Laboratory for experimental botany

Sucrose synthase isozyme SUS1 in the maize root cap is preferentially localized in the endopolyploid outer cells

Ale Kladnik, Barbara Vilhar, Prem S. Chourey and Marina Dermastia
Canadian Journal of Botany 82: 96-103 (2004)
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Corresponding author: Marina Dermastia, Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, SI-1001 Ljubljana, Slovenia. Tel: +386 1 4233388. Fax: +386 1 2573390. E-mail: marina.dermastia{at}bf.uni-lj.si

Abstract: The structure of the maize (Zea mays L.) root cap was studied to quantitatively evaluate the relationship among the size of the cells, their endopolyploidy level, and the abundance of the sucrose synthase isozyme SUS1. Median longitudinal root cap sections were analysed using immunolocalization, quantitative DNA staining, and image cytometry. Both the immunolocalization signal for the SUS1 protein and the endopolyploidy level increased from calyptrogen towards the root cap periphery and were thus the highest in the outer cells. These cells had a nuclear DNA content of mostly 8C or higher and the largest volumes of all root cap cells. The high amount of SUS1 protein in the outer, endopolyploid cells suggests an association between endoreduplication and the abundance of this enzyme. The outer cells are involved in mucilage production; hence, there is a possibility that sucrose synthase provides monosaccharide precursors for mucilage synthesis.

Key words: nuclear DNA amount, endoreduplication, immunolocalization, image cytometry, Zea mays L.

Figures:



Figure 1. Endopolyploidy level and immunolocalized sucrose synthase isozyme SUS1 in the maize root cap. (A) Median longitudinal section quantitatively stained with the Feulgen reaction; the size of individual images recorded for measurement of integrated optical density of the nuclei is indicated by a dashed rectangle. (B) Frequency polygon of relative nuclear DNA amounts in the root cap cells shown in A, expressed in C-value units. The cells were separated into C-value classes corresponding to different endopolyploidy levels (2C, 4C, 8C, 16C+) by setting the class limits at midpoints between the consecutive peak modes in the frequency polygon (dashed vertical lines). (C) The endopolyploidy level of individual cells in the longitudinal root cap section shown in A. (D) Median longitudinal section immuno-stained with the SUS1 antiserum, with inset showing the negative control treated with the non-immune serum. (E) Frequency polygon of the minimal grey values of cells in the immuno-stained root cap section shown in D. The cells with the minimal grey value lower than the threshold value (dashed vertical line) were classified as containing a high amount of the SUS1 protein (darkly stained cells), and cells with the minimal grey value higher than the threshold as containing a low amount of the SUS1 protein (lightly stained cells). Threshold was set so that the volume occupied by cells with either strong or weak SUS1 signal equalled 50% of the total root cap volume. (F) The level of immunolocalization staining in individual cells in the longitudinal root cap section shown in D.




Figure 2. Frequency distribution of maize root cap cells in relation to the cell volume. Data were obtained with the 3D rotational model of the root cap (Bengough et al. 2001, Annals of Botany 87). (A) Number of cells with different endopolyploidy levels. (B) Contribution of cells with different endopolyploidy levels to the total root cap volume. Inset - the relationship between the endopolyploidy level and the median cell volume; the mean standard error is shown (number of root caps N = 6) (C) Number of cells with strong / weak signal for SUS1 protein. (D) Contribution of cells with strong / weak signal for SUS1 protein to the total root cap volume. The shaded areas around the frequency polygons in A - D represent the standard errors for six (A, B) and three (C, D) root caps analysed.




Figure 3. Relationship between the endopolyploidy level and the amount of the SUS1 protein in the cells of the maize root cap. Data were obtained with the 3D rotational model of the root cap (Bengough et al. 2001, Annals of Botany 87). The endopolyploidy and immuno-staining data were linked on the positional basis of cells. The mean percentage of cells with a strong SUS1 signal was calculated for each endopolyploidy level from data for nine caps; error bars represent the standard error.