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First published online 8 April 2003
doi: 10.1242/jcs.00437

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Chromatin of endoreduplicated pavement cells has greater range of movement than that of diploid guard cells in Arabidopsis thaliana

Biotech Center for Agriculture and The Environment, Rutgers The State University of New Jersey, New Brunswick, NJ 08901, USA

View larger version (12K): [in a new window]   Fig. 1. Detailed genomic structure characterization of the T-DNA inserts in transgenic Arabidopsis thaliana line EL702C. (a) Schematic construct map of the T-DNA region in the binary vector pEL702. The plasmid was designed such that the DNA between the right and left borders (T-DNA) can be transferred into plant nuclei via Agrobacterium. Thus, when stable transgenic Arabidopsis plants are treated with the synthetic glucocorticoid dexamethasone (Dex), the expressed fusion proteins can localize to the integrated loci by association with the lac operator array. The numbers on the top indicate the size of DNA construct in kbp. The arrow indicates the orientation of T-DNA from the left border to right border. Colored areas indicate gene expression cassette: blue, glucocorticoid receptor expression; yellow, hygromycin phosphotransferase expression; green, GFP-LacI/NLS induced expression. Abbreviations and symbols: blue dot rectangle, pea rbcS-E9 terminator; blue triangle, cauliflower mosaic virus 35S promoter; green dot rectangle, pea rbcS-3A terminator; green rectangle, 6XGal4 UAS and TATA box; yellow dot rectangle, nopaline-synthase gene terminator; yellow triangle, nopaline-synthase gene promoter; GVG, glucocorticoid binding domain/VP16 acidic activation domain/Gal4 DNA-binding domain; HPT, hygromycin phosphotransferase; LacO, 256mer of lac operator arrays; LB, left border; RB, right border. (b) Molecular characterization of the integrated loci with T-DNAs. The number of integrated loci was first characterized by Southern blot analyses, and the borders of the insertion sites were subsequently defined by subcloning and sequencing of the respective regions for the different sites using various strategies. The solid circle indicates the location of the centromere. The entire length of the chromosome and the distance between the two insertion loci are indicated in Mbp. Arrows indicate the direction of T-DNAs from LB to RB. Shaded rectangles represent neighboring reading frames around the T-DNA inserts.

View larger version (51K): [in a new window]   Fig. 2. Representative images of Arabidopsis thaliana EL702C seedling, guard cells, pavement cells and nuclei observed in this study. (a) A live seedling used to observe chromatin movements. Bar, 1 mm. (b) Microscopically observed stomatal guard cells on a cotyledon. Two guard cells symmetrically located. Bar, 50 µm. (c) Microscopically observed pavement cells on a hypocotyl. Elongated rectangular cells fill the surface of the hypocotyl. Bar, 50 µm. (d) Guard-cell nuclei accumulating GFP-LacI/NLS in two guard cells. Three spots can be seen in the left nucleus and two spots in the right nucleus. Bar, 10 µm. (e) A pavement-cell nucleus accumulating GFP-LacI/NLS. Six spots can be seen in this nucleus. The nucleus elongates to opposite sides, parallel to the long axis of the hypocotyl. Bar, 10 µm.

View larger version (10K): [in a new window]   Fig. 3. Nuclear properties of guard cells and pavement cells in homozygous EL702C seedlings. (a) GFP spot numbers observed in guard-cell nuclei (open column) and in pavement-cell nuclei (closed column). Spot numbers were counted in a total of nine guard-cell nuclei and ten pavement-cell nuclei whose chromatin movements were measured. The mean spot numbers per nucleus are shown with the standard deviations from the mean indicated. The number of samples analysed is indicated by `n' on the graph. (b) Relative nuclear DNA contents of guard-cell nuclei (open column) and pavement-cell nuclei (closed column). The samples were stained with DAPI and the nuclear intensities in guard cells (total of 17 in three seedlings) and pavement cells (total of 15 in three seedlings) were compared with that in root-tip cells (whose ploidy is known to be 2C). The mean relative intensities are shown with the standard deviations from the mean. The number of samples analysed is indicated by `n' on the graph. (c) Nuclear volumes in guard-cell nuclei (open column) and pavement-cell nuclei (closed column). The nuclear volumes in a total of nine guard-cell nuclei and ten pavement-cell nuclei whose chromatin movements were measured were calculated by measuring the non-lac-operator-binding GFP signals in nuclei. The mean volumes per nucleus are shown with the standard deviations from the mean indicated. The number of samples analysed is indicated by `n' on the graph.

View larger version (18K): [in a new window]   Fig. 4. Chromatin dynamics in guard-cell and pavement-cell nuclei. Overall mean-square differences in distance between GFP spots <d2> plotted against elapsed time intervals <t> in guard-cell () and pavement-cell () nuclei of homozygous EL702C seedlings. Each data point represents an average of data collected from at least nine independent experiments. Open circles (, guard-cell nuclei) and closed circles (•, pavement-cell nuclei) represent data from fixed seedlings, which were imaged under identical conditions with data points representing averaged values from at least six independent experiments and nine nuclear observations. Vertical bars at each time point show the standard errors for the particular set of data collected. If the motion were not restricted, <d2> would continue to increase with increasing time. The longer the time interval between measurements, the greater the distance between two diffusion points is expected to change.