1021-4437/04/5102- © 2004
Russian Journal of Plant Physiology, Vol. 51, No. 2, 2004, pp. 241–248. Translated from Fiziologiya Rastenii, Vol. 51, No. 2, 2004, pp. 269–277.
Original Russian Text Copyright © 2004 by Andreev, Spiridonova, Kunakh, Solov’yan.
The basic mechanisms of aging and the ways to con-
trol this process invariably appeal to many researchers.
The present study considers some aspects of seed
aging. It is a process of special interest because it pro-
ceeds in the system of low metabolic activity and does
not involve the activation of characteristic genetic pro-
grams participating in cell differentiation and pro-
grammed cell death.
Seed aging is accompanied with the gradual decline
in the germination rate as the integral result of many
biochemical and physiological events. In particular, the
efﬁciency of cell reparation systems and antioxidant
mechanisms declined in deteriorating seeds [1–3],
resulting in lipid peroxidation and protein damage.
Apparently, the activation of hydrolytic enzymes,
which accounts for protein and carbohydrate cleavage
and enhanced fragmentation of nuclear DNA, is one of
the factors of declining seed viability [4–6]. However,
the processes occurring in cell nuclei in the course of
seed aging are basically unknown.
Previously we worked out an approach to studying
the speciﬁc organization of nuclear DNA in high-order-
packed chromatin. After agarose-embedded cell nuclei
or cells were treated with protein-denaturing agents,
such as SDS or proteinase K, the fractionation of such
preparations produced high-molecular-weight (HMW)
DNA fragments (50–100 kb). These fragments were
shown to arise following DNA cleavage at the sites of
nuclear matrix attachment, the residual protein back-
bone structures resistant to extraction with 2 M NaCl;
this fragmentation is catalyzed by matrix-associated
nuclease similar to DNA topoisomerase II [7, 8]. The
pattern of such HMW DNA fragments seems to expose
the speciﬁc chromatin organization into loop domains
that develop due to the contacts between DNA and the
nuclear matrix proteins. We also established that the
developmental changes in the functional state of plant
cells proceeded in parallel with the changes in the pattern
of nuclear HMW-DNA fragmentation and assumed that
the latter are of functional meaning .
In the present study, we used the previously devel-
oped approach to assessing DNA cleavage into HMW
fragments in order to elucidate the processes that take
place in the nuclei of aging seed embryos and the prob-
able causes of this phenomenon.
MATERIALS AND METHODS
In this study we used seeds of rye (
cv. Boguslavka) collected in 1995, 1997, and 2000 and
obtained from the Institute of Plant Physiology and
Genetics, National Academy of Sciences of Ukraine.
Accelerated aging was performed using the technique
Aging and Loss of Germination in Rye Seeds Is Accompanied
by a Decreased Fragmentation of Nuclear DNA
at Loop Domain Boundaries
I. O. Andreev, E. V. Spiridonova, V. A. Kunakh, and V. T. Solov’yan
Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine,
ul. Zabolotnogo 150, Kiev, 03143 Ukraine;
fax: (38-044) 2666-0759; e-mail: firstname.lastname@example.org
Received September 25, 2002
—To investigate the processes that occur in the embryo cell nuclei in the course of natural and accel-
erated aging of rye seeds, nuclear DNA structural organization into chromatin loop domains was studied. The
loss of germination was shown to be accompanied by a decreased excision of chromatin loop domains. The
study of chromatin accessibility to DNase I did not reveal any considerable changes in chromatin architecture
that would explain the decreased DNA fragmentation at matrix attachment regions. A soluble nuclear protein
of ca. 31 kD was found to manifest nuclease activity, which declined with the loss of germination. The study of
DNA fragmentation in histone-depleted nuclei (nucleoids) disclosed a nuclease activity resistant to 2 M NaCl
extraction and sensitive to the speciﬁc inhibitors of DNA topoisomerase II; the latter activity also declined with
aging. The authors conclude that the changes in DNA fragmentation patterns in aging seeds were primarily
caused by a decreased activity of the enzymes accounting for the excision of chromatin loop domains.
Key words: Secale cereale - seed aging - accelerated aging - DNA fragmentation - nuclear nucleases - DNA
: FIGE—ﬁeld inversion gel electrophoresis;
HMW—high molecular weight; MAR(s)—matrix-attachment
region(s) of DNA; PMSF—phenylmethylsulfonyl ﬂuoride.