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Embryonic control of epidermal cell patterning in the root and hypocotyl of Arabidopsis

Department of Biology, University of Michigan, Ann Arbor, Michigan 48109, USA

View larger version (153K): [in a new window]   Fig. 1. Accumulation of GL2 transcripts during embryo development. Median longitudinal sections of embryos were hybridized to GL2 antisense (A-D) or sense (E-H) RNA probes labeled with digoxigenin-UTP. The low GL2 hybridization signal in D does not necessarily reflect a reduction in RNA abundance, because this in situ technique is less sensitive with sections from mature embryos (Y. L. and J. S., unpublished observations). (A,E) Globular stage embryos. Bar, 5 µm. (B,F) Heart stage embryos. Bar, 10 µm. (C,G) Torpedo stage embryos. Bar, 20 µm. (D,H) Mature embryos. Bar, 25 µm.

View larger version (73K): [in a new window]   Fig. 2. GL2 gene expression in the root and hypocotyl of 3-day-old plants bearing the GL2::GUS or GL2::GFP reporter constructs. Postembryonic expression of GL2::GUS (blue staining) and GL2::GFP (green fluorescence) occurs preferentially in epidermal cells located outside periclinal cortical cell walls. Propidium iodide staining (red) was performed on plants in D and E to visualize cell boundaries. (A) Hypocotyl bearing the GL2::GUS transgene, stained for GUS activity (blue). Bar, 100 µm for A and B. (B) Hypocotyl bearing the GL2::GFP transgene, viewed by fluorescence microscopy (green). (C) Surface view of root tip from seedling with the GL2::GUS transgene, stained for GUS activity. Bar, 50 µm for C and D. (D) Surface view of root tip from seedling with GL2::GFP, viewed by fluorescence microscopy. The inset shows the arrangement of the underlying cortical cells, with an arrow marking the anticlinal cortical cell wall. (E) Median longitudinal optical section of root tip with the GL2::GFP transgene, viewed by fluorescence microscopy. Bar, 50 µm.

View larger version (97K): [in a new window]   Fig. 3. The pattern of GFP accumulation (green) in embryos from plants bearing the GL2::GFP transgene viewed by fluorescence microscopy. The red background in the images is due to autofluorescence. Owing to the progressive increase in the level of GL2::GFP expression during embryogenesis, the GFP signal was amplified in embryos at earlier stages (e.g. B and C) relative to later stages (e.g. I and J), so accurate comparisons of signal intensity between the stages cannot be made with these images. Propidium iodide staining (red) was performed with the mature embryos shown in K and L to visualize cell boundaries. (A) Median longitudinal optical section from a globular stage embryo. Bar, 50 µm. (B,C) Surface view of two different embryos from the late-triangular/early-heart stage. Bar, 50 µm for B-D. (D) Median longitudinal optical section from a late-triangular/early-heart stage embryo. (E) Surface view of a heart stage embryo. Bar, 50 µm for E and F. (F) Median longitudinal optical section from a heart stage embryo. (G) Surface view of a torpedo stage embryo. Bar, 50 µm for G and H. (H) Median longitudinal optical section from a torpedo stage embryo. (I) Surface view of a mature embryo. Bracket indicates the location of the root-hypocotyl junction region. Bar, 40 µm for I and J. (J) Median longitudinal optical section from a mature embryo. (K) Magnified view of the hypocotyl region of a mature embryo. (L) Magnified view of the root region of a mature embryo. The insets in K and L show optical sections of the underlying cortical cell layer, revealing that epidermal cells outside the anticlinal cortical cell walls (marked by arrows) lack GFP expression. Bar, 15 µm for K and L.

View larger version (72K): [in a new window]   Fig. 4. GL2 gene expression and cell organization in the root-hypocotyl junction region of Arabidopsis embryos and seedlings. Brackets in A-E mark the region of the root-hypocotyl junction. Propidium iodide staining (red) was performed with the mature embryo in C and the seedling in D to visualize cell boundaries. (A-D) Root-hypocotyl junction region from torpedo stage embryo (A), curled cotyledon stage embryo (B), mature embryo (C) and 2-day-old seedling (D) bearing the GL2::GFP transgene, viewed by fluorescence microscopy. Note the numerous root hairs (red) visible in D. Bars in A and D, 50 µm; in B, 45 µm; in C, 20 µm. (E) Root-hypocotyl junction region from 2-day-old seedling bearing the GL2::GUS transgene and stained for GUS activity. Numerous root hairs are visible in the junction region. Bar, 50 µm. (F-I) Transverse sections from the root (F), root-hypocotyl junction region (G,H) and hypocotyl (I) from 3-day-old seedlings. Sections were stained with a fluorescent cell wall dye and viewed by fluorescence microscopy. The epidermis (ep), cortex (c), outer cortex (oc), inner cortex (ic), and endodermis (en) layers of the root and hypocotyl sections are indicated. Note the unusual cell anatomy in the ground tissues in G and H, where cells occupying the incomplete middle-ground-tissue layer are indicated by asterisks. Bars in F,G,I, 50 µm; bar in G also applies to H.

View larger version (121K): [in a new window]   Fig. 5. The pattern of GFP accumulation in mutant embryos and seedlings bearing the GL2::GFP transgene viewed by fluorescence microscopy. Propidium iodide staining (red) was performed on the seedling roots in E, F, K, L, Q and V to visualize cell boundaries. (A-F) The ttg-1 mutant. (G-L) The wer-1 mutant. Surface view of heart-stage embryo (A,G), torpedo stage embryo (B,H), mature embryo with the fluorescence signal amplified (C,I), mature embryo without amplification of the signal (D,J) (compare to Fig. 3I), seedling root tip with fluorescence signal amplified (E,K), and seedling root tip without amplification of the signal (F,L) (compare with Fig. 2D). A GFP-expressing cell is indicated by an arrow in K. (M-Q) The cpc mutant. (R-V) The gl2-1 mutant. Surface view of late-triangular/early-heart stage embryo (M,R), heart stage embryo (N,S), torpedo stage embryo (O,T), mature embryo (P,U) and seedling root tip (Q,V). Bar in A (G,M,N,R,S) 15 µm; in B (H,O,T) 50 µm; in C (D,I,J,P,U) 40 µm; in E (F,K,L,Q,V) 50 µm.