Maize Genetics Cooperation Newsletter vol 84 2010
Please Note: Notes submitted to the Maize Genetics
Cooperation Newsletter may be cited only with consent of authors.
PIACENZA, Italy
Institute of Agronomy, Genetics and Field Crop, Universit� Cattolica S.
Cuore (UCSC)
LJUBLJANA, Slovenia
Department of Biology, Biotechnical Faculty, University of Ljubljana
Analysis of cell size
and endopolyploidy level in the mutant defective
endosperm-18 (de18) of maize
Lanubile, A; Kladnik, A; Marocco, A.
The maize mutant defective endosperm-18 (de18) accumulates less dry matter in the
endosperm tissue and it shows a level of indole-acetic acid (IAA) at least 15
times lower than its normal counterpart. The addition of synthetic auxins to
the developing de18 grains rescues
the wild type phenotype (Torti et al., 1986 Theor Appl Genet 72:602-605). The
analysis of differentially expressed genes indicates that auxin methabolism
pathway is impaired in the de18
mutant. The plant hormones auxins and cytokinins are involved in regulation of
mitosis and endoreduplication, cellular key processes during seed development.
In fact enlargement of the maize endosperm relies upon cell division and cell expansion,
which is in turn linked to endoreduplication of nDNA (Kondorosi et al., 2000
Curr Opin Plant Biol 3:448-492). Endoreduplication begins at 10 DAP (days after
pollination) (Kowles and Phillips, 1985 Proc Natl Acad Sci USA 82:7010-7014;
Kowles and Phillips, 1988 Int Rev Cytol 112:97-136). Cells with highly
endopolyploid nuclei occupy a major part of the volume of the starchy endosperm
(Vilhar et al., 2002 Plant Physiol 129:23-30) and the highest nDNA amount,
expressed as C value, is tipically 96C to 192C, according to data obtained by
measuring nuclear volume (Tschermak-Woess and Enzenberg-Kunz, 1965 Planta
64:149-169), Feulgen cytophotometry, and flow cytometry (Kowles et al., 1992
Genome 35:68-77; Schweizer et al., 1995 Proc Natl Acad Sci USA 92:7070-7074;
Larkins et al., 2001 Exp Bot 52:183-192; Settler and Flannigan, 2001 Exp Bot
52:1401-1408).
.To investigate whether the reduced
endosperm of de18 is due to impaired
cell division and endoreduplication process, as a consequence of the low auxin
levels during seed development, wild-type B37 and de18 kernels were analyzed at 8, 12 and 16 DAP. The collected seeds
have been fixed, embedded in Paraplast and sectioned for microscopy analysis.
Nuclear endoreduplication level, number and size of cells have been measured
with the optical miscroscope and computer image analysis using the 3D model
developed for maize endosperm. Observations of cells distribution with
different ploidy levels in both genotypes, showed that at 8 DAP most of cells
in the endosperm were 3C and 6C cells and they were restricted mainly to the
outermost layers. Endoreduplication began in the nuclei of the central starchy
endosperm cells (12C) and proceeded basally and outward until 16 DAP, where 96C
and 192C nuclei were localized in the central part of endosperm. The most
significant differences between de18
and B37 were detected at 12 DAP, where the mutant showed a deficiency in the
ploidy level, number and volume of cells. These results suggest that the mutant
is characterized by a defective cellular proliferation, associated to reduced
cell volumes, contributing to a decreased endosperm development. Next step of
this study will be to analyze the correlation between starch content and ploidy
level in de18.