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First published online June 28, 2004
doi: 10.1242/10.1242/dev.01182

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Stage-specific markers define early steps of procambium development in Arabidopsis leaves and correlate termination of vein formation with mesophyll differentiation

University of Toronto, Department of Botany, 25 Willcocks Street, Toronto, Ontario M5S 3B2, Canada

View larger version (116K): [in a new window]   Fig. 2. Expression of ET1335-GUS in correlation to procambium formation. DIC micrographs of (A,B) lateral views, (C-H) abaxial views. (A,C,E,G) Congruence of ET1335-GUS expression and anatomically recognizable procambium in leaves of various ages as indicated. (B,D,F,H) Enlargements of A,C,E,G, respectively (boxed in C,E,G). Outlined cell boundaries illustrate our recognition criterion for procambium: procambial cells (red) are narrow and approximately twice the length of surrounding GM cells (yellow). Numbers refer to vein orders. Upper right: primordium age in days after germination (DAG). Scale bars: (A,B,D,F,H) 10 µm; (C,E,G) 25 µm.

View larger version (32K): [in a new window]   Fig. 1. Vascular development in the Arabidopsis first leaf. (A-C) Lateral view. (D-K) Abaxial view. Colors in A-J: procambium, blue, xylem, brown. Colors in K illustrate our application of the classification of vein orders by Hickey (Hickey 1973): 1st, yellow; 2nd, red; 3rd, green; 4th, black; 5th, purple. Veins of the 3rd and higher order can be formally classified as either branching from a next lower-order vein or connecting only lower-order veins. Only examples of these veins are shown in H-K. Numbering of 2nd order vein loops and of the enclosed intercostal areas (IAs) from distal to proximal. lp, leaf primordium, SAM, shoot apical meristem. DAG, days after germination.

View larger version (146K): [in a new window]   Fig. 3. Expression of ET1335-GUS during development of the first leaf. (A,B) Lateral view, (C-L) abaxial view; (A-J,L) DIC microscopy, (K) dark-field optics. Arrow in F indicates transient expression in GM cells at the tip of 3.5-5.5 DAG primordia. Upper right: primordium age in days after germination (DAG). Lower left: fraction of primordia showing nearly identical features. Scale bars: (A-F) 25 µm; (G) 50 µm; (H,I) 100 µm; (J,K) 500 µm; (L) 1 mm.

View larger version (128K): [in a new window]   Fig. 4. Expression of Athb8-GUS during development of the first leaf. (A-D) Lateral view. (E-P) Abaxial view. (A-P) DIC microscopy. Arrow in E points to a single Athb8-GUS expressing cell at the onset of 2nd order loop formation. Upper right: primordium age in days after germination (DAG). Lower left: fraction of primordia showing nearly identical features. Scale bars: 25 µm.

View larger version (48K): [in a new window]   Fig. 6. (A) Schematic summary of vein-associated temporal expression profiles. Green bars indicate the duration of maximum expression of each marker, ramped termini indicate gradual onset or decline of marker gene expression. (B) Termination of vein formation by differentiating mesophyll (distal, top; proximal, bottom; arrows connect consecutive stages of leaf development). Mesophyll differentiation (green) progressing from distal to proximal interferes with polar acquisition of preprocambial cell fates (light blue) at different stages of vein ontogeny, resulting in the formation of a freely ending vein distally and a connecting vein proximally.

View larger version (115K): [in a new window]   Fig. 5. Experimental manipulation of vein formation and mesophyll development. (A-O) Athb8-GUS expression, abaxial view, DIC microscopy. (B,E,H,K,N) Enlargements of boxed areas in A,D,G,J,M, respectively. (C,F,I,L,O) Enlargements of boxed areas in B,E,H,K,N, respectively. Upper right: primordium age in DAI (days after primordium initiation) and position in IA (intercostal area). Lower left: growth condition (A, B or C, as outlined in Materials and methods). Red numbers indicate vein order. Arrows point to examples of chloroplasts in mesophyll cells. (P,Q) Polygonal cells classified as GM (P) and mesophyll cells (Q) from leaves expressing the LTI6b::YFP plasma membrane marker. Left panel: cleared leaf, abaxial view, DIC microscopy; right panel: LTI6b::YFP expression (green) and chloroplast autofluorescence (red), abaxial view, CLS microscopy. CLS micrographs in P and Q were taken with identical microscope settings. Scale bars: (A,D,G) 50 µm; (B,E,H,J,K,M,N) 25 µm; (C,F,I,L,O,P,Q) 10 µm.

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