Chromatin diminution during endosperm development in Allium atroviolaceum Boiss. (Alliaceae).

Page 1

Bull. Georg. Acad. Sci
Bull. Georg. Acad. Sci Bull. Georg. Acad. Sci
Bull. Georg. Acad. Sci. 166, 3 : 537

Chromatin diminution during endosperm development
in Allium atroviolaceum Boiss. (Alliaceae)
537

. 166, 3 : 537. 166, 3 : 537
. 166, 3 : 537- - - -540
540540
540; 2002
; 2002; 2002
; 2002

G. Gvaladze, N. Nadirashvili, M. Akhalkatsi

Institute of Botany, Georgian Academy of Sciences
Presented by corr.-member of the GAS G. Nakhutsrishvili


Abstract

The chromatin diminution phenomenon during endosperm formation in Allium
atroviolaceum was confirmed by cytophotometric method. Triploid endosperm nuclei, originated by
the triple fusion, lose heavily condensed chromosome set beginning with the 8-nucleate stage. This
chromosome set is arisen by pycnotic chalazal polar nucleus participating in the triple fusion. As a
result, endosperm nuclei at a cellular stage possess only diploid genome. In spite of this fact, the
endosperm of the investigated plant species is functioning normally.


Key words: embryology, endosperm, chromatin, Allium.

Endosperm in Allium species is originated by triple fusion, a typical second unit of the
double fertilization occurring in angiosperms. During this process, two polar nuclei of the central
cell of a female gametophyte fuse with sperm nucleus and, as a result, a triploid endosperm is
formed (Gvaladze, 1976). In contrast with the other species of a genus Allium, micropylar and
chalazal polar nuclei of a central cell of the female gametophyte of Allium atroviolaceum and A.
rotundum reveal difference in structural organizations. In spite of the fact that both of them are
haploid and contain similar sets of chromosomes, chalazal polar nucleus is smaller than the
micropylar and possesses Feulgen-positive chromatin determining its pycnotic nature (Gvaladze,
1962, 1966). The similar phenomenon is described in A. porrum (Sokolov, 1968).
This structural difference of the polar nuclei in these species plays definite role during
endosperm development, when beginning with the 8-nucleate stage, the diminution of the
heterochromatinised DNA of the chalazal nucleus occurs. Chromatin diminution was observed
visually on cytological preparations, when condensed Feulgen-positive DNA exits from endosperm
nuclei via the nuclear envelope and eliminates in the hyaloplasm (Gvaladze, 1962, 1966). It was
supposed (Gvaladze, 1962, 1976) that this process caused completely or partially elimination of
chromosome set of the chalazal polar nucleus from the entire triploid genome of the endosperm
nuclei. The similar observation was done in Erythronium japonicum, where the elimination of a
complete genome of the chalazal polar nucleus proceeds after the first division of the primary
endosperm nucleus (Oikawa, 1953). In general, DNA diminution is a rare phenomenon in the plant
kingdom and is qualified as intraspecific unorthodox genome size variation, i.e. “plastic genome”
(Greilhuber, 1998).
Current data on elimination of the heterochromatinised DNA of the chalazal polar nucleus in
A. atroviolaceum and A. rotundum are based only on visual observations on cytological
preparations and no experimental confirmation of the fact, does DNA content really diminish in
endosperm nuclei as a result of elimination of the chalazal polar nucleus genome or not, is provided.
The objective of this investigation concerns the experimental confirmation of elimination of
the chalazal polar nucleus genome from the endosperm nuclei in A. atroviolaceum. For this

Page 2

purpose, cytophotometric measurements of DNA content in endosperm nuclei were conducted to
determine the occurrence of DNA diminution phenomenon during endosperm development.
Investigation was carried out in the Laboratory of Cytology at the Institute of Zoology of the
Georgian Academy of Sciences. DNA content was determined by scanning cytophotometer,
Reichert, Austria, with one-wave cytophotometric method (wave length was 550 µm) on sections
after Feulgen staining. Probe diameter was 0,63 µm. All nuclei were scanned with the same
magnification (ocular x15, objective x40). DNA content was determined using the formula C=VA,
where C is DNA content in a single nucleus, V – volume of a nucleus, A – DNA concentration on
the section of a nucleus. Error of the method is 5%. Obtained data were processed statistically
(Brodskii, 1956).
DNA content of the integument cell nucleus (number of measured nuclei x=30) in the ovule
of A. atroviolaceum was used as reference for species-specific diploid DNA content (2n=16). DNA
content in endosperm nuclei was measured at late cellular stage of the endosperm development
(x=50).
Cytologically, the structure of the somatic interphase nuclei of the integument tissue is
euchromatic and contains more or less uniformly distributed chromocenters, which should
correspond to the constitutive heterochromatin. Endosperm nuclei at early stages of the endosperm
development, when endosperm is nuclear, possess heavily condensed chromatin region, which by
position and structure corresponds to the genome originated by pycnotic chalazal polar nucleus
during triple fusion. It should be noted that at later stages, when endosperm becomes cellular, most
endosperm nuclei are deprived of such heterochromatinised DNA region.
Determination of DNA content at the late cellular stage of the endosperm development,
when cell formation is almost completed, have revealed that DNA content in the endosperm nuclei
is close to 2C DNA content in nuclei of the integument cells (Tab. 1). This is indicative that most of
the endosperm nuclei contains diploid set of the chromosomes. Measurements have shown that
some endosperm cells still continue division at this stage determining the occurrence of both G1 and
G2 phases. Therefore DNA content corresponds to 2C or 4C.

Table 1. DNA content in nuclei of the integument and the endosperm cells of A. atroviolaceum.

Nucleus class Number of
nuclei (x)

Integument
Endosperm
50

Diminution of the chalazal polar nucleus genome during endosperm development in A.
atroviolaceum should be conditioned by the fact that already at the megagametogenesis proceeds
inactivation of this chromosome set, which becomes heterochromatinised, resulting in the lack of a
function and determining its elimination.
Firstly, phenomenon of heavily chromatinisation of the chalazal polar nucleus was found in
Gagea species possessing tetrasporic Fritillaria type embryo sac (Darlington, 1947; Geitler, 1950;
Romanov, 1961). In these plants, the triploid chalazal polar nucleus depending on the condensation
rate may participate or not in a triple fusion. Although, elimination of the heterochromatinised DNA
was not observed during endosperm development in these species. In Gagea lutea, where the
pycnotic triploid chalazal polar nucleus participates in the formation of pentaploid endosperm,
cytophotometric measurements have shown that 3:2 relationship of heterochromatic and
euchromatic chromosome sets is maintained up to the cellular endosperm (Greilhuber et al., 2000).
Occurrence of pycnotic chalazal nuclei in Fritillaria type of embryo sac was interpreted as
“depression” of the chalazal pole of a female gametophyte i.e. progressive pycnosis (Romanov,
1961; Gvaladze, 1976). Although, electron-microscopic study (Gvaladze, Akhalkatsi, 1992)
538

DNA content C
(mean ± standard deviation)

30

2,06± ± ± ±0,3
2,24± ± ± ±0,6

Page 3

revealed that “depression” has no effect on normal functioning and structure of surrounding
cytoplasm. Nowadays, it is experimentally confirmed (Bužek et al., 1998) that chromatin
condensation in the chalazal polar nucleus proceeds differently than it is the programmed cell death,
apoptosis (Wyllie, 1980). This phenomenon may be characterized as transformation of the entire
genome into facultative heterochromatin (Bužek et al., 1998; Greilhuber et al., 2000).
For long time, it has remained largely unknown that facultative heterochromatin does occur
in plant kingdom (Greilhuber et al., 2000). According to the classical definition heterochromatin
represents chromosome segments or chromosomes that form chromocenters, i.e. condensed
chromatin bodies, which after telophase do not decondense like as euchromatin does (Heitz, 1933).
In difference with the constitutive heterochromatin, which is characterized by a permanently
condensed state, facultative heterochromatin became inactivated during a certain developmental
stage (Brown, 1966). There are two well-known examples of cytologically observable facultative
heterochromatinisation in eucaryotes: X-chromosome inactivation in female mammals (Barr,
Bertram, 1949), and an inactivation of the whole paternally derived genome in male coccids
(Khosla et al., 1996). Nowadays, immunostaining experiments have demonstrated that
heterochromatinisation of the chalazal polar nucleus in Gagea lutea is similar to the inactivated
human X chromosome and is determined by deacetylation of lysine of a histone H4 in the absence
of global hypermethylation of cytosin occurring at the formation of constitutive heterochromatin
(Bužek et al., 1998).
On the base of these data one should be suggested that the genome of the chalazal polar
nucleus of A. atroviolaceum undergoes complete or partially inactivation already at the
megagametogenesis by transforming into facultative heterochromatin. During endosperm
formation, genome of the chalazal polar nucleus should not play any essential role in the
functioning of the endosperm nuclei and proceeds its elimination from the triploid endosperm
genome originated by the triple fusion. Although, participation of the chalazal polar nucleus in the
formation of the primary endosperm nucleus supports earlier hypothesis (Gvaladze, 1976) that a
role of the chalazal polar nucleus of A. atroviolaceum in a triple fusion is restricted only to the
initiation of earlier development of the endosperm in comparison with the embryo. Beginning with
the 8-nucleate stage, in spite of the elimination of the chalazal polar nucleus genome from the
endosperm nuclei, the development of diploid endosperm proceeds similar to the triploid
endosperm of the other Allium species that do not possess pycnotic chalazal polar nucleus and are
not characterized by DNA elimination from the endosperm nuclei.

Acknowledgement
We thank Dr. G. Kvinikhidze for providing kind help to conduct cytophotometric
investigation.

References

1. Brodskii, 1959. Cytophotometry. In: Uspekhi Sovr. Biol. 42:87-107. (Russ.).
2. Gvaladze G.E. 1962. Development of generative organs and embryogeneses of some species of
Allium. Thesis. (Russ.).
3. Gvaladze G.E. 1966. Study of peculiar structure of the endosperm. Proc. Georg. Bot. Soc. 3:3-8.
(Russ.).
4. Gvaladze G.E. 1976. Chalazal polar nucleus of the central cell of an embryo sac of
angiosperms. Tbilisi, Metsniereba. (Russ.).
5. Gvaladze, G., Akhalkatsi, M. 1992. On depression of chalazal part of an embryo sac in
Angiosperms. Bot. Zhurn. 77, 7:71-75. (Russ.).
6. Romanov I.D. 1961. Origin of peculiar structure of endosperm nuclei in Gagea. DAN SSSR
141, 4:984-986. (Russ.).
539

Page 4

7. Sokolov I.D. 1968. Cytoembryological study of some cultivated onion species (Allium cepa L.,
A. porrum L., A. fistulosum L.). Thesis. (Russ.).
8. Barr M.L., Bertram E.G. 1949. A morphological distinction between neurones of the male and
female, and the behaviour of the nucleolar satellite during accelerated nucleoprotein synthesis.
Nature 163:676-677.
9. Brown S.W. 1966. Heterochromatin. Science 151:417-425.
10. Bužek J, Ebert I., Ruffini-Castiglione M., Široky J., Vyskot B., Greilhuber J. 1998. Structure
and DNA methylation pattern of partially heterochromatinised endosperm nuclei in Gagea lutea
(Liliaceae). Planta 204:506-514.
11. Darlington C.D. 1947. Nucleic acids and the chromosomes. Symp. Soc. Exp. Biol. 1:252-269.
12. Geitler L. 1950. Notizen zur endomitotischen Polyploidisierung in Trichocyten und Elaiosomen
sowie über die Kernstrukturen bei Gagea lutea. Chromosoma 3:271-281.
13. Greilhuber J. 1998. Intraspecific variation in genome size: A critical reassessment. Ann. Bot.
82:27-35.
14. Greilhuber J., Ebert I., Lorenz A., Vyskot B. 2000. Origin of facultative heterochromatin in the
endosperm of Gagea lutea (Liliaceae). Protoplasma 212:217-226.
15. Heitz E. 1933. Die Herkunft der Chromozentren. Planta 18:571-636.
16. Khosla S., Kantheti P., Brahmachari V., Chandra H.S. 1996. The specific nuclease-resistant
chromatin fraction in the male Planococcus lilacinus. Chromosoma 104:386-392.
17. Oikawa K. 1953. Endosperm development in Erythronium japonicum. Bull. Liberal Arts Dept.,
Mil. Univ. 10:23-31.
18. Wyllie A.H. 1980. Cell death: the significance of apoptosis. Int. J. Cytol. 68:251-306.
540