.
Fuhrer J, Kaur-Sawhney R, Shih LM and Galston AW. Kaur-Sawhney R, Shih Flores HE. and Galston AW.
Effects of exogenous 1,3-diaminopropane and Relation of polyamine synthesis and titer to aging and
spermidine on senescence of oat leaves. II. Effects of senescence in oat leaves. Plant Physiol. 69: 405-410,
polyamines on ethylene biosynthesis. Plant Physiol. 1982.
70: 1597-1600, 1982. Kaur-Sawhney R, Tiburcio AF and Galston AW. Spermidine
Galston AW and Tiburcio AF (Ed). Lecture Course on and flower bud differentiation in thin-layer explants of
Polyamines as Modulators of Plant Development 257: tobacco. Planta. 173: 282-284, 1988.
Fundacion Jaun Madrid, March, 1991. Kumar A, Taylor MA, Mad Arif SA and Davies HV. Potato
Galston AW and Kaur-Sawhney R. Polyamines as plants expressing antisense and sense SAMDC
endogenous growth regulators. In: Plant Hormones, transgenes show altered levels of polyamines and
Physiology, Biochemistry and Molecular Biology ethylene: Antisense plants display abnormal phenotypes.
(2nd edn). Davies PJ (Ed). Kluwer Academic Publishers, Plant J. 9: 147-158, 1996.
Dordrecht, The Netherlands. 158-178, 1995. Kumar A, Altabella T, Taylor MA and Tiburcio AF. Recent
Galston AW, Kaur-Sawhney R, Altabella T and Tiburcio AF. advances in polyamine research. Trends Plant Sci.
Plant polyamines in reproductive activity and response 2: 124-130, 1997.
to abiotic stress. Bot Acta. 110:197-207, 1997. Kumar A and Minocha SC. Transgenic manipulation of
Gerats AGM, Kaye C, Collins C and Malmberg ML. polyamine metabolism. In: Transgenic Plant Research.
Polyamine levels in Petunia genotypes with normal and Lindsey K (Ed). Academic Publishers. Harwood.
abnormal floral morphologies. Plant Physiol. 86: 390- 187-199, 1998.
393, 1988. Lepri O, Bassie L, Safwat G, Thu-Hang P, Trung-Nghia P,
Hafner EW, Tabor CW and Tabor H. Mutants of E.coli that Hölttä E, Christou P and Capell T. Over-expression of
do not contain 1,4-diaminobutane (putrescine or the human ornithine decarboxylase cDNA in transgenic
spermidine). J Biol Chem. 254: 12419-12426, 1979. rice plants alters the polyamine pool in a tissue-specific
Hamasaki-Katagiri N,Tabor CW and Tabor H. Spermidine manner. Mol Gen Genet. 266:303-312, 2001.
biosynthesis in S. cereviseae: Polyamine requirement of Lockhart DJ and Winzeler EA. Genomics, gene expression
a null mutant of the SPE3 gene (spermidine synthase). and DNA arrays. Nature. 405: 827-836, 2000.
Gene 187:35-43. 1997. Macrae M, Plasterk RHA and Coffino P. The ornithine
Hamasaki-Katagiri N, Katagiri Y, Tabor CW and Tabor H. decarboxylase gene of Caenorhabditis elegans-cloning,
Spermine is not essential for growth of S. cereviseae: mapping and mutagenesis. Genetics. 140:517-525, 1995.
Identification of the SPE4 gene (spermine synthase) and Malmberg RL and Rose DJ. Biochemical genetics of
characterization of a spe4 deletion mutant. Gene. resistance to MGBG in tobacco: Mutants that alter SAM
210: 195-201, 1998. decarboxylases or polyamine ratios and floral
Hanzawa Y, Takahashi T, Michael AJ, Burtin D, Long D, morphology. Mol Gen Genet. 207: 9-14, 1987.
Pineiro M, Coupland G and Komeda Y. ACAULIS5, an Malmberg RL, Watson MB, Galloway GL and Yu W.
Arabidopsis gene required for stem elongation, encodes Molecular genetic analyses of plant polyamines. Critical
a spermine synthase. EMBO J. 19: 4248-4256, 2000. Rev Plant Sci. 17: 199-224, 1998.
Heimer YM and Mizrahi Y. Characterization of ornithine Martin-Tanguy J. The occurrence and possible function of
decarboxylase of tobacco cells and tomato ovaries. hydroxy-cinnamoyl acid amides in plants. Plant Growth
Biochem J. 201: 373-376, 1982. Regul. 3: 383-399, 1985.
Hibasami H, Tanaka M, Nagai J and Ikeda T. Martin-Tanguy J. Metabolism and function of polyamines in
Dicyclohexylamine, a potent inhibitor of spermidine plants: Recent development (new approaches). Plant
synthase in mammalian cells. FEBS Letters. 116: 99- Growth Regul. 34: 135-148, 2001.
101, 1980. Masgrau C, Altabella T, Farrás R, Flores D, Thompson AJ,
Icekson I, Goldlust A and Apelbaum A. Influence of ethylene Besford RT and Tiburcio AF. Inducible overexpression
on S-adenosylmethionine activity in etiolated pea of oat arginine decarboxylase in transgenic tobacco
seedlings. J Plant Physiol. 119: 335-345, 1985. plants. Plant J. 11: 465-473, 1997.
Kakkar RJ and Rai VK. Plant polyamines in flowering and Mehta RA, Handa A, Li N and Mattoo AK. Ripening-
fruit ripening. Phytochemistry. 33: 1281-1288, 1993. activated expression of S-adenosylmethionine
Kallio A, McCann PP. and Bey P. DL-α (Difluoromethyl) decarboxylase increases polyamine levels and influences
arginine: A potent enzyme-activated irreversible ripening in transgenic tomato fruits (abstract no. 134).
inhibitor of bacterial arginine decarboxylase. Plant Physiol. 114: S-44, 1997.
Polyamines in plants 11

Mehta RA, Cassol T, Li N, Ali N, Handa AK and Mattoo AK. Roy M and Wu R. Arginine decarboxylase transgene
Engineered polyamine accumulation in tomato enhances expression and analysis of environmental stress
phytonutrient content, juice quality and vine life. Nat tolerance in transgenic rice. Plant Sci. 160: 869-875, 2001.
Biotech. 20: 613-618, 2002. Sasaki Y, Asamizu E, Shibata D, Nakamura Y, Kaneko T,
Milton KW, Tabor H and Tabor CW. Paraquat toxicity is Awai K, Amagai M, Kuwata C, Tsugane T, Masuda T,
increased in E. coli defective in the synthesis of Shimada H, Takamiya K, Ohta H and Tabata S.
polyamines. P roc Natl Acad Sci USA. 87: 2851-2855, Monitoring of methyl jasmonate-responsive genes in
1990. Arabidopsis by cDNA macroarray: self-activation of
Minocha SC and Sun D. Stress tolerance in plants through jasmonic acid biosynthesis and crosstalk with other
transgenic manipulation of polyamine biosynthesis phytohormone signaling pathways. DNA Res. 8: 153-
(abstract no. 1552). Plant Physiol. 114: S-297, 1997. 161, 2001.
Mirza JI and Iqbal M. Spermine-resistant mutants of Schaffer R, Langraf J, Pérez-Amador MA and Wisman E.
Arabidopsis thaliana with developmental abnormalities. Monitoring genome-wide expression in plants. Curr
Plant Growth Regul. 22:151-156, 1997. Opin Biotechnol. 11: 162-167, 2000.
Montague MJ, Koppenbrink JW and Jaworski EG. Schena M, Shalon D, Davis RW and Brown PO. Quantitative
Polyamine metabolism in embryogenic cells of Daucus monitoring of gene expression patterns with a
carota. Plant Physiol. 62: 430-433, 1978. complementary DNA microarray. Science. 270: 467-470,
Muhitch MJ, Edwards LA and Fletcher JS. Influence of 1995.
diamines and polyamines on the senescence of plant Slocum RD, Kaur-Sawhney R and Galston AW. The
suspension cultures. Plant Cell Rep. 2: 82-84, 1983. physiology and biochemistry of polyamines in plants.
Neubauer G, Gottschalk A, Fabrizio P, Seraphin B, Arch Biochem Biophys. 325: 283-303, 1984.
Luhrmann R, and Mann M. Identification of the proteins Slocum RD and Galston AW. Changes in polyamine
of the yeast U1 small nuclear ribonucleoprotein complex biosynthesis associated with post-fertilization growh and
by mass spectrometry. P roc Natl Acad Sci USA. development in tobacco ovary tissue. Plant Physiol.
94: 385-390, 1997. 79: 336-343, 1985.
Ozturk ZN, Talame V, Deyholos M, Michalowski CB, Slocum RD and Galston AW. Inhibition of polyamine
Galbraith DW, Gomukirmizi N, Tuberosa R and biosynthesis in plants and plant pathogenic fungi. In:
Bohnert HJ. Monitoring large-scale changes in transcript Inhibition of Polyamine Metabolism. Biological
abundance in drought- and salt-stresses barley. Plant Mol Significance and Basis for New Therapies. McCann PP,
Biol. 48: 551-573, 2002. Pegg AE and Sjoerdsma A (Ed). Academic Press,
Panicot M, Masgrau C, Borrell A, Cordeiro A, Tiburcio AF New York. 305-316, 1987.
and Altabella T. Effects of putrescine accumulation in Slocum RD. Polyamine biosynthesis in plants. In:
tobacco transgenic plants with different expression of oat Biochemistry and Physiology of Polyamines in Plants.
arginine decarboxylases. Physiol Plant. 114:281-287, Slocum RD and Flores HE (Ed). CRC Press, Boca
2002a. Raton, FL, USA. 22-40, 1991a.
Panicot M, Minguet E, Ferrando A, Alcázar R, Blázquez MA, Slocum RD. Tissue and subcellular localisation of
Carbonell J, Altabella T, Koncz C and Tiburcio AF. polyamines and enzymes of polyamine metabolism. In:
A polyamine metabolon involving aminopropyl Biochemistry and Physiology of Polyamines in Plants.
transferases complexes in Arabidopsis. Plant Cell. Slocum RD and Flores HE (Ed). CRC Press, Boca
2002b. In press. Raton, FL, USA. 93-103, 1991b.
Pérez-Amador MA, León J, Green PJ and Carbonell J. Smith TA and Marshall JHA. The di and polyamine oxidases
Induction of arginine decarboxylase ADC2 gene of plants. In: P rogress in Polyamine Research (Advances
provides evidence for the involvement of polyamines in in Experimental Biology and Medicine, 250) Plenum
the wound response in Arabidopsis. Plant Physiol. Press, New York. 573-587, 1988.
In press. Soyka S and Heyer AG. Arabidopsis knockout mutation of
Rafart-Pedros A, Mac Leod MR, Ross HA, McRae D, ADC2 gene reveals inducibility by osmotic stress. FEBS
Tiburcio AF, Davies HD and Taylor M. Manipulation of Lett. 458: 219-223, 1999.
the S-adenosylmethionine decarboxylase transcript level Srere PA. Complexes of sequential metabolic enzymes.
in potato tubers. Over-expression leads to an increase in Annu Rev Biochem. 56: 89-124, 1987.
tuber number and a change in tuber size distribution. Steglich C and Schefler IE. Selection of ornithine
Planta. 209: 153-160, 1999. decarboxylase-deficient mutants of Chinese hamster
Rajam MV, Dagar S, Waie B, Yadav JS, Kumar PA, Shoeb F ovary cells. Methods Enzymol. 94: 108-111, 1983.
and Kumria R. Genetic engineering of polyamine and Sugumaran M, Nellaiappan K, Amaratunga C, Cardinale S
carbohydrate metabolism for osmotic stress tolerance in and Scott T. Insect melanogenesis. III. Metabolon
higher plants. J Biosci. 23:473-482, 1998. formation in the melanogenic pathway. Regulation of
12 Ravindar Kaur-Sawhney et al.

phenoloxidase activity by endogenous dopachrome Williams KL and Humphery-Smith I. Progress with


isomerase. Arch Biochem Biophys. 378: 393-403, 2000. gene-product mapping of the Mollicutes: Mycoplasma
Suttle JC. Effect of polyamines on ethylene production. genitalium. Electrophoresis. 16: 1090-1094, 1995.
Phytochemistry. 20: 1477-1480, 1981. Watson MB, Emory KK, Piatak RM and Malmberg RL.
Tabor CW and Tabor H. Polyamines. Annu Rev Biochem. Arginine decarboxylase (polyamine synthesis) mutants
5: 749-790, 1984. of Arabidopsis thaliana exhibit altered root growth.
Thu-Hang P, Bassie L, Safwat G, Trung-Nghia P. Christou P Plant J. 13: 231-239, 1998.
and Capell T. Expression of a heterologous S- Whitney P and Morris D. Polyamine auxotrophs of
adenosylmethionine decarboxylase cDNA in plants S. cereviseae. J Bacteriol. 134: 214-220.
demonstrates that changes in SAMDC activity determine Williams-Ashman HG and Schenone A. Methyl-glyoxyl-bis
levels of the higher polyamines spermidine and (guanylhydrazone) as a potent inhibitor of mammalian
spermine. Plant Physiol. 129:1744-1754, 2002. and yeast S-adenosylmethionine. Biochem Biophys Res
Tiburcio AF, Kaur-Sawhney R and Galston AW. Polyamine Commun. 46: 288-295, 1972.
metabolism. In: Intermedatory Nitrogen Metabolism. Wisman E and Ohlrogge J. Arabidopsis microarray service
16, The Biochemistry of Plants. Miflin BJ. and Lea PJ facilities. Plant Physiol. 124: 1468-1471, 2000.
(Ed). Academic Press. 283-325, 1990. Young ND and Galston AW. Are polyamines transported in
Tiburcio AF, Campos JL, Figueras X and Besford RT. Recent etiolated peas? Plant Physiol. 73: 912-914, 1983.
advances in the understanding of polyamine functions Zhu H, Bilgin M, Bangham R, Hall D, Casamayor A,
during plant development. Plant Growth Regul. 12: 331- Bertone P, Lan N, Jansen R, Bidlingmaier S, Houfek T,
340, 1993. Mitchell T, Miller P, Dean RA, Gerstein M and
Tiburcio AF, Altabella T, Borrell A and Masgrau C. Snyder M. Global analysis of protein activities using
Polyamine metabolism and its regulation. Physiol Plant. proteome chips. Science. 293: 2101-2105, 2001.
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 N1-
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,
Journal of Cell and Molecular Biology 2: 13-18, 2003. 13
Haliç University, Printed in Turkey.

P h e n o l i c c y c l e i n p l a n t s a n d e n v i ro n m e n t
Valentine I. Kefeli1, Maria V. Kalevitch2* and Bruno Borsari3
1
Slippery Rock Watershed Coalition, 3016 Unionville Rd., Cranberry Twp., PA 16066, USA; 2Robert
Morris University, 881 Narrows Run Rd., Moon Township PA 15108, USA; 3Slippery 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.

Key words: Allelopathy, chloroplasts, humus, phenolics, soil micelle

B i t k i l e r d e f e n o l i k d ö n g ü v e ç e v re

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.

Anahtar sözcükler: Allelopati, kloroplastlar, humus, fenolikler, toprak miseli

I n t ro d u c t i o n and their fragments contribute to the mineralization of


soil nitrogen and humus formation. Thus, humus
Phenolics are very stable products in plant organisms. participates actively in fulfilling plants nutritional
Generally, they are characterized by a benzene ring needs and growth. Light enhances the biosynthesis of
and one hydroxyl group (-OH). They can be converted phenolic substances in plant chloroplasts and these
into lignin which is the main phenolic polymer in constitute in addition to soil micelles (humus) a second
plants. Microorganisms break down these molecules formation site for this diverse group of organic
14 Valentine I. Kefeli et al.

molecules. It should be mentioned however, that


COOH CHO
phenolics tend to accumulate in plant vacuoles in I
HCOH
C— O
relatively high amounts, or they deposit in the I HCOH
CH2
secondary cell wall as lignin. CH2O
Phosphoenol- Dehydroquinic acid
pyruvic acid 5-Dehydroshikimic acid Erythrose-4-phosphate
2-keto- COOH
3-desoxy-
C h l o ro p l a s t s a s c e n t e r s o f p h e n o l i c s b i o s y n t h e s i s 7-phospho- Shikimic acid
D-Araboheptonic acid
OH OH
Experiments with chloroplasts of willow (Salix spp.) OH
HOOC CH2-CO-COOH
leaves showed that the synthesis of phenol-carboxylic COOH
Chorismic acid
acids and flavonoids is strongly stimulated by light
exposure. Metabolic inhibitors that depress
OH NH2
photosynthetic activity (simazine, diurone, Prephenic acid Anthranilic acid
chloramphenicol), affect negatively the biosynthesis of
CH2-CH-COOH CHOH-CHOH-CH2O
flavonoids. Leaves chloroplasts have the capability to
localize phenol compounds, some of which are N
specific to these organelles only. The chloroplasts of
Phenylalanine Indolylglycerophosphate
spring willow leaves contain more phenols than the
chloroplasts of the same leaves in the autumn. Light is CH-CH-COOH
CH2-CO-COOH
a mandatory condition to initiate phenolics synthesis
and this is indicated also by the lack of such molecules N
I
in the protoplastids of etiolated willow shoots (Kefeli OH
H Indolyl-acetic acid
p-Coumaric acid IAA
and Kalevitch, 2002). Light appears also to induce
flavonols synthesis in the chloroplasts and cytoplasm. F i g u re 1: Phenol-propanoids in metabolic bifurcation.
Chalcone and phenolcarbonic acid present in etiolated
willow shoots can be considered metabolic precursors
of light-synthesized flavonols. In certain cell Muzafarov and collaborators (1992) investigated
compartments (vacuoles and cell wall) phenols are the functions of some phenolics in chloroplasts. They
contained in significant amounts (Lewis and assumed that the essence of the relationship between
Yamamoto, 1990). However, it is not clear yet how photosynthesis and phenolics biosynthesis is that
phenols are translocated within plant cells and how phenolics exert a direct and an indirect effect on the
they affect the function of cell organelles such as process of solar accumulation itself. From our point of
ribosomes and mitochondria. Phenolic substances that view, flavonoids as polyfunctional compounds in
inhibit plant growth (hydroxy derivatives of cinnamic green plastids fulfill three major functions as:
acid, coumarin and naringenin) are synthesized • substrates (use polyphenols and their catabolic
similarly to other phenolics. The synthesis of growth products for other kinds of biosynthesis);
inhibitor derivatives of hydroxycinnamic acids follows • energy sources (electron and proton transport, ion
the pathway: shikimic acid-chorismic acid-prephenic exchange and membrane potential, radicals
acid-cinnamic acid and p-coumaric acid. A theory of formation);
metabolism bifurcation among phenolic substances, • regulators (involvement in enzyme reactions as
some of which can inhibit growth and synthesis of inhibitors or activators).
indolic compounds has been proposed. According to During photosynthesis under light, flavonoids
this new approach, indolil-3-acetic acid (IAA) change the rate of electron transport and
becomes the main natural auxin (Kefeli, 1978; Kefeli photophosphorylation, bringing about the change of
and Dashek, 1994; Kefeli and Kalevitch, 2002). ATP/NADPH ratio. In the reactions of carbon
Therefore, indole auxins (IAA, indoleacetonitrile) as metabolism they can shift the dynamic equilibrium of
well as phenolic inhibitors (p-coumaric acid, pentosephosphate reduction cycle to enhance the
coumarin, naringenin and others) are derived from the synthesis of certain metabolites both due to the change
common precursors, shikimic and chorismic acids in energy substrate intake and to the interaction with
(Figure 1). enzymes of the cycle. Additionally, flavonoids
Phenolic cycle 15

exercise a feedback control over their own


OH
biosynthesis, although this phenomenon is not clearly 2 3
6´ C CH = CH ´OH
understood. This questionable situation remains as the 5´


2´ O
1
6 5
4

HO 3´
biosynthesis of the entire flavonoid structure within O O6H10O5
Isosalipurposide
plastids has not been explained, nor the complete 1 2´, 4´, 6´, 4-tetroxychalcone-
enzymatic package of their biosynthesis has been -2´-glucoside of chalconarin-
O H genin (chalcone)
discovered yet. Lack of direct eveidence of flavonoids HO 8 1 C
2´ 3´
1´ 4´ OH
7 2
transport within the cell and through the whole plant 6
5 4
3
CH2
6´ 5´

constitues another challenge to a more accurate O


C
O
description of their functions. Noneless, a variety of C 6H 10O 3
Salipurposide
phenolic compounds, present simultaneously within naringenin-5-glucoside
cells appear to be capable of influencing the rate and 2 (Flavanone-glycoside)
direction of plants metabolic activities. Thus, any
change in the flavonoid structure, or qualitative O H
2´ 3´
HO 8 1 C OH
composition of the phenol complex result in a change 7
6
2
3
1´ 4´
6´ 5´
5 4 CH2
of the mechanism of its effect upon the processes of OH
C
cell energy exchange. O
3 Naringenin
Chalcone and phenolcarbonic acids present in (Flavanone)
etiolated willow shoots can be viewed as the potential Eriodictyol
precursors of light synthesized flavonoids. However, (Flavanone) 4
the use of paper chromatography to investigate Luteolin
isosalidpurposide transformation products did not (Flavone)

reveal the presence of any flavonols sensitive to


F i g u re 2: Flavonoid biosynthesis.
conventional reagents. Therefore, the transformation
of chalcone (isosalpurposide) in lightless vitro appears
to terminate at a second stage. The synthesis of P h e n o l i c s u b s t a n c e s s e c re t e d b y ro o t s a n d l e a c h e d
eriodyctiol and luteolin that occurred in willow leaves f rom leaves
evidently took place in vivo and under light exposure.
It should be pointed out however, that phloridzin and Plants contain and secrete a diverse group of growth
isosalipurposide were decomposed from aglycone and inhibiting substances that may affect other plants
that phloridzin and phloretin produced yellow stains development, if grown in their vicinity (allelopathy).
on the chromatogram as well as flavonoids. It is known Leaf exudates of willow species such as Salix rubra or
that flavonoid glycosides are revealed as dark spots on Salix viminalis, contain phenolic inhibitors like
chromatograms exposed to UV light. Therefore, our naringenin derivative isosalipurposide. Other species
yellow stains were classified as flavanones, since they instead like apple trees (Malus spp.) contain
did not react with AlCl3, nor Na2CO3 like flavonols, phloridzin, which is a strong respiratory inhibitor.
that also form yellow spots. At the same time, similar Roots and leaves of the wild plant Nanaphyton native
to chalcones and aurones, these floridzin to semi-desert regions of Mongolia contain also strong
transformation products are yellow colored and they phenolic inhibitors. Seed as well may secrete
turn into orange-pink when exposed to Na2CO3 or allelochemicals. Tobacco seed (Nicotiana tabacum)
NH4OH. Relatively easy transformations of for example suppress germination of its own seed
isosalipurposide and phloridzin into compounds of when leachates come in contact with the seeds (Kefeli
other classes (flavanones, chalcones, or aurones) and Kalevitch, 2002). Although the inhibition of
evidenced the role of these products in the general germination was observed at various levels of
metabolism of flavonoids (Figure 2). 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.
16 Valentine I. Kefeli et al.

Hence, molecular genetics becomes a tool, which may excreted substances had an allelopathic nature and
help to regulate the level of secondary metabolites in were involved in developing ecological relationships
plants. Therefore, there is a need continue the search with adjacent plants of different species.
for botanical herbicides as a rise of ecological During the composting process water extracts
concerns has clearly identified the environmental contain many inhibiting substances that might form
impact of herbicides of synthesis. toxic exudates (Kefeli et al., 2001). Paper
Root exudates affect the germination of seeds of chromatography reveals the presence of phenolic acids
different crops: monocots and dicots (Table 1 and and coumarins in water extracts. The highest
Table 2). However, it must be pointed out that only concentrations of these inhibitors was measured in
some phenolics were studied in the exudates of willow abscised leaves of red maple (Acer rubrum L.). One
roots (1) which have no analogues in the roots (2) and gram of dry leaves was mixed in 29 ml of water to
leaves found among the common allelopathogens. prepare the extracts. The pH of the solution was
Although some of these substances could be retained between 5.4 and 5.6 and the extracts were incubated
by willow roots, others where excreted into an external for a week at room temperature while the pH raised to
medium. Chromatography of these water exudates and 7.2. Further observations revealed that during
a subsequent investigation of their chromatograms composting the amount of phenolics was drastically
with UV-B light showed that most of these substances reduced. Seed germination tests were performed with
are polyphenols such as coumarin, or phenolic acids. these water extracts and pure water (control) on lettuce
The phenolic substances retained by cells had different and wheat seeds. Germination rate and seedling
chemical properties than those located in the root lengths were measured to demonstrate that phenolics
exudates. Thus, the data confirm the hypothesis that decreased inhibiting properties after dilution, or after

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

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
Phenolic cycle 17

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


Photosynthesis 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
Secondary Plant biomass
substances was also high in potassium, low in available nitrogen,
in living plants
and medium in phosphates content, while very high
was the microbial activity. Horizon E (28-52 cm) was
Active secretion
from roots ochric in color, it contained more loam, less organic
into the Biomass of matter, lower microbial activity and pH=7.7. Horizon
water and soil dead plants
B (52-62 cm) had no organic matter, microbial activity
was the lowest and pH=7.8. Water permeability was
Microorganisms Composting process also measured for each horizon to evaluate penetration
Composting process (phenolics and N-sources) 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
Transformed phenolics and To alumo-silicate horizon B. Soil fertility conditions were also assessed
other secondary substances matrix with a wheat/clover germination test. A sand substrate
was used as control, which yielded 30-50%
Humus formation
germination. Horizon A had a germination of 80-82%,
horizon B 40-60%, horizon E/B (with lowest microbial
F i g u re 3: Secondary substances, plant biomass accumula- activity) yielded 30-70% germination rate (Kalevitch
tion and humus formation during allelopathic effects. et al., 2002). The results of these experiments appear to
indicate that topsoil (for its highest microbial activity)
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).

S o i l - m i c ro b i a l c o m p l e x f o r p h e n o l i c d e c o m p o s i t i o n

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 carbon-
nitrogen 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 F i g u re 4: Phenolic cycle.
18 Valentine I. Kefeli et al.

is an effective medium usable to facilitate composting Biochem Physiol Pflanzen. 184: 363-369, 1989.
of maple and sumac leaves, contaning nature phenolic Yamamoto T, Yokotani-Tomita K, Kosemura S, Yamada K
compounds. and Hasegava K. Allelopathic substances exuded from a
serious weed. J Plant Growth Reg. 18: 65-67, 1999.

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.

R e f e r ences

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


microflora and fabricated soils. American Society for
Microbiology. 103rd 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.
Journal of Cell and Molecular Biology 2: 19-23, 2003. 19
Haliç University, Printed in Turkey.

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


Tülin Aktaç1*, Ayflegül Kabo¤lu1, Elvan Bakar1 and Hamiyet Karakafl2
1
University of Trakya, Faculty of Arts and Sciences, Department of Biology, 22080, Edirne-Turkey;
2
University 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 (p<0.001), generally an insignificant increase were
observed in the organ weights (liver: p>0.05, 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.

Key words: Citric acid, food preservative, enzymes, mouse, liver

F a re l e r d e s i t r i k a s i d i n t e k t o k s i k d o z u n u n k › s a s ü re l i e t k i l e r i

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 (p<0.001), organ a¤›rl›klar›nda (karaci¤er: p>0.05 , 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.

Anahtar sözcükler: Sitrik asit, besin koruyucu, enzimler, fare, karaci¤er

I n t ro d u c t i o n substances are antioxidants which are used as food


preservatives. However, peroxides of saturated fats
Humans are exposed daily to complex mixtures of and their secondary oxidation products, can be toxic
chemical compounds in their food. One of these and impair food quality (Würtzen, 1990). Thus despite
20 Tülin Aktaç et al.

their economic importance, they can have negative physiological saline to control group mice. 10 days
effects on living organisms. Xenobiotics entering the after the injection, the mice were killed by cervical
organism are held by intestine, kidney and liver cells dislocation and then the necessary studies were
for detoxification. These cells contain important commenced. The livers, kidneys and spleens dissected
detoxification enzymes. During the detoxification of out, weighed, liver samples were seperated for
xenobiotics, free radicals are produced in microscopical examination. Blood samples were also
oxidation/reduction reactions, and these radicals can taken for enzyme assays. The serum levels of enzymes
have destructive effects on tissues. were determined using a Merck Mega 600
The toxic effects of many food preservatives on autoanalyser with the aid of Diasis Kits. Data were
living organisms have been studied by many analyzed by M.Whitney U test for multiple
researchers (Makoveç and Sindelar, 1984; Daniel, comparisons for the differences between the control
1986; Cabel et al., 1988; Kagan et al., 1990; Jung et and treated groups. For histological examination, liver
al.,1992; Nijhoff and Peters, 1992; Fujitani, 1993; samples were fixed with 10% buffered formalin,
Weemaes et al., 1997; Mc Farlene et al., 1997; Safer processed and stained hematoxylin-eosin.
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 Results
food industry, there is no report on more detailed
effects of citric acid (or its salts) in liver. In addition, The effects of citric acid injection on the body weight
soft drinks, cosmetics and drugs, in which citric acid is and liver, kidney, spleen weights was shown Table 1
approved for use, are consumed by most of humans and 2. Although the liver, spleen and kidney weights
every day. were not changed significantly (p>0.05), the body
A way of analysing harmfull effects of foreign weights were decreased significantly (p<0.001). CK,
materials entered to organism is to determine the LDH, ALT and AST activities were not changed
effects of the chemicals on the enzymes. Enzymes statistically (p >0.05) as shown in Table 2. The results
have a very important role in the metabolical process of the microscopic investigation showed that liver
since they are biological catalysts. Thus, their abnormal of mice treated with citric acid has necrotic
serum levels indicates various diseases. Among these changes, compare to the control group (Figure 1-6).
enzymes are, creatine kinase (CK), lactate These changes were slightly degeneration of tissue
dehydrogenase (LDH), alanine aminotransferase (ALT) (Figure 2), cytoplasmic vacuolisation, nuclear
and aspartate aminotransferase (AST) which are the membran invaginations (Figure 3, 4) and picnotic nuclei
most important. Therefore, we studied short term (Figure 5). In addition, we observed degeneration of the
treatment of citric acid (10 days) in mice. In these blood vessel endothelium (Figure 6).
experiments, firstly we tested total body weigths,
organ weights (liver, kidney, spleen), and determined
the serum levels of creatin kinase (CK), lactate Discussion
dehydrogenase (LDH), alanin aminotransferase (ALT)
and aspartat aminotransferase (AST), and secondly the The effects of xenobiotics in living organisms can
liver tissue was investigated histopathologically. investigate in various ways. Among these are, short-
term toxicity tests which are used very commonly. In
these methods, many parameter are used to test the
Material and Methods effects of xenobiotics. Some of these parameters are
body weight, organ weights, blood profile, and
Male mice (Balb/C albino) weighing 25-30 g were histopathological examination. In this study, the short-
used in our experiments. Five mice were used control term effects of citric acid applied intraperitoneally
group and ten mice were used the citric acid-treated were investigated. It was reported that the body weight
group. Animals were fed by pellet baits and water. decreases in mouse (Würtzen, 1990), and in rats
LD25 dose (480 mg/kg.bw.) of citric acid (Merck; in (Nijhoff and Peters, 1992) by the effects of phenolic
physiological saline) were injected intraperitoneally to antioxidant butylated hydroxytoluene (BHT) and
experiment group mice, and the same amount of butylated hydroxyanisole (BHA) in chronic studies. In
Short-term effects of citric acid 21

F i g u re 1: The control group of the liver tissue, bar F i g u r e 2: Citric acid group. Distortion of general
representes 20 µm. histological structure of the liver, v: blood vessel, bar
representes 10 µm.

F i g u re 3: Citric acid group. Nuclear invaginations (arrows),


vacuolisation (v), and damaged nucleus (n) in necrotic cells, F i g u re 4: Citric acid group. Invaginations of hypertrophic
bar representes 4 µm. cell nucleus (arrow), bar representes 4 µm.

F i g u re 5: Citric acid group. Picnotic nuclei (arrows) in F i g u re 6: Citric acid group. Degenerated endothelium
hepatocytes, bar representes 10 µm. (arrows) of blood vessel (v), bar representes 10 µm.

contrast, any significant change was seen in body of sodium benzoate) for ten days by Fujitani (1993).
weight in F344 rats (0.2, 2.5 and 3.0 % of sodium Similarly, Kabo¤lu and Aktaç (2002) were determined
benzoate) and in B6C3F1 mice (1.81, 2.09 and 2.4 % 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.

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 (p<0.001).
(**) not significant (p>0.05).

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).

sodium benzoate. Also, at the present study we not significant to compare with the control. These
determined a significant decrease of body weight in results were similar with findings obtained in F344 rats
mice by the effect of citric acid (Table 1). and B6C3F1 mice by Fujitani (1993).
Some autors have shown that food preservatives Although the organ weights and serum levels of
had increasing effects to organ weight. The effects of enzymes were not changed significantly, the
BHT and BHA on the increasing of the liver and examination by light microscopy revealed
thyroid weights were demonstrated in mice by pathological changes in liver of mice, such as
Würtzen (1990). Similarly, the effects of BHT on vacuolisation and glassy cytoplasm in the hepatocyte,
increasing of the liver weight in rats was also shown nuclear membrane invaginations, picnotic nuclei.
by Mc Farlene et al. (1997) and Safer and Nughamish Similarly, with the effect of sodium benzoate in the
(1999). Fujitani (1993) was also obtained significant rats and mice, high vacuolisation and glassy
increasing of the liver and kidney by the effects of appearance in hepatocyte cytoplasm was explained
sodium benzoate in male rats. In our previous studies, (Fujitani, 1993). Again, similar findings were obtained
increasing of the total liver weight were seen oral in the rats with oral treatment of BHT (Mc Farlene et
treatment of sodium benzoate (Kabo¤lu and Aktaç, al., 1997; Safer and Nughamish, 1999), and with
2002) and citric acid (Aktaç et al., 2002) but it was not sodium benzoate, benzoic acid and citric acid in mice
significant. Additionally, in the present study, we could (Kabo¤lu and Aktaç, 2002; Aktaç et al., 2002). The
not find any significant change the liver, kidney and results of present study suggested that citric acid has
spleen weights by the intraperitoneal injection of citric hepatotoxic effects and long term exposure may
acid (Table 2). According to our results, serum CK, induce severe damage in liver of mice. However, the
LDH, AST and ALT levels in the treated animals were mechanism of damaging effects of citric acid need to
Short-term effects of citric acid 23

be clarified by more detailed studies.


Finally, we can conclude that consumption of the
foodstuffs containing preservatives is important for the
human health.

R e f e r ences

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.
Journal of Cell and Molecular Biology 2: 25-30, 2003. 25
Haliç University, Printed in Turkey.

C h a r a c t e r i s a t i o n o f R P P 7 mutant lines of the col-5 ecotype of


Arabidopsis thaliana
Canan Can1, Mehmet Özaslan1*, Eric B. Holub2
1
University of Gaziantep, Faculty of Science & Arts, Department of Biology, 27310 Gaziantep; 2Plant
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.

Key words: Arabidopsis thaliana, Col-5, Hiks-1, Peronospora parasitica

Arabidopsis thaliana ’ n › n C o l - 5 e k o t i p i n d e n e l d e e d i l e n m u t a n t h a t l a r d a n R P P 7 g e n i n i n
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.

Anahtar sözcükler: Arabidopsis thaliana, Col-5, Hiks-1, Peronospora parasitica

I n t ro d u c t i o n rice wheat, tomato, pepper and some other important


crops are isolated and the mechanism of resistance is
Following the isolation of the Pseudomonas syringae determined (Richter and Ronald, 2000). The mutant
resistance genes (R-gene) from tomato, the research on lines with lack of R-genes have a potential importance
isolation and characterization of R-genes against plant in this type of work (Mc-Dowel et al., 1998).
pathogens has been improved (Hammond-Kosack and Arabidopsis thaliana is a member of cruciferae
Jones, 1997). Recently many R-genes conferring family and it is known to have a small genome size of
resistance to fungi, bacteria, nematode and viruses in 120 Mb. It is a flowering plant and is a best model for
26 Canan Can et al.

the working on genome analyses, growth regulation, with many isolates (Century et al., 1995; Aarts et al.,
hormons, flowering, disease resistance and 1998). Similarly, in (Ethylene Intensitive) mutant lines
embryogenesis. Arabidopsis and tomato were used to do have the ethylene synthesis. But it was found that
determine the mechanisms of disease resistance the P. syringae f.s.p. tomato resistance continued in
(Thomas et al., 1997; Botella et al., 1998). It is also the this mutant lines. This study showed that ethylene was
host of many pests which attacks to crop plants. Many not important for A. thaliana and bacteria relationships
genes that provides resistance to bacteria and fungi (Bent et al., 1994; Dong, 1998). Lsd (Lesions
disease have been isolated and characterized from A. Simulating Disease resistance response) and acd
thaliana (Dangl and Jones, 2001; Feys and Parker, (Accelerated Cell Death) mutant lines produce
2000). Hypersensitive Resistance (HR) like symptoms
Peronospora parasitica is a causal agent of mildew without a pathogen infection. These symptoms are
disease in the genus cabbage, turnip etc. of cruciferae formed by the influence of external factors like heat
family. R-genes that determines resistance to P. and light (Lam et al., 1999). So, it is accepted that lsd
parasitica (RPP) were isolated and characterized and acd loci are negative regulators for HR formation
(Holub and Beynon, 1997; Parker et al., 1997; Botelli (Dietrich et al., 1994). In general, the presence of
et al., 1998; McDowell et al., 1998; Bittner-Eddy et al., different resistance mechanisms in A. thaliana which
2000). The researches on RPP genes have shown that are directed by RPP genes was found by the
these genes are at the specific regions at certain places characterization of mutant lines (Glazebrook et al.,
of each chromosome called as “Major recognition 1997; McDowell et al., 2000).
complexes-MRC” (Can, 1997; Holub and Beynon, In this study, the mutant lines of the Col-5 ecotype
1997). of A. thaliana were characterized, to understand the
RPP7 gene is present in Col-5 ecotype and mechanisms of RPP7 gene that confers resistance to
recognized by the Hiks-1 isolate of P. parasitica. This Hiks-1 isolate of P. parasitica.
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., Material and methods
1995; Can, 1997). The Hiks1 isolate also recognizes
the RPP1 gene which is present in Nd-1 ecotype, and Plant and fungus material
has an epistatic effect on the RPP7 gene (Tor et al.,
1994). In this study, Col-5 ecotype of A. thaliana lines having
The mutant lines that lack the R-genes were MRC-B, MRC-C and MRC-H regions (Can, 1997;
studied in detail and has a wide area of interest such as Holub and Beynon, 1997) were used as wild type
molecular and classical genetics. However, in order to ecotype. The Fast Neutron (FN) applied mutant lines
study the relationships between A. thaliana and the were obtained commercially and the selections of
RPP genes and to investigate the genome mutant lines were performed by using the Hiks-1
organizations, some mutant lines were used (Parker et isolate of P. parasitica. The Hiks-1 isolate recognizes
al., 1996). The mutant lines lacking the RPP genes the RPP7 gene which is in the MRC-B region of wild
were obtained from Ws-O that contain RPP14 gene Col-5 ecotype, and 7 days after inoculation it induces
exhibiting resistance to No-Co2 isolate by using Ethyl a resistance which is defined with HR. Four mutant
Methane Sulfate (EMS). The lines were then used to lines were used in this study denoted as FN3922,
separate the RPP10 and RPP1 genes, which were FN3928, FN3929 and FN3930. The HR does not occur
allelic to RPP14 that is on the third chromosome. It in mutant lines, and the pathogen completes its life
was found that the WsEDS line was susceptible to all cycle by sexual and asexual sporulation.
P. parasitica isolates tested and that the WsEDS locus
was necessary for the function of the RPP genes Regeneration of Hiks-1 isolate from oospore
(Parker et al., 1996; Bittner-Eddy and Beynon, 2001; population
Falk et al., 1999). The npr (Non expressor of PR
protein) mutant lines of A. thaliana synthesize the The Hiks-1 isolate was regenerated by using oospore
proteins which are related with pathogenesis. So, population. To do this, the seeds of A. thaliana that
systemic resistance is not seen following inoculation were susceptible to the Hiks-1 isolate were sown into
Characterisation of RPP7 gene 27

little plastic pots containing 4:1:1 (torf: perlit: sand) of Microscopic analysis
mixture for 40-50 seeds each. The pots were irrigated
to wet the seeds and 1-2 x 105 oospore/ ml were added Fungal development in plant tissue was examined
to the pots. The containers were held at 4 °C for 1-2 under light and fluorescence microscope. The infected
weeks to break the dormancy. Following this, the leaves were taken and put absolute methanol for 5-6
containers were placed into the climated room at 18-20 hours followed by saturated chloral hydrate solution
°C, 10 h light and 14 hour dark period. Within 10-15 for 4-5 hours. Then, tissues were placed in 50 %
days following seed germination, some seedlings glycerol solution for microscopic analyses.
having sporulation was collected and placed into
eppendorf tubes containing 200 µl dH2O. The DNA analysis
eppendorf tubes were shaked gently to allow the
conidia to pass to water. The conidia suspension was Total plant genomic DNA was isolated with some
used to inoculate 7 days old seedlings of EBH3529 and modifications by using the methods of Ausubel et al.,
Ksk-1 ecotypes, and the plants were placed into the (1994). Five to eight grams of plant material was
climate room. By this way, the regeneration of the grounded in N2 and transferred to the tubes containing
Hiks-1 isolate was done by subculturing 3-4 times. The 15 ml buffers (100 mM Tris-HCl, 50 mM EDTA, 500
conidia were stored at –20 °C and were used when mM NaCl, 10 mM Mercaptoethanol, %25 SDS) with
needed. 100 mg/lt proteinase K. The solution was kept at 55 °C
The same procedure were applied for, Ahco-1, for 1 hour. At the end of this time period, 5 ml of 5 M
Ahco-2, Ahco-7, Wand-1, Cand-5, Hind-2 and Hind-4 potassium acetate was added and held in ice for 20
isolates using Nd-1 and Col-5 ecotypes (Can, 1997). minutes, and the solution was centrifuged at 17000
rpm for 25 minutes. The supernatant was mixed with
Characterization of P. parasitica isolates by using 0,6 volume of isopropanol and held at -20 °C for
different A. thaliana ecotypes minutes and the DNA was precipitated. Phenol-
chloroform was used to wash the DNA and a second
Regenerated P. parasitica isolates were inoculated into precipitation was done. The DNA was dissolved in
Col-5, Ksk-1, Nd-1, Ws-3, Tsu-1, Ler-1, Oy-1 and dH2O and stored at -20 °C. The isolated DNA was
Wei-1 ecotypes in order to do phenotypic diluted in such to 50-100 ng/µl to use in polimerase
characterization. The A. thaliana ecotypes were chain reactions (PCR). For PCR reactions, the closest
obtained from Dr. Eric Holub (HRI- UK) marker to the RPP7 gene was used (Can, 1997). To do
The conidia suspension was adjusted to 4-5 x 104 this, the solution which contains 0.05 mm primer, 2
conidia/ml concentration for plant inoculations. The mm dNTPs, 25 mm MgCl2, 1 x Taq buffer and IU Taq
cotyledons of 7-8 days of the A. thaliana ecotypes DNA polymerase was completed to 25 ml volume. The
were inoculated in such a way that it would be one PCR reactions were performed at 94 °C for 5 min
drop to each cotyledon. The plants were placed into followed by 94 °C for 1 minute, 56 °C for 1 min, 72 °C
climated room with 18-20 °C, 10 h light, 14 h darkness for 13 minute (35 cycles) and 72 °C for 10 min. The
conditions after the inoculation and the plants were samples were electrophoresed at 80 W for 4 hours.
checked at the end of 3. and 7. days. The evaluation
was done regarding the pathogen sporulation and
hypersensitive reaction types (the interaction Results and discussions
phenotypes). Phenotypic reactions were examined
under the fine group as; pitting with no pathogen Characterization of P. parasitica isolates
sporulation (PN), flecking with no pathogen
sporulation (FN), flecks with delate and moderate In order to determine the changes at the RPP7 locus in
pathogen sporulation, 1-20 sporangiophorus per each the mutant lines, Ahco-1, Ahco-2, Ahco-7, Wand-1,
cotyledon (DM), flecking with delate pathogen Cand-5, Hind-2 and Hind-4 isolates were used. Ahco-
sporulation, 5-10 sporangiophorus per each cotyledon 1, Ahco-2, Ahco-7 recognize MRC-B region which is
(FDL), early and heavy pathogen sporulation, 20> located at the first chromosome in the Nd-1 ecotype
sporangiophores per cotyledon (EH), (Holub et al., (Can, 1997), and these isolates were presumed to
1994). recognize RPP7 allele of Nd-1 ecotype. Wand-1,
28 Canan Can et al.

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

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).

Cand-5, Hind-2 and Hind-4 isolates recognize MRC-B Phenotypic characterization of mutant lines
and MRC-C region which present at the second
chromosome in the Col-5 ecotype. The isolates P. parasitica isolates were used to inoculate the Col-5
regenerated from the oospore populations were lines. The results were shown in Table 2.
inoculated on different A. thaliana ecotypes (Col-5, As indicated in Table 1, DM and EH phenotypes
Ksk-1, Nd-1, Ws-3, Ler-1, Oy-1 and Wei-1) to developed, following inoculation of FN3922, FN3928,
determine if they were original. The results are shown FN3929 and FN3930 mutant lines with the Hiks-1
in Table1. isolate. These results revealed that the RPP7 gene is
As it could be seen in Table 1, the isolates not present in the mutant lines. However, Ahco-1,
generated from the oospore populations were found to Ahco-2 and Ahco-7 isolates exhibited the EH
be as original, and there was no variation (Can, 1997). phenotype compared to DM in the wild Col-5 ecotype.
Therefore these isolates were used to inoculate the This result showed that absence of the RPP7 gene
mutant lines recovered through inoculation with the increased susceptibility. The important point in here
Hiks-1 isolate. 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
Ahco-2 DM FDL FN EH EH EH EH
Ahco-7 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).
Characterisation of RPP7 gene 29

F i g u re 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.

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
F i g u re 1: Microscopic reactions occurred in Col-5 ecotype mutant lines with Wand-1, Hind-2, Hind-4 resulted FN
following inoculation with the Hiks-1 isolate. (A) Six hours phenotype. This results shows that the RPP7 gene has
after inoculation. a. Haustorium formed in the mesofil cells. no correlation with the RPP genes present in seconder
b. Cell death and (B) Twelve hours after inoculation. chromosome at the MRC-C region (Table 2).
Hypersensitive reaction and cell death.
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
750 bp → susceptible genotypes and the mutant lines allowed the
500 bp → penetration and mycelial development without any cell
(Figure 1, 2). Fungal development in the mutant lines
supported the macroscopic results (Mc Dowell et al.,
F i g u re 3: Distribution of g2a markers in mutant lines. 2000).
M indicates the 1 kb DNA marker.
DNA analyses of mutant lines
may also show that when the RPP genes are allelic
they do function in coordination. nga280 and g2a markers present at the lower arm of
Cand-5 isolate recognizes MRC-B region which is the first chromosome of A. thaliana were detected to
present in Col-5 and determined with FN interaction give less recombination with the RPP7 loci (Can,
phenotype. FN3922 mutant line exhibited the EH 1997). Therefore the mutant lines were subjected to
phenotype with Cand-5 and FN with other isolates PCR analyses with these markers. Results are given in
tested (Table 2). This result may show that FN3922 Figure 3.
may be a Col-5 contamination. On the other word it is The distributions of the bands in mutant lines were
possible that in this line, many RPP genes present in similar to those in the wild type Col-5. This result may
the MRC-B loci (Can, 1997) could be mutated. suggest that no deletions may occurred in the mutant
Therefore, FN3922 may not be a specific mutant of the lines and that the loss of function could be due to a
RPP7 gene. As it is known the Fast Neutron point mutation.
30 Canan Can et al.

R e f e r ences homology to eukaryotic lipases. P roc Natl Acad Sci


USA. 96: 3292-3297, 1999.
Aarts N, Metz M, Holub E, Staskawicz BJ, Daniels M and Feys BJ and Parker JE. Interplay of signaling pathways in
Parker JE. Differential requirements for EDS1 and plant disease resistance. Trends Genet. 16: 449-455,
NDR1 by disease resistance genes define at least two R 2000.
gene-mediated pathways in Arabidopsis. Proc Natl Acad Glazebrook J, Rogers EE and Ausubel FM. Use of
Sci USA. 95: 10306-10311, 1998. Arabidopsis for genetic dissection of plant defense
Ausubel F, Brent R, Kingston RE, Moore DA, Seiddman JG, responses. Annu Rev Genet. 31: 547-569, 1997.
Smith JA and Struhl K. Current Protocols in Molecular Hammond-Kosack KE and Jones JDG. Plant disease
Biology. John Wiley and Sons. 1994. resistance genes. Annu Rev Plant Physiol Plant Mol
Bent AF, Kunkel BN, Dahlbeck D, Brown KL, Schmidt R, Biol. 48: 575-607, 1997.
Giraudat J, Leung J and Staskawicz BJ. RPS2 of Holub EB, Beynon J and Crute I. Phenotypic and genotypic
Arabidopsis thaliana, A leucine-rich repeat class of plant characterisation of interactions between isolates of
disease resistance genes. Science. 265 (5180): 1856- Peronospora parasitica and accessions of Arabidopsis
1860, 1994. thaliana. Mol Plant-Microbe Interacts. 7 (2): 223-239,
Bittner-Eddy PD, Crute IR, Holub EB and Beynon JL. 1994.
RPP13 is a simple locus in Arabidopsis thaliana for Holub EB. and Beynon J. Symbiology of mouse-ear cress
alleles that specify downy mildew resistance to different (Arabidopsis thaliana) and oomycetes. Adv Bot Res.
avirulence determinants in Peronospora parasitica. The 24: 227-273, 1997.
Plant Journal. 21(2): 177-188, 2000. Lam E, Pontier D and Pozo O. Die and let live-programmed
Bittner-Eddy PD and Beynon JL. The Arabidopsis downy cell death in plants. Curr Opin in Plant Biol. 2: 502-507,
mildew resistance gene, RPP13-Nd, functions 1999.
independently of NDR1 and EDS1 and does not require Mc-Dowel JM, Dhandaydham M, Long TA, Aarts MGM,
the accumulation of salicylic acid. Mol Plant-Microbe Goff S, Holub EB and Dangl JL. Intragenic
Interactions. 14: 416-421, 2001. recombination and diversifying selection contribute to
Botella MA, Parker JE, Frost LN, Bittner-Eddy PD, the evolution of downy mildew resistance at the RPP8
Beynon JL, Daniels MJ, Holub EB and Jones JDG. locus of Arabidopsis. Plant Cell. 10: 1861-1874, 1998.
Three genes of the Arabidopsis RPP1 complex McDowel JM, Cuzick A, Can C, Beynon J, Dangl JL and
resistance locus recognize distinct Peronospora Holub EB. Downy mildew (Peronospora parasitica)
parasitica avirulence determinants. Plant Cell. 10: 1847- resistance genes in Arabidopsis vary in functional
1860, 1998. requirements for NDR1, EDS1, NPR1 and salicylic acid
Can C. Bittner-Eddy P, Tör M, Williams K, Gunn N, Bakht S, accumulation, The Plant Journal. 22 (6): 523-529, 2000.
Atkinson L, Debener T, Chimot P, Crute I, Beynon J and Parker JE, Holub EB, Frost LN, Falk A, Gunn ND and
Holub EB. Revealing the organization of RPP loci in the Daniels MJ. Characterization of eds1, a mutation in
Arabidopsis thaliana genome, Poster abstract in 6th Arabidopsis supressing resistance to Peronospora
International Conference on Arabidopsis Research, parasitica specified by several different RPP genes.
Medison, Winconsin. 7-11 June, 1995. Plant Cell. 8 (11): 2033-2046, 1996.
Can C. Genomic organisation of pathogen recognition genes Parker JE, Coleman MJ, Szabo V, Frost LN, Schmidt R, Van
in Arabidopsis thaliana to Peronospora parasitica, der Biezen EA, Moores T, Dean C, Daniels MJ and
Ph. D. Thesis, University of London, Wye Collage, UK. Jones JDG. The Arabidopsis downy mildew resistance
1997. gene RPP5 shares similarity to the Toll and Interleukin-1
Century KS, Holub EB and Staskawicz BJ. NDR1, a locus of receptors with N and L6. Plant Cell. 9: 879-894, 1997.
Arabidopsis thaliana that is required for disease Richter TE and Ronald PC. The evolution of disease
resistance both a bacterial and a fungal pathogen. P roc resistance genes. Plant Molecular Biology. 42: 195-204,
Natl Acad Sci USA. 92 (14): 6597-6601, 1995. 2000.
Dangl JL and Jones JDG. Plant pathogens and integrated Thomas CM, Jones DA, Parniske M, Harrison K, Balint-
defense responses to infection. Nature. 411: 826-833, Kurti P, Hatzixanyhis K and Jones JDG. Characterization
2001. of the tomato Cf-4 gene for resistance to Cladosporium
Dietrich RA, Richberg MA, Schmidt R, Dean C and fulvum identifys sequences that determine recognitional
Dangl JL. A novel zinc finger protein is encoded by the specificity in Cf-4 and Cf-9. Plant Cell. 9: 2209-2224,
Arabidopsis LSD1 gene and functions as a negative 1997.
regulator of plant cell death. Cell. 88: 685-694, 1994. Tor M, Holub EB, Brose E, Mussker R, Gunn N, Can C,
Dong X. SA, JA, ethylene, and disease resistance in Crute I and Beynon JL. Map positions of three loci in
plants. Curr Opin Plant Biol. 1: 316-323, 1998. Arabidopsis thaliana associated with isolate-specific
Falk A, Feys BJ, Frost LN, Jones JDG, Daniels MJ and recognition of Peronospora parasitica (downy mildew).
Parker JE. EDS1, an essential component of R gene- Mol Plant-Microbe Interac. 7 (2): 214-222, 1994.
mediated disease resistance in Arabidopsis has
Journal of Cell and Molecular Biology 2: 31-34, 2003. 31
Haliç University, Printed in Turkey.

The effect of meta - t o p o l i n o n p r o t e i n p r o f i l e i n r a d i s h c o t y l e d o n s


Serap Ça¤1 and Narçin Palavan-Ünsal2*
1
Istanbul University, Department of Biology, Botany Section, Süleymaniye 34460, Istanbul-Turkey;
2
Haliç 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.

Key words: Cotyledon growth, meta-topolin, protein

Meta - t o p o l i n i n t u r p k o t i l e d o n l a r › n d a p r o t e i n p r ofiline 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.

Anahtar sözcükler: Kotiledon büyümesi, meta-topolin, protein

I n t ro d u c t i o n Mok, 1994; Binns, 1994).


Bioassays are used to establish the relative
Cytokinins, N6-substituted adenine derivatives are a biological activity of plant hormones compared with
class of plant hormones that were first identified as others. The cytokinin bioassays used most frequently
factors that promoted cell division (Miller et al., 1955; depend on growth of tissues in sterile culture (Letham
1956) and have been implicated in many other aspects 1967). Such methods are extremely sensitive but it
of plant growth and development including shoot needs at least 3 weeks to get final results. Letham
initiation and growth, apical dominance, senescence (1971) described a rapid bioassay for cytokinins based
and photomorphogenetic development (Letham, on the ability of these compounds to promote
1971; Thimann, 1980; Mok and Mok, 1994). markedly the expansion of radish cotyledons excised
Although the physiological effects of cytokinins have soon after seed germination.
been well documented, the molecular mechanisms To date the effects of common cytokinins i.e.
underlying cytokinin action remain obscure (Mok and kinetin, benzyladenine (BA) and its riboside have been
32 Serap Ça¤ and Narçin Palavan-Ünsal

documented in radish cotyledons. A new active room temperature. The gels were diffusion-destained
aromatic cytokinin meta-topolin (mT) have been by repeated washing in the solution containing 7.5 %
determined by Strnad et al. (1997) in poplar. The acetic acide, 5 % methanole and 87.5 % distilled water.
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 Results and discussion
soluble protein contents in radish cotyledons that has
not been studied before. The early observations revealed that cytokinins exert
parallel effects in maintain protein or nucleic acid
levels while inhibiting senescence. Cytokinins
Material and methods stimulate both structural and enzymatic protein
synthesis. They are selectively increasing the levels
Plant material and bioassay of certain enzymes associated generally with
photosynthetic process (Feierabend, 1969). It is not
Radish (Raphanus sativus L.) seeds were germinated in clear whether the enhanced activity is due to greater
darkness for 4 days at 25 °C on moist filter paper in 5 synthesis, inhibition of degradation or activation of the
cm petri dishes. Cotyledons were excised excluding enzymes.
petiole tissues and four cotyledons were placed in each We already observed that new aromatic cytokinin
petri dish after measuring the fresh weight. The mT at 0.25 to 1 mM concentration range delayed
cotyledons were placed with their adaxial sides down the senescence in excised wheat leaf segments
on the paper. They were incubated in a growth chamber (Palavan et al., 2002). This concentration range was
at 25°C ± 2°C and 12 h light-dark photoperiods. Three high for radish cotyledon growth therefore lower
ml mT was applied per petri dish at 0.05, 0.1, 0.25, 0.5 concentrations were examined (0.05 to 1 mM) in
and 1.0 mM concentrations. Cotyledon growth was addition.
measured by determining fresh and dry weights 3 days Cotyledon growth increased with the treatments of
after the application (Letham, 1971) and the data mT significantly (Figure 1). Stimulation of cotyledon
presented here representative of 15 experiments. growth was closely related with increasing
concentrations of mT; 0.05 to 1 mM mT increased the
Measurement of soluble protein content cotyledon growth about two fold in fresh weight basis
(p<0.05), while dry weights of cotyledons during the
Soluble protein content was determined as in Bradford growth were not effected by mT application (Palavan-
(1976) using bovine serum albumin as standard. Each Ünsal et al., 2002).
experiment was repeated four times and each treatment Cytokinins promote cell enlargement in certain
included three replicates. tissues and organs. This effect is most clearly seen in
cotyledons. The expansion of cotyledons is resulted
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 F i g u re 1: The effect of meta-topolin on cotyledon growth in
with Coomassie brilliant blue solution for overnight at radish (Values are average of 30 cotyledons).
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
F i g u re 2: The effect of meta-topolin on soluble protein con-
occurred with mT treatments. Analysis of the soluble
tent during the growth of radish cotyledons. Values are aver-
age of 4 experiments. 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.
F i g u re 3: SDS-PAGE analysis of soluble proteins from Results obtained in this study showed that, total
meta-topolin treated radish cotyledons. Gel was stained with soluble protein content in radish cotyledons not
Coomassie blue. Lane 1: Control, Lane 2: 0.05 mM mT, effected from exogenously applied mT. On the other
Lane 3: 0.1 mM mT, Lane 4: 0.25 mM mT, Lane 5: 0.5 mM
hand, when protein profile was examined
mT, Lane 6: 1 mM mT treatments. Molecular mass (kDa) of
markers are indicated on left hand margin. electrophoretically additional bands were observed in
mT treated samples. These can be explained by the fact
that mT stimulate new protein synthesis without
from cell enlargement during cotyledon growth. effecting total protein content.
Cytokinin treatment promotes additional cell In conclusion, natural aromatic cytokinin mT has
expansion with no increase in the dry weight of the an important role in the control of cotyledon growth
treated cotyledons (Huff and Ross, 1975). and this response closely associated with protein
Letham (1971) reported the ability of cytokinins to profile. The results of this research are exhibited mT as
promote markedly the expansion of radish cotyledons a promising plant growth regulators in physiological
and explained this response by the promotion of cell studies.
enlargement. mT also as a most active aromatic
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).

R e f e r ences

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.
Journal of Cell and Molecular Biology 2: 35-38, 2003. 35
Haliç University, Printed in Turkey.

T h e e f f e c t o f e l e c t romagnetic fields on oxidative DNA d a m a g e


Serkan ‹fller1 and Günhan Erdem2*
1
Department of Biology, Institute of Applied Sciences, Çanakkale Onsekiz Mart University, Çanakkale,
Turkey; 2College 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, low-
level 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.

Key words: Electromagnetic fields, DNA damage, ascorbic acid, vitamin C, oxidative stress

E l e k t ro m a n y e t i k a l a n › n o k s i d a t i f D N A h a s a r › ü z e r i n d e k i e t k i s i

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.

Anahtar sözcükler: Elektromanyetik alan, DNA hasar›, askorbik asit, C vitamini, oksidatif stres
36 Serkan ‹fller and Günhan Erdem

I n t ro d u c t i o n DNA samples was spectrophotometrically determined.


All DNA samples were free from proteins, RNAs and
There are many reports on the biological effects of solvents used for extraction.
electromagnetic fields (EMF) and there have been
many attempts to develop a theoretical explanation of Oxidative DNA cleavage reactions
this phenomenon. Some epidemiological studies have
suggested that exposure to ambient, low-level 50/60 Cleavage reactions were carried out in a medium
Hz EMF increases risk of disease including cancer containing 0.5 µg DNA, 20 mM Tris-HCl (Sigma) pH
such as leukemia among children who live close to 7.8, 0.25 mM ultra-pure ascorbic acid (Merck) and
power lines or among men whose jobs expose them to CuCl2 (Sigma) in the final concentrations of 2.5, 5, 7.5
EMF (Wertheimer and Leeper, 1979; Tomenius, 1986; and 10 µM, in a final volume 10 µl. Other antioxidants
Savitz et al., 1988; London et al., 1991). EMF firstly (glutathion, cystein and dithiothreitol, Sigma) and
affects the cell membrane. Some ion channels such as metal chelator (EDTA, Merck) were added to the
Na-K ATPase have been affected according the level reaction mixtures at a final concentration of 0.5 mM.
of EMF. The alteration in the activity of these proteins The mixtures were incubated at room temperature for
causes an increasing or decreasing intracellular 10, 20 and 30 minutes. Adding EDTA at a final
concentration of many ions such as Na+, K+, Mg2+ and concentration of 25 mM stopped reactions. DNA
Ca2+ which plays very important roles in cell signaling. cleavages in the reaction mixtures were analyzed on
Therefore, the biological effects of EMF expand the 1% agarose gel (Promega) electrophoresis.
among the cellular systems (Goodman et al., 1995).
Although the several types of biological effects of EMF exposure system
EMF have been shown, the molecular mechanisms of
these effects have not been explained yet. Some Electromagnetic fields were applied by using the
studies have suggested that EMF exposure could be Helmholtz coil. The coil system was constructed by
due to both the increase in the concentration (Jajte, using the polyester sphere that was surrounded by
2000) and oscillating of free radicals (Scaiano et al., copper wire with 0.75 cm diameter (Galt et al., 1995).
1995). EMF is known to affect radical pair The diameter and height of the sphere were 16 and 26
recombination and they may increase the cm, respectively. 50 Hz, 4.5 V electricity was applied
concentration of oxygen free radicals in living cells to coil system. As a result, 0.3 mT EMF was generated
(Jajte, 2000). Increasing the concentration of free at the center of the coil system which includes handles
radicals creates oxidative stress and some biological for the sample tubes.
reactions such as DNA damage occur under this
condition. Metabolic energy production or effects of
chemicals and radiation can form oxidative stress. Results and discussion
In this study, oxidative stress was formed by the
oxidation of ascorbic acid with Cu2+ ions and the effect The oxidative DNA damage was induced by the
of 50 Hz, 0.3 mT EMF on the oxidative DNA damage concentration of cupric ions (Figure 1). In the constant
was investigated. 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
Materials and methods breakage at this condition (Figure 1, lanes 4 and 10).
Therefore, main DNA band in the lane 10 of Figure 1
DNA isolation is thinner than the lane 4. In the presence of high
cupric ions concentration, excess scission of DNA
High molecular weight (app. 10 kb) human genomic molecules occurred at the EMF when compared to
DNA was isolated from the white blood cells with the normal conditions (Figure 1, lanes 6 and 12).
modified method of Poncz et al. (1982) by using MBI Electromagnetic fields did not have an effect on the
Fermentas genomic DNA isolation system. Molecular oxidation of ascorbic acid in the absence of cupric ions
weight and purity of DNA samples were controlled by (specific data was not shown, but the sample in Figure
agarose gel electrophoresis. The concentration of 3, lane 12 had reflected this result, because EDTA was
EMF and oxidative DNA damage 37

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

F i g u re 1: The effect of EMF and Cu(II) concentrations on F i g u re 2: The effect of incubation time on oxidative DNA
oxidative DNA damage. All lanes include 0.5 µg DNA. The damage depends in electromagnetic fields. All lanes include
DNA samples in the lanes from 1 to 6 were incubated under 0.5 µg DNA. Incubation times were 30 min from 1 to 4, 20
normal condition, 7 to 12 were incubated in EMF at room min from 5 to 8 and 10 min from 9 to 12. Ascorbic acid
temperature in the presence of 0.25 mM ascorbic acid concentration were 0.25 mM in all lanes. Cu(II)
except the 1 and 7 which were control lanes. Cu(II) concentrations were 2.5 µM in 1, 3, 5, 7, 9 and 11 while
concentrations were 1.25 µM in 2 and 8, 2.5 µM in 3 and 9, 5 µM in 2, 4, 6, 8, 10 and 12. The samples in 1, 2, 5, 6, 9 and
5 µM in 4 and 10, 7.5 µM in 5 and 11, 10 µM in 6 and 12 10 were incubated in EMF. Other samples were incubated at
lanes. Incubation time was 30 min for all samples. normal conditions.

chelating cupric ions and eliminated their oxidative In the presence of EDTA as a cationic metal
effects). chelator, oxidative DNA damage was not observed.
The results of this study showed that the oxidative This result showed that ascorbate oxidation and
DNA damage depends on the incubation time (Figure 2). oxidative DNA damage depend on cupric ions as an
DNA breakages could be observed at the 20th minute of oxidizing agent (Figure 3, lanes 6 and 12). As an
incubation time (Figure 2, lanes 6 and 8). EMF antioxidant, cystein did not block the oxidative DNA
exposure enhances the oxidative DNA damage after damage (Figure 3, lanes 4 and 10). Glutathione
the 20th minute (Figure 2, lanes 2 and 4). reduced the oxidative stress. Therefore, the DNA
damage was formed as aggregation rather than
fragmentation in the presence of glutathione (Figure 3,
1 2 3 4 5 6 7 8 9 10 11 12 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
F i g u re 3: The effect of some antioxidants (glutathione, fragmentation or aggregation. The degree of oxidative
cystein, dithiothreitol) and metal chelator (EDTA) on
DNA damage varies in the levels and reactivity of free
excessive oxidative DNA damage in EMF. All lanes include
radicals produced in the reaction medium. In the
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 presence of oxygen, the hydroxyl and peroxyl radicals
lanes 1 and 7, respectively. Lanes 2 and 8 were scission such as superoxide anion and hydroperoxyl radical are
controls. Glutathione (3 and 9), cystein (4 and 10), produced by the reaction between radical form of
dithiothreitol (5 and 11) and EDTA (6 and 12) ascorbic acid (ascorbyl radical) and molecular oxygen
concentrations were 0.5 mM. The samples in 1 to 6 were (Fuchs et al., 1990).
incubated at normal condition. The others were incubated in These radicals attack to electrophilic nuclei on the
EMF. Incubation time was 30 min for all samples. targets and create secondary carbon radicals. At the
38 Serkan ‹fller and Günhan Erdem

high level or high reactivity of these radicals, excess R e f e r ences


formation of secondary carbon radicals on the same
DNA molecule causes a reaction between each other Erdem G, Öner C, Önal AM, K›sakürek D and Ö¤üfl A. Free
and then the DNA damage occurs as fragmentation. radical mediated interaction of ascorbic acid and
ascorbate/Cu(II) with viral and plasmid DNAs. J Biosci.
Therefore, DNA size became smaller and gave the 19: 9-17, 1994.
smeared patterns on gel electrophoresis (lanes 5 and 6 Eveson RW, Timmel CR, Brocklehurst B, Hore PJ and
in Figure 1) However, at the low level or low reactivity McLauchland KA. The effects of weak magnetic fields
of oxygen species, the oxidative DNA damage results on radical recombination reactions in micelles. Int J
in aggregation of the DNA molecules with the Radiation Biol. 76: 1509-1522, 2000.
intermolecular reaction of the secondary carbon Fuchs J, Mehlhron RJ and Packer L. Assay for free radical
radicals. Thus, the DNA samples became heavier and reductase activity in biological tissue by electron spin
resonance spectroscopy. Methods in Enzymology.
were retarded on the gel electrophoresis (lanes 3 and 9 186: 670-674, 1990.
in Figure 3). Galt S, Whalstrom J, Hamnerius Y, Holmqvist D and
Our results showed that extremely low-frequency Johannesson T. Study of effects of 50 Hz magnetic fields
EMF enhanced the effect of oxidative stress on DNA on chromosome aberration and growth-related enzyme
damage and supported the idea obtained from previous ODC in human amniotic cells. Bioelectrochemistry and
studies on an increasing effect of EMF on the Bioenergetics. 36: 1-8, 1995.
concentration and the life-time of free radicals (Jajte, Goodman EM, Greenebaum B and Marron MT. Effects of
electromagnetic fields on molecules and cells. In: Int
2000; Scaiano et al., 1995; Jajte and ZmySlony, 2000).
Rew Cytology, A Survey of Cell Biology. Jean KW and
Especially the comparisons of lane 2 to lane 4 in Jarvik J (Ed). Academic Press. 158: 279-338, 1995.
Figure 2 and lane 5 to lane 11 in Figure 3, indicate that Jajte J and ZmySlony M. The role of melatonin in the
the degree of the oxidative stress under the EMF is molecular mechanism of weak, static and extremely low
greater than the normal condition. frequency (50 Hz) magnetic fields (ELF). Medycyna
In the brain cells of rats, an increase in DNA Pracy. 51: 51-57, 2000.
single- and double-strand breaks had been found after Jajte JM. Programmed cell death as a biological function of
electromagnetic fields at a frequency of (50/60 Hz).
acute exposure to a sinusoidal 60 Hz magnetic field.
Medycyna Pracy. 51: 383-389, 2000.
When the experiment was carried out in the presence Lai H and Singh NP. Melatonin and N-tert-butyl-alpha-
of melatonin or a radical scavenger compound N-tert- phenylnitrone block 60-Hz magnetic field-induced DNA
butyl-alpha-phenylnitrone (PBN), the effect of single and double strand breaks in rat brain cells.
magnetic fields on brain cell DNA was not observed J Pineal Res. 22: 152-62, 1997.
(Lai and Singh, 1997). Melatonin is a neurohormone London SJ, Thomas DC, Bowman JD, Sobel E, Cheng TC
and it is also an antioxidant and a free radical and Peters JM. Exposure to residential electric and
scavenger. Therefore, this hormone could protect magnetic fields and risk of childhood leukemia. Am J
Epidemiol. 134: 923-37, 1991.
biological systems against oxidative damage. The Poncz M, Solowiejczyk D, Harpel B, Mory Y, Schwartz E
increasing effect of EMF on the concentration of free and Surrey S. Construction of human gene libraries from
radicals has been suggested that melatonin suppression small amounts of peripheral blood: Analysis of ß-like
in humans may increase the probability of mutagenic globin genes. Hemoglobin. 6: 27-36, 1982.
and carcinogenic risk (Jajte and ZmySlony, 2000). Savitz DA, Wachtel H, Barnes FA, John EM and Tvrdik JG.
EMF (≥1 mT) increases the concentration of free Case-control study of childhood cancer and exposure to
radicals that escape from the alkyl sulphate and 60-Hz magnetic fields. Am J Epidemiol. 128: 21-38, 1988.
Scaiano JC, Cozens FL and Mohtat N. Development of a
sulphonate micelles. The effect of extremely low- model and application of the radical pair mechanism to
frequency EMF on the radicals formed from singlet radicals in micelles. Photochemistry and Photobiology.
precursors is larger than triplet precursors. Some 62: 818-829, 1995.
radicals such as hydroxyl and peroxyl radicals Tomenius L. 50-Hz electromagnetic environment and the
generated in the biological reactions are formed from incidence of childhood tumors in Stockholm County.
singlet precursors (Eveson et al., 2000). Bioelectromagnetics. 7: 191-207, 1986.
In conclusion, the results obtained from our study Wertheimer N and Leeper E. Electrical wiring configurations
and childhood cancer. Am J Epidemiol. 109: 273-284,
suggest that the effects of extra low frequency EMF on
1979.
the concentration of free radicals and the Zareie MH, Erdem G, Öner C, Öner R, Ö¤üfl A and Piflkin E.
recombination of radical pairs might trigger the Investigation of ascorbate-Cu (II) induced cleavage of
carcinogenesis in the populations living close to the DNA by scanning tunneling microscopy. Int J Biol
overhead electric power distribution lines. Macromol. 19: 69-73, 1996.
Journal of Cell and Molecular Biology 2: 39-42, 2003. 39
Haliç University, Printed in Turkey.

C h ro m o s o m e s o f a b a l a n c e d t r a n s l o c a t i o n c a s e e v a l u a t e d w i t h
a t o m i c f o r c e m i c r oscopy
Zerrin Y›lmaz1*, Mehmet Ali Ergun2, Erdal Tan3
1
Department of Medical Biology and Genetics, Baskent University, Faculty of Medicine, 06570,
Maltepe, Ankara, Turkey; 2Department of Medical Biology and Genetics, Gazi University, Faculty of
Medicine, 06510, Besevler, Ankara, Turkey; 3Materials 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.

Key words: Balanced translocation, chromosome polymorphism, atomic force microscopy

D e n g e l i t r a n s l o k a s y o n v a k a s › n d a k ro m o z o m l a r › n a t o m i k g ü ç m i k roskobu ile
de¤erlendirilmesi

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.

Anahtar sözcükler: Dengeli translokasyon, kromozom polimorfizmi, atomik güç mikroskobu

I n t ro d u c t i o n cellular level. Human cytogenetics is almost always


concerned with light microscope studies of
Cytogenetics is the study of genetic material at the chromosomes. Staining procedures which provide a
40 Zerrin Y›lmaz et al.

uniform unbanded appearance to chromosomes are genitourinary, endocrinological and other organ
referred to solid or covential staining. They can, systems; also laboratory findings were normal.
however, be useful for studies on chromosome
breakage as scoring gaps and breaks can be difficult in Light microscopy analysis
lightly stained chromosome bands. Giemsa banding
(G-banding) has become the most widely used Metaphase chromosome preparation was obtained
technique for the routine staining of human from peripheral blood lymphocytes using standard
chromosomes. The chromosome banding patterns techniques (Verma and Babu, 1995). Conventional
obtained reflect both the structural and functional cytogenetic analysis was carried out using GTG-
composition of chromosomes. Consititutive banding and C-banding techniques (Benn and Perle,
heterochromatin is the structural chromosomal 1992). The chromosome images were captured by
material seen as dark staining material in interphase as computer imaging (Cytovision system, Image
well as during mitosis. It includes both repetetive analysis, Applied Imaging, Saunderland, UK).
DNA, satellit DNA and some non-repetetive DNA. For each patient we analysed 20 metaphases, and
C-banding can be used to demonstrate the repetetive C-banding procedure was performed while
DNA (Benn and Perle, 1992). investigating 14 cenh+.
Atomic force microscopy (AFM) is a diagnostic
tool for detecting detailed structures of the Atomic force microscopy and analysis
chromosomes and the surface topography of human
chromosomes can be examined using this new The AFM used in this study was TopoMetrix
technique (Binning et al., 1986; Musio et al., 1997). TMX2000 Explorer, operating in contact mode and air.
AFM could be considered as a tool for further Throughout the surface analysis, we have used
chromosomal studies. standard pyramidal tip (1520-00) with the radius of
In our previous studies using AFM, we showed curvature of approximately 1000 A°. During the
that, unbanded human metaphase chromosomes surface analysis, the metaphase region was primarily
displayed a banding pattern similar to G-bands, and for determined and addressed by light microscopy. Later,
the first time we have provided an AFM imaging of the region under consideration was scanned via AFM
chromosomes in trisomy 13, 21 and Klinefelter at various scan ranges changing from 150 µm down to
Syndrome patients (Ergun et al., 1999). Besides, G and 10 µm or less to image the chromosomes in a good
C-banding patterns of chromosomes were also manner. The applied force and the image resolution
investigated (Sahin et al., 2000; Tan et al., 2001). were between 1 and 3 nN and 400x400 pixels (or
In this study, we used AFM in order to analyse the higher) respectively for each image acquisition. The
surface topography of derivative chromosomes of a raw data gathered were analysed by using the software
female patient whose daughter was referred to our of the microscopy system in two or three-dimensional
Genetics department with the chief complaints of patterns.
abortions. 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
Materials and methods performed on derivative chromosomes.

Case presentation
Results
In this study we evaluated the chromosomes of a
family. This family was referred to our genetics The karyotype of the male revealed 46, XY, while the
department for cytogenetic analysis because of two cytogenetic analysis of his wife was karyotyped as
previous abortions during the first trimester. They had 46, XX, t (7;12) (p 21;q 14); a balanced translocation.
no live-born children after a marriage of 5 years. The She also had 14cenh+. In order to understand the origin
male was 37 years old and healthy, and his non- of this translocation chromosome, her mother was
consanguineous wife was 36 years old. Her physical karyotyped and she was also found to be a translocation
examination revealed no abnormalities in carrier; 46, XX, t (7;12) (p 21; q14) and 14 cenh+.
AFM in chromosome evaluation 41

F i g u re 1: The partial karyotype of the daughter (a) and the


mother (b); derivative chromosomes 7 and 12 and F i g u re 3: AFM image of chromosome 14 indicated by an
chromosomes 14 with C-banding images are shown. arrow.

Discussion

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
F i g u re 2: AFM image of derivative chromosome 7 and line (Mullinger and Johnson, 1987). It was thought that,
measure analysis of the gap region. these regions correspond to high-order structural
aberrations resulting from an incomplete or irregular
composition of chromatid fibres induced by a
The karyotypes of the mother and the daughter translocation of a chromosomal fragment. The gap
were similar; 46, XX, t (7;12) (p 21; q 14). The partial regions were the results of chromosomal
karyotype of the daughter (Figure 1a) and the mother rearrangements (Uehara et al., 1996).
(Figure 1b) with the derivative chromosomes 7, 12 and Our second analysis was on the satellite region of
chromosomes 14 with C-banding images are shown. the chromosome 14 of the mother. First of all, GTG-
We performed detailed measurements on the banding revealed an increase in the heterochromatin
derivative chromosomes of the mother and detected region of the short arm of chromosome 14. Then, we
the attached chromosome fragment from derivative performed C-banding procedure to understand if this
chromosome 12 to the derivative chromosome 7. Our region was belonging to a constitutive heterochromatin
measurements revealed a 0.6 µm gap region (Figure 2). region, or to an extra banding region. The results of
Besides we also analysed the satellite region of C-banding confirmed that these regions were
chromosome 14 (Figure 3) of the mother. belonging to heterochromatin region. Our 3-
dimensional 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 Uehara S, Sasaki H, Takabayashi T and Yajima A. Structural
of chromosomes 1, 9, and 16 and on the distal arm of aberrations of metaphase derivative chromosomes from
Y chromosome (Burkholder and Duczek, 1980; Cook, reciprocal translocations as revealed by scanning
1995). Our AFM image also helps us to understand electron microscopy. Cytogenet Cell Genet. 74: 76-79,
1996.
that these regions were not belonging to G- banding
Verma RS and Babu A Banding techniques. In: Human
regions, as there was not a banding pattern (Tan et al., Chromosomes Principles and Techniques. Verma RS and
2001). Babu A (Ed). McGraw-Hill Inc. New York. 72-133,1995.
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.

R e f e r ences

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.
Journal of Cell and Molecular Biology 2: 43-48, 2003. 43
Haliç University, Printed in Turkey.

E f f e c t o f e p i r u b i c i n o n m i t o t i c i n d e x i n c u l t u r ed L-cells
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<0.05 - p<0.001).

Key words: Epirubicin, L-cell, transformed fibroblast, mitotic index, in vitro

E p i r u b i s i n i n k ü l t ü r d e k i L - h ü c re l e r i n d e m i t o t i k i n d e k s e e t k i s i

Özet

Kanser kemoterapisi, yayg›n tümörlerin tedavisinde cerrahi uygulama ve radyoterapinin yan›nda gerçeklefltirilen ek
bir uygulamad›r. Antrasiklin türevi bir antibiyotik olan epirubisin (EPI) klinik olarak kullan›lan antineoplastik
ilaçlardan birisidir. Bu çal›flmada, EPI in sitotoksik etkileri, transforme edilmifl fare fibroblastlar›nda (L-hücreleri)
araflt›r›ld›. EPI in 0.001 µg/ml, 0.01 µg/ml ve 0.1 µg/ml konsantrasyonlar› 2, 4, 8, 16 ve 32 saat süresince hücrelere
uyguland›. Sonuçlar uygulanan zaman ve konsantrasyona ba¤l› olarak EPI in L-hücrelerinin mitotik indeks
de¤erlerini düflürdü¤ünü gösterdi. Bu düflüfl kontrol grubu ile karfl›laflt›r›ld›¤›nda, her bir deney grubunda istatistik
olarak anlaml› bulundu (p<0.05 - p<0.001).

Anahtar sözcükler: Epirubisin, L-hücreleri, transforme edilmifl fibroblast, mitotik indeks, in vitro

I n t ro d u c t i o n 1989; Zuckerman et al., 1993). This drug is commonly


used since it has an equivalent spectrum of antitumor
Of the cancer drugs in clinical use, the anthracyclines action as doxorubicin but with less systemic and
have a spectrum of antitumor activity and are clearly cardiac toxicity (El-Mahdy Sayed Othman, 2000).
the most useful cancer drugs among the natural The mechanism of anti-tumour action for EPI has
product (Chabner and Mayers, 1993). not been completely elucidated. Various studies have
Epirubicin (EPI) is the epimer of the anthracycline revealed that EPI enters the cells rapidly and is
antibiotic doxorubicin, with inversion of the 4’- localised in nuclei and forms a complex with DNA by
hydroxyl group on the sugar moiety, and has been used intercalation between DNA strands (Di marco, 1984).
alone or in combination with other cytotoxic agents in DNA replication and transcription have been shown to
the treatment of a variety of malignancies (Young, be inhibited by this intercalation (Lollini et al., 1989;
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 F i g u re 1: Structural formulae of EPI.
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 1943). Transformed L-cells obtained from mouse
inhibition of HeLa cell colony formation (Di marco et subcutaneous connective tissue in 1943. They were
al., 1976). Similarly, EPI was as active as doxorubicin supplied by Dr. P.P. Dendy of Department of
on mouse embryo fibroblast proliferation (Di marco et Radiotherapeutics, Cambridge University, in 1975.
al., 1977), but was taken up in greater amount than The cells were grown in Medium-199 (M-199, Gibco
doxorubicin by L1210 leukemia cells in vitro (Wilson lab.) containing 10% foetal bovine serum (FBS, Gibco
et al., 1981). lab.), 100 µg/ml streptomycin and 100 IU/ml
There have been few studies about the effect of EPI penicillin, and were passaged twice a week in
on mitotic index of rapidly proliferating cells. In this appropriate number of 25 cm2 flasks and the volume of
study, we have therefore studied the effect at EPI, the complete medium in each flask was completely to
employed in the concentrations of 0.001 µg/ml, 12 ml. Cells were removed from the surface of culture
0.01 µg/ml and 0.1 µg/ml for a period of 2 to 32 hours, flasks by addition of 0.25% trypsine (Gibco lab.) and
on proliferation of transformed L-cells in culture centrifuged for 3 minutes at 1500 cycle/min.
which was investigated by measuring mitotic index in Following the addition of M-199 on the cell
order to investigate the effectiveness of this drug in precipitate, the cells became ready for the experiment.
chemotherapy. 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 3.104 cells/ml in petri
Material and methods dishes and incubated for 24 hours with 95% air and 5%
CO2 containing medium at 37°C with pH 7.2 in a
Chemical dessicator. At the end of this incubation medium was
removed, replaced with medium containing EPI
EPI (4’-epidoxorubicin), an anthracycline antibiotic, is concentrations.
a doxorubicin stereoisomer, possessing the L-arabino
instead of the L-lyxo configuration of the sugar moiety Drug application
(Figure 1). In EPI therefore the hydroxyl group on the
sugar moiety, possessing the stable 1C4 conformation, Epirubicin (Farmorubicin, Carlo Erba) was dissolved
has an equatorial orientation (Plosker and Faulds, immediatedly before use in sterile medium (M-199) to
1993). give the required concentration. We used 0.001 µg/ml,
0.01 µg/ml and 0.1 µg/ml concentrations of EPI. Cells
Cell line were treated with these doses for 2, 4, 8, 16 and 32
hours.
The cells used in this study were derived from mouse
fibroblast by in vitro malign transformation (Earle,
Epirubicin effect on mitotic index 45

Mitotic index analysis Statistical analysis

Mitotic index were studied by the methods of Feulgen. Mitotic index values which obtained from experiments
Before the cells were treated with Feulgen, they were were calculated to evaluate the statistical analysis. The
prepared with 1 N HCl at room temperature for 1 differences between the percentage distrubition of M
minute and then hydrolized with 1 N HCl for 10.5 phase of the various treatment groups and control were
minutes at 60°C. After slides were treated with compared by the Student-t test (n=25).
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 Results

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 L-
cells 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
F i g u re 2: Mitosis in L-cells under the light microscope time the differences among the effects of various drug
(3.3x100). 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,
buffer. After staining, the slides were rinsed in distilled 4 and 8 hours EPI applications in Table 1 especially
water. And then the slides were air dried. At last with EPI concentration of 0.1 µg/ml. However, in the
mitotic index were calculated by counting metaphases, treatment of 0.1 µg/ml concentration, mitotic
anaphases and telophases for each tested drug inhibition reached a maximum at 32 hours application.
concentration and control (Figure 2). At least three The values of mitotic index of the cells treated with
thousands cells were examined from each slide for EPI for 32 hours showed that mitotic index decreased
mitotic index. 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 devia-
tion (SD).

Mitotic index (%)

EPI 2 hours 4 hours 8 hours 16 hours 32 hours


concentrations

Control 1.44 ± 0.12 SD 1.93 ± 0.14 3.04 ± 0.08 3.39 ± 0.15 3.84 ± 0.21
0.001 µg/ml 1.35 ± 0.09 a 1.80 ± 0.10 a 2.72 ± 0.07 a 2.96 ± 0.04 b 3.10 ± 0.30 b
0.01 µg/ml 1.29 ± 0.11 a 1.79 ± 0.05 b 2.61 ± 0.06 b 2.70 ± 0.13 b 2.99 ± 0.16 b
0.1 µg/ml 0.94 ± 0.02 c 1.04 ± 0.01 c 1.77 ± 0.09 c 1.85 ± 0.08 c 1.02 ± 0.22 c
a
: p < 0.05, b: p < 0.01, c: p < 0.001
46 Gül Özcan Ar›can and Mehmet Topçul

EPI significantly decreased the mitotic index in Although, in vitro studies with antitumour agents,
cultures of L-cells. The results show that EPI and with anthracyclines in particular, have not shown
decreased the mitotic index at significant level p<0.05 to predict the antitumour activity in vivo (Sinha and
- p<0.01 for lower drug concentrations 0.01 µg/ml and Politi, 1990; Nistico et al., 1999), in some studies,
0.001 µg/ml, at highly significant level p< 0.001 for significant correlations have been detected between the
0.1 µg/ml drug concentration when compared with the in vitro activity of EPI and other anthracyclines against
control. various tumour specimens, and therapeutic response
In addition, the reductions in mitosis of the cells (Bartkowiak et al., 1992; Plosker and Faulds, 1993).
following different treatment times (2, 4, 8, 16 and 32 Anthracyclines, including EPI, appear to result in
hours) with 0.1 µg/ml EPI concentration were maximal cell death in the S and G2 phases of the cell
statistically significant (p<0.001) from each other. cycle, but cytotoxic effects may occur in the G1 and M
However, this level of significance for the different phases at higher drug concentrations (Plosker and
treatment times was not observed with 0.01 µg/ml and Faulds, 1993; Topçul et al., 2002). Maximal lethal
0.001 µg/ml concentrations of EPI, respectively. effects of EPI were demonstrated in the S and G2
phases of the cell cycle in murine and human tumour
cell lines (Hill and Whelan, 1982).
Discussion An important comparison between EPI and
doxorubicin in vitro was carried out by Hill and
Anthracycline antibiotics have been used extensively Whelan (1982). The studies were performed on a wide
in the treatment of wide variety of malignancies, and range of murine and human cell lines: NIL8 (Syrian
are a standard component of many combination golden hamster cells); four human tumor lines (COLO-
chemotherapy regimens. EPI has been used alone or in 206 and LOVO, derived from colon carcinomas; SCC-
combination with other antineoplastic agents in the T/G, derived from a squamous cell carcinoma from the
treatment of a broad range of neoplasms. Studies in tongue; CHP 100, derived from a neuroblastoma);
understanding the mechanism of EPI have suggested L5178Y lymphoblastoid cell sub-lines. In the all cell
that EPI forms a complex with DNA by intercalation lines tested, both drugs showed comparable
between DNA strands, thus inhibiting DNA replication cytotoxicity, which increased exponentially with drug
and transcription (Özcan et al., 1997; Stewart et al., concentration and with duration of exposure. Of high
1997), and it increases DNA strand brekage (Cantoni et interest was the study carried out on NIL8 cells
al., 1990). synchronized by mitotic selection and treated for 1
EPI induced differentiation of human hour with the drug. Results showed that maximum cell
neuroblastoma cells in vitro, possibly related to a kill was observed with both drug in the S phase, some
reduction in the growth of surviving cells, thus kill during early G2, but no kill in G1 and M if low
allowing activation of intrinsic differentiation concentrations were used. Data from flow
mechanisms. Following culture of human microfluorimetry analyses and monitoring of mitotic
neuroblastoma cell lines with EPI 10 or 100 nmol/L for indices suggested population arrest in G2 for
24 hours, outgrowth of neurite processes was doxorubicin (Casazza and Giuliani, 1984).
detectable 3 or 4 days after exposure, and maximal In the present study, treating L-cells with various
morphological differentiation was achieved after 5 or 6 concentrations of EPI for 2, 4, 8, 16 and 32 hours,
days (Rocchi et al., 1987). decreased mitosis. The results showed that EPI
EPI has also been shown to be effective in diminished mitotic index of L-cells, depending upon
inhibiting basement membrane degradation, a property time and applied concentrations. When compared to
deemed necessary to prevent development of the control, this decrease was found statistically
metastases (Plosker and Faulds, 1993). In addition, EPI significant in each group (p<0.05-p<0.001). The values
has been shown to inhibit proliferation of all of mitotic index reached a minimum at EPI
neuroblastoma cell lines by 69 to 78 % relative to concentration of 0.1 µg/ml with increasing treatment
controls (Rocchi et al., 1987), of a human alveolar time. Increased concentrations resulted in a decrease
rhabdomyosarcoma cell clone (Lollini et al., 1989), on the values of mitotic index, being statistically
and of haemopoietic progenitor cells from several significant (p<0.001). Briefly, EPI concentration of 0.1
human leukaemic cell lines in liquid culture (Bagnara µg/ml showed to possess relatively more effect on
et al., 1987). proliferation of L-cells. Thus, the results of our study
Epirubicin effect on mitotic index 47

seem to be concordant with the above mentioned Di marco A. Epirubicin: Mechanism of action at the cellular
studies suggesting that cytotoxic effects of EPI might level. In: Advances in Anthracycline Chemotherapy:
occur in the G1 and M phases at higher drug Epirubicin. Bonadonna G (Ed). Masson, Milano-Italy.
41-47, 1984.
concentration.
Earle WR. Production of malignancy in vitro. IV. The mouse
In our study, decreases in the mitotic index of cells fibroblast cultures and changes seen in the living cells.
with increasing both treatment time and EPI J Nat Cancer Inst. 4: 165-212, 1943.
concentration have confirmed that EPI is an effective El-Mahdy Sayed Othman O. Cytogenetic effect of the
inhibitor of mitosis. anticancer drug epirubicin on Chinese hamster cell line
In conclusion, the results of this study declared the in vitro. Mutation Res. 468: 109-115, 2000.
cell kinetics and cytotoxic effects of the anticancer Haldane A, Finlay GJ, Baguley BC. A comparison of the
drug, EPI, in treated cultures of L-cell line. Although effects of aphidicolin and other inhibitors on
EPI has less systemic and cardiac toxicity than topoisomerase II-directed cytotoxic drugs. Oncol Res.
5 (3): 133-138, 1993.
doxorubicin and other anthracyclines with an
Hill BT and Whelan RDH. A comparison of the lethal and
equivalent spectrum of antitumor action, it still has kinetic effects of doxorubicin and 4’-epidoxorubicin
cytotoxic effects. in vitro. Tumori. 68: 29-37, 1982.
Lollini PL, De Giovanni C, Del Re B. Myogenic
differentiation of human rhabdomyosarcoma cells
Acknowledgment induced in vitro by antineoplastic drugs. Cancer
Research. 49: 3631-3636, 1989.
We would like to thank Prof. Dr. Atilla Özalpan for his Nistico C, Garufic C, Barni S, Frontini L, Galla DA,
Giannaarelli D, Vaccaro A, Dottovio AM, Terzoli E.
kind help and critics.
Phase II study of epirubicin and vinorelbine eith
granulocyte colony-stimulating factor: A high-activity,
dose-dense weekly regimen for advanced breast cancer.
R e f e r ences Ann Oncol. 10 (8): 937-942, 1999.
Özcan G and R›dvano¤ullar› M. The effect of epirubicin on
Bagnara GP, Rocchi P, Bonsi L. The in vitro effects of the cell cycle of L-cells. 13th National Congress of
epirubicin on human normal and leukemic hemopoietic Biology. Istanbul, Turkey. 3: 267-276, 1996.
cells. Anticancer Research. 7: 1197-1200, 1987. Özcan FG, Topçul MR, Y›lmazer N, R›dvano¤ullar› M.
Bartkowiak D, Hemmer J, Rottinger E. Dose dependence of Effect of epirubicin on 3H-thymidine labelling index in
the cytokinetic and cytotoxic effects of epirubicin in cultured L-strain cells. J Exp Clin Cancer Res. 16(1): 23-
vitro. Cancer Chemother Pharmacol. 30 (3):189-192, 27, 1997.
1992. Plosker GL and Faulds D. Epirubicin. A review of its
Cantoni O, Sestili P, Cattabeni F. Comparative effects of pharmacodynamic and pharmacokinetic properties, and
doxorubicin and 4’-epidoxorubicin on nucleic acid therapeutic use in cancer chemotherapy. In: Drugs.
metabolism and cytotoxicity in a human tumor cell line. Chrisps P (Ed). 0012-6667. 45 (5): 788-856, 1993.
Cancer Chemother and Pharmacol. 27: 47-51, 1990. Robert J and Gianni L. Pharmacokinetics and metabolism of
Casazza AM and Giuliani FC. Preclinical properties of anthracyclines. Cancer Surv. 17: 219-252, 1993.
epirubicin. In: Advances in Anthracycline Chemotherapy: Rocchi P, Ferreri AM, Simone G. Epirubicin-induced
Epirubicin. Bonadonna G (Ed). Masson, Milano-Italy. differentiation of human neuroblastoma cells in vitro.
31-40, 1984. Anticancer Research.7: 247-250, 1987.
Chabner BA and Myers CE. Antitumor antibiotics, Sinha BK and Politi PM. Anthracyclines. Cancer chemother
In: Cancer: Principles and Practice of Oncology. De vita apy. Biol Response Modif. 11: 45-57, 1990.
VT (Ed). AJF Lippincott, Philadelphia. 374-385, 1993. Skladanowski A and Konopa J. Interstrand DNA
Di marco A, Casazza AM, Gambetta R, Supino R, Zunino F. crosslinking induced by anthracyclines in tumour cells.
Relationship between activity and aminosugar Biochem Pharmacol. 47 (12): 2269-2278, 1994.
stereochemistry of daunorubicin and adriamycin Stewart DJ, Cripps MC, Dahrouge RGS, Yau J, Tomiak E,
derivates. Cancer Res. 36: 1962-1966, 1976 Huan S, Soltys K, Prosser A, Davies RA. Pilot study of
Di marco A, Casazza AM, Dasdia T, Necco A, Pratesi G, multiple chemotherapy resistance modulators plus
Rivolta P, Velcich A, Zaccara A, Zunino F. Changes of epirubicin in the treatment of resistant malignancies.
activity of daunorubicin, adriamycinand stereoisomers Cancer Chemother Pharmacol. 41: 1-8, 1997.
following the introduction or removal of hydroxyl Topçul MR, Ar›can Özcan G, Erensoy N, Özalpan A. Effect
groups in the amino sugar moiety. Chem Biol Interac. of epirubicin and tamoxifen on labelling index in FM3A
19: 291-302, 1977. cells. J Cell and Mol Biol. 2: 81-85, 2002
48 Gül Özcan Ar›can and Mehmet Topçul

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.
49

L e t t e r to editor

P ro s t a t e c a n c e r a n d i m p o r t a n c e o f Determination of the free PSA (i.e., the percentage of


PSA that is unbound to serum proteins) has also been
t u m o r m a r k e r studies suggested as a means of distinguishing malignancy
from benign hyperplasia. PSA has revolutionized the
P ro s t a t k a n s e r i v e t ü m o r m a r k › r l a r › i l e management of prostate cancer since its development
ilgili çal›flmalar›n önemi in the 1980s. For unclear reasons, PSA derived from
malignant epithelial cells tends to bind more avidly to
Prostate cancer is the most commonly new diagnosed serum proteins. Thus, in men with an elevated serum
noncutaneous malignancy in men in USA. In the year PSA level, cancer is more likely to be present when the
2002, according to the health statistics 189,000 men in percentage of free PSA is low. Because the relative
the United States are expected to be diagnosed with the sensitivity versus specificity varies, depending on the
disease and 30,200 men are expected to die of it. free PSA cutoff, the optimal cutoff value for free PSA
Incidence varies greatly, with African Americans is still under debate. Prostate specific antigen (PSA)
having the highest incidence in the world (224 cases represents the best serum marker for prostatic
per 100,000 population). The incidence of prostate carcinoma and is considered as most perfect tumor
cancer in African Americans stands in stark contrast to marker available today. Nevertheless, the use of PSA
the incidence in white Americans (150 per 100,000) to detect prostate cancer is clinically imprecise since
and that in men in Western Europe (39.6 per 100,000), benign and malignant prostate disease can cause
Japan (8.5 per 100,000), and China (1.1 per 100,000). elevations in PSA. It is sensitive but spesificity is not
Tumor markers are biological molecules that good to show tumor agressiveness, and so does not
indicate the presence of malignancy. They are benign prostatic hypertropy from invasive cancer.
potentially useful in cancer screening, aiding Age-spesific cut-offs have been suggested to improve
diagnosis, assessing prognosis, predicting in advance a spesificity, but there is still substantial overlap
likely response to therapy, and monitoring patients between normals and those with cancer. Further
before and after diagnosis. Because of low prevalance markers of tumor agressiviness, either measured in
of most cancers in the general population and the serum or needle biopsy specimens are needed to
limited sensivity and spesificity of avaible markers, determine which patients are in need of curative
these tests alone are generally of little value in treatment.
screening for cancer in healthy subjects. Currently, S e r u m a c i d p h o s p h a t a s e : Serum acid phosphatase
however, prostate spesific antigen (PSA) in (ACP) served as the only serum tumor marker for
combination with digital rectal examination (DRE) are prostate cancer between the 1930s and 1980s. more
undergoing evaluation as screening modalities for sensitive serum tumor marker in detection of localized
prostate cancer. Because of a lack of sensitivity and disease and in monitoring response to therapy. In the
spesificity markers are rarely of use in early diagnosis past two decades, the use of ACP has diminished
of cancer. Also they can be used as monitoring disease because of problems with lack of sensitivity and
evaluation with therapy. The goal of future research specificity and because of the discovery of prostate-
should be that development of the most specific, cheap specific antigen (PSA), a is an independent predictor
and easy markers for common cancer types as prostate of biochemical recurrence in men who undergo
cancer. surgery. ACP level is independently predictive of
P rostate spesific antigen: Screening prostate cancer biochemical recurrence following radical retropubic
provides a dilemma unique among cancer sites. The prostatectomy (RRP), when adjusted for other
best strategy is determination of the ratio of the predictive variables.
prostate serum antigen (PSA) to the volume of the G r a n i n s : The nomenclature for chromogranin-A
prostate gland in prostate cancer diagnosis. continues to evolve; for simplicity,it is referred as
50

granin-A (GRN-A). GRN-A is a 49-kilodalton protein such as the amount of tumor angiogenesis, and
that is produced exclusively by endocrine and immunohistochemical levels of various markers,
neurondocrine (NE) cells. It is costored and cosecreted including Ki-67, Bcl-2, p53, and E-cadherin. E-
with the resident hormones of these cells, such as cadherin as a biomarker to predict prognosis in
catecholamines and calcitonin (CT). Although the patients at risk of disease recurrence after radical
function of GRN-A is not known, it can serve as a prostatectomy is warranted.
tissue and serum marker for a variety of endocrine Serum total homocystein: Homocystein (Hcy) as a
cells and tumors. There are several major cancer types tumor marker targets to reveal chemotherapy effects
are characterized by NE differentiation. Recently, the on patients. It is largely derived from cellular
importance of NE differentiation and the attendant methionine, an essential amino acid drawn from
expression of chromogranin-A has become dietary intake. Intracellular homocysteine is normally
appreciated for prostate cancer. Clinical and basic secreted extracellularly, at rapid rates. In the
roles of chromogranin-A in human prostate cancer are circulating blood, the majority of the homocysteine
still studied. Although the function of GRN-A is not binds to albumin, forming a disulfide linkage.
known, several theories have emerged about its role: Approximately 10% to 20% of the Hcy also exists as a
(1) that it participates and perhaps regulates the storage mixed disulfide with cysteine or with homocysteine
and secretion of its coresident hormones in secretory itself . Very little Hcy is present in the circulating
vesicles; (2) that it inhibits proteolytic cleavage blood in a free reduced form (approximately
enzymes; (3) that it binds calcium and thus regulates 1%).Elevated serum tHcy (total homocysteine, free
the biologic effects of this ion; and (4) that it is a and protein-bound) are detectable in patients with
precursor for peptides that have unique biologic effects malignant diseases. Finding increased circulating tHcy
on the function and growth of its resident cells. in tumor cells may also be related to the so-called
Function notwithstanding, the production of GRN-A in ‘‘methionine dependency’’ of many, but not all, tumor
NE prostate cancers has resulted in the availability of cells. Many tumor cells are methionine dependent
a new serum and tissue marker for the tumors. The because of their inability to convert homocysteine
clinical potential of GRN-A as a serum and tumor (Hcy) to methionine by way of the remethylation
marker in prostate cancer. It is now wellestablished reaction. On the other hand, normal cells have no
that GRN-A can be a marker for advanced disease. problem obtaining methionine from homocysteine.
More importantly, GRN-A may be a marker for early Folate is critical to the remethylation reaction. Any
and recurrent disease, even in the absence of abnormal folate deficiency will result in the impairment of
PSA. GRN-A serum levels may also have prognostic function of the remethylation reaction, causing
significance, especially for androgen-independent accumulation of Hcy. Therefore, it was generally
prostate cancer. believed that the rapid proliferation rate of tumor cells,
E - c a d h e r i n : In attempts to determine which cancers such as in prostate cancer and in the so-called
of patients with clinically localized disease who methionine dependency of tumor cells, was due to the
undergo radical prostatectomy will recur, the most depletion of folate by the rapid growing tumor cells
well-characterized and accepted predictors are model and changing levels of fLV (a form of folate) in 24 h
equations that take into account preoperative serum after therapy. In other words, with a better
prostate-specific antigen (PSA), final Gleason score, understanding of the effects of various drugs, the rise
and final pathologic stage. Prediction of regression for and fall of circulating tHcy could be used as a new
the individual patient using these statistical models, tumor marker to monitor cancer patients during
however, is still not precise, and these models could therapy, complementing commonly used tumor
still be improved on. Thus, additional markers are markers. The general impression that elevated tHcy is
needed to more accurately target high-risk patients for detectable in cancer patients derives from the fact that
inclusion in clinical trials involving investigational many cancer patients take anti-folate drugs such as
therapies for locally advanced prostate carcinoma. methotrexate. It is important to know that the level of
Several other approaches show promise in this regard, tHcy reflects the tumor cell proliferation rate.
including nuclear morphometry, where the results have Regardless of the folate status, it is very likely given
been quite consistent. Other more controversial our results and others that the rapid proliferation of
markers include DNA ploidy and other biomarkers, tumor cells is one of the major reasons that elevated
51

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
53

Book reviews

Ç e t i n ALGÜNEfi, Radyasyon Biyofizi¤i, Trakya The book is a valuable guide of radiation


Üniversitesi Yay›nlar›, Edirne, 135 sayfa, ISBN: 975- biophysics. On the other hand, this book is a good
374-051-4, 2002. document because there are very limited Turkish
publication in this area. For this reason, I
Kitapta atom ve çekirde¤inin yap›s›, karars›z recommended this book for the biologists, physisists,
çekirdekler, iyonizan radyasyon tipleri ve özellikleri, agriculturists and veterinarians who apply radiation on
radyasyonun madde ile etkileflmesi ve radyasyon living organisms for several purposes.
birimleri, iyonizasyona sebep olmayan radyasyonlar,
iyonizan radyasyonlar›n biyolojik etkileri ve
radyasyondan korunma konular› tart›fl›lm›flt›r. Atilla ÖZALPAN
Bölümlerin ayr›nt›l› incelenmesinde, kitab›n Haliç University,
diziliminin geleneksel tarzda, flekil ve tablolar›n Department of Molecular Biology and Genetics
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.

Atilla ÖZALPAN
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.
55

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and ideas will be accepted. Manuscripts should not
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56

by superscript small letters and type footnotes of the XRCCS gene and its role in DNA repair and
below the table. Each table must be referred to in V (D) J recombination. Science. 265: 1442-1445,
the text. 1994.
2. Fine drawings can either submitted as original Ohlrogge JB. Biochemistry of plant acyl carrier
drawings ready for print or as clean and high proteins. In: The Biochemistry of Plants: A
contrast glossy black and white photographs. Comprehensive Treatise. Stumpf PK and Conn EE
3. Photographs must be supplied as black and white, (Ed). Academic Press, New York. 137-157, 1987.
high contrast, glossy prints, trimmed at right angles. Weaver RF. Molecular Biology. WCB/Mc
4. Captions for figures should be typed Graw-Hill.1999.
double-spaced, on a separate sheet. Each caption 2. Only papers published or in press should be cited in
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R e f e re n c e s :

1. Citation in the text should take the form: Smith and


Robinson (1990) or (Smith and Robinson, 1990). If
several papers by the same author in the same
year are cited, they should be lettered in sequence
(1990a), (1990b), etc. When papers are by more
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al. (1990) or (Smith et al., 1990).
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order. The following models for the reference list
cover all situations. The punctuation given must be
exactly followed.
Redford IR. Evidence for a general relationship
between the induced level of DNA double-strand
breakage and cell killing after X-irradiation of
mammalian cells. Int J Radiat Biol. 49: 611- 620,
1986.
Taccioli CE, Cottlieb TM and Blund T. Ku 80: Product
Journal of Cell and Molecular Biology
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 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

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