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Identification of a developmental transition in plasmodesmatal function during embryogenesis in Arabidopsis thaliana

Insoon Kim, Frederick D. Hempel, Kyle Sha, Jennifer Pfluger and Patricia C. Zambryski^*

Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, CA 94720, USA
Present address: Mendel Biotechnology, 21375 Chabat Blvd, Haywood, CA 94545, USA

View larger version (49K): [in a new window]   Fig. 1. Schematic representation of stages of embryo development in Arabidopsis. The zygote divides to from an embryo composed of a filamentous suspensor (S) that maintains contact with maternal tissues, and a terminal embryo proper (EP). The embryo becomes green at the heart stage of development when chlorophyll and chloroplasts first form. The suspensor degenerates during the torpedo stage. This diagram was adapted from more detailed descriptions of embryo development (Meinke, 1994; West and Harada, 1993) and simplified to highlight stages related to the study reported here. A, apical cell; B, basal cell; C, cotyledon; PE, protoderm; PV, provascular tissues; H, hypocotyl; R, radicle; SC, seed coat; En, endosperm.

View larger version (37K): [in a new window]   Fig. 2. Genetic screen for ise mutants Mature Arabidopsis siliques contain about 60 seeds; roughly half are drawn for simplicity. Dark green seeds contain wild-type embryos, and light green seeds contain defective embryos. Embryos were released from their seed coats (sc) and viewed under bright-field illumination. In the example diagrammed here, wild-type is dark green and has reached the mid-torpedo stage, while the mutant is light green and retarded in morphological development (see Fig. 6B,C,D for in vivo results). Embryos were incubated with 10 kDa F-dextran for 5 minutes at room temperature, washed extensively, then viewed by fluorescence microscopy. Wild-type (wt) and most morphologically defective embryos emit red autoflorescence due to cholorophyll, and are unable to transport 10 kDa dextran (left bottom panel). A small fraction of embryo-defective lines (called increased size exclusion of plasmodesmata, ise) take up and allow movement of 10 kDa F-dextran, and thus exhibit green fluorescence (right bottom panel).

View larger version (93K): [in a new window]   Fig. 3. Characterization of cell-to-cell transport in Arabidopsis wild-type embryos. Ler embryos at different stages of development are loaded with either HPTS (A-D, I-L) or 10 kDa F-dextran (E-H). All cells in embryos allow the movement of HPTS, indicating that the embryo constitutes a single symplastic domain, from early heart (A), late heart (B), early torpedo (C), to mid-torpedo (D) stages of embryo development. Cellular localization of HPTS shows the tracer in the cytoplasm as well as the nuclei (arrowhead in A) but excluded from apoplastic regions (arrow in A). (I) Higher magnification of part of the root of the embryo in D showing HPTS distribution. (J-L) Detail of two single cells from I. Chlorophyll autofluorescence (c) marks the cytoplasm. HPTS is found both in cytoplasm, indicated by chloroplasts, and nuclei (n). L combines the images from J and K. In contrast to HPTS, 10 kDa F-dextrans move only in early heart (E) and mid heart (F) embryos. Early (G) and mid (H) torpedo embryos do not allow the movement of corresponding dextran. Instead, small numbers of cells are loaded at the tip of radicle (arrowhead) and the region where cotyledons join the hypocotyl (arrow). The images in A-D and I-L are optical sections captured by confocal laser scanning microscopy. Images in E-H were obtained by epifluorescence microscopy, and are therefore less highly resolved (see Materials and Methods for detail). Scale bars, 50 µm in A-I; 5 µm in J-L.

View larger version (60K): [in a new window]   Fig. 4. Uptake and subsequent movement of symplastic tracers in cells of wild-type mid-torpedo embryos. (A) When embryos are removed from their seed coats, physical damage occurs in a subset of cells. As a result, small regions of cell walls and plasma membranes are broken to a sub-lethal level to provide an initial entrance site for uptake of symplastic tracers such as HPTS and F-dextran, which do not usually cross plasma membranes. Yellow jagged lines indicate the most common site of damage in our loading assay (see Results). Black jagged lines the random abraded sites on the protodermal layer, which are observed less frequently. co, cotyledon; ra, radicle; sc, seed coat. (B) A small number of cells at the base of the detached cotyledons from mid-torpedo embryos are cytoplasmically loaded with 10 kDa F-dextran (asterisks). Yet further movement to neighboring cells does not occur so that rest of the cells show only chlorophyll auto-fluorescence (red). Scale bar, 50 µm. (C) A typical example of loaded cells in a region at the edge of the protodermal layer where abrasion has occurred (marked as black jagged lines in A). Individual cells in the protodermal layer take up 10 kDa F-dextrans (green arrows) and show cytoplasmic localization of the probe. However, subsequent movement of the probe is inhibited (yellow arrows with red X). Scale bar, 5 µm. (D) A diagram showing that a partially broken cell wall and plasma membrane (jagged edge) may provide the initial entrance site for uptake (green arrow) of symplastic tracers, F-dextran or HPTS (green circles). Further symplastic transport (yellow arrows) is then determined by the SEL of plasmodesmata and the size of symplastic tracers introduced.

View larger version (53K): [in a new window]   Fig. 5. Tracer loading and movement in wild-type embryos. Mid-torpedo embryos were incubated with HPTS for a shorter amount of time (1-3 minutes versus 5 minutes in other experiments) to allow observation of partially loaded embryos. (A) A detached cotyledon reveals the movement of HPTS only in a small number of cells at the base. Arrow indicates the direction of probe movement. (B) A detached cotyledon partially loaded in the center (c), and the tracer has subsequently spread into ground tissues (g). (C) A detached cotyledon showing complete loading with HPTS. (D) The same cotyledon as in C viewed with a different filter set allowing visualization of combined chlorophyll and FITC fluorescence. Loaded region with HPTS appears yellowish green owing to the additive effect between red autofluorescence from chlorophyll and green emission from HPTS. (E) A whole embryo, fully loaded with HPTS in its radicle (ra) and hypocotyl (hy), but partially loaded at the base (*) of cotyledons (co). (F) The same embryo as in E monitored with a different filter set allowing visualization of combined chlorophyll and FITC fluorescence. Images were obtained by using Chroma FITC (HPTS fluorescence; A,B,C,E) and Zeiss FITC (chlorophyll plus HPTS fluorescence; D,F) filter sets on a compound epifluorescence microscope (see Materials and Methods). Scale bars, 50 µm.

View larger version (51K): [in a new window]   Fig. 6. Cell-to-cell movement of fluorescent tracers in ise1 mutant embryos of Arabidopsis. (A) Immature siliques of both wild-type and +/ise1 heterozygous plants contain seeds developing synchronously during early embryo development, up to the heart stage. (B) A later stage silique of +/ise1 heterozygous plants contains dark green seeds (arrowhead) and light green seeds (arrow) in a ratio of 3:1. Seeds are 0.5 mm in length. (C,D) Embryos from the same silique; dark green seeds contain mid-torpedo wild-type embryos (C) and light green seeds contain ise1/ise1 mutant embryos (D), both showing red chlorophyll autofluorescence. (E) A keule embryo at torpedo stage loaded with HPTS. (F) A ise1/ise1 embryo allow the movement of 10 kDa F-dextran. The hypocotyl and the radicle show uniform and complete loading, and the cotyledons show partial loading at their bases. (G) Detail of the hypocotyl in F. 10 kDa F-dextran localizes in the cytoplasm as well as nuclei (arrowhead), but not in the cell wall in apoplastic regions (arrow). (H) A ise1/ise1 embryo fully loaded with HTPS, showing that the ise1 embryo constitutes a single symplast with open plasmodesmata. Scale bars, 50 µm.

View larger version (116K): [in a new window]   Fig. 7. Light micrographs of mid-torpedo embryos. At the mid-torpedo stage, wild-type (A,C) and sibling ise1 mutant (B,D) embryos are similar in cell size and regular patterns of cell division (arrows) both in the hypocotyl (A,B) and the radicle (C,D) viewed with Nomarski optics. Toluidine Blue staining of hypocotyl thin sections of wild-type (E) and mutant (F) embryos shows that cells in the provascular regions (pv) are narrow and elongated in both. Vacuoles (v) in mutant cells (F) are larger in size and fewer in number than in cells of wild-type (E). Scale bars, 40 µm.

View larger version (110K): [in a new window]   Fig. 8. Postgermination phenotype of ise1 mutant. (A) Wild-type (wt) and sibling ise1 seedlings 18 days postgermination on culture plates. At this stage the wild-type seedling has 4 true leaves with trichomes as well as an elongated primary root, but the mutant has only a pair of macroscopic cotyledons and a very short primary root. (B) Scanning electron micrograph of the root epidermis of wild-type 2 days postgermination shows cell files with root hairs (red circles) and without root hairs (yellow X). In the ise1 root epidermis (C), however, all cell files produce root hairs (red circles) indicating a homogenization of cell fate. Scale bars, 50 µm.

View larger version (16K): [in a new window]   Fig. 9. The molecular genetic map of ISE genes on chromosome I. ISE1 gene maps between CAPS markers, NCC1 and GAPB on chromosome I. The black bar represents chromosome I and numbers above the bar indicate the genetic location of corresponding markers shown below. ISE1 maps 0.51 cM apart from NCC1 and its approximate location is marked as a white block on the bar. ISE2 maps between GAPB and ADH on chromosome I and its approximate location is marked as a vertical line. The genetic map positions given are taken from the Lister and Dean recombinant inbred (RI) map from The Arabidopsis Information Resource (TAIR, http://www.Arabidopsis.org/search/marker_search.html) as well as Nottingham Arabidopsis Stock Centre (NASC) RI map (http://nasc.nott.ac.uk/new_ri_map.html). The number of recombinants and the total number of chromosomes tested are noted with a slash under the corresponding markers.