DEV01182 Supplemental Data

Files in this Data Supplement:

• Supplemental Table (PDF) - Origins of marker lines
• Supplemental Figure 1 -

  Fig. S1. Expression of GT5211-GUS in Arabidopsis first leaf development. (A-D) Lateral view. (E-P) Abaxial view. (A,C,D,F,K,M,N) Dark-field optics. (B,E,G-J,L,O,P) DIC microscopy. Arrows in F and K indicate domains of weak, but somewhat elevated GT5211-GUS expression presaging the position of higher-order procambial strands. (P) Enlargement of the area boxed in O. Upper right: primordium age in DAG (days after germination). Lower left: fraction of primordia showing nearly identical features. Scale bars: (A-H) 25 mm; (I-K) 50 mm; (L-N,P) 100 mm; (O) 1 mm. Expression of GT5211-GUS. Earliest GT5211-GUS expression was detected in the center of the 2.5 DAG primordium (C). At 3 DAG, GUS expression was associated with the emergence of the primary procambial strand (D). From 4 DAG, two clearly distinguishable levels of GUS expression could be assigned to distinct cell types: a strong expression level was stably associated with procambium, while weak expression was observed in nearly all GM cells but stronger close to emerging procambium. These two main levels of expression could easily be distinguished, since weak expression could only be visualized as a pink label under dark-field illumination. At 4 DAG, GT5211-GUS was strongly expressed in the 1^st order procambial strand (E) and weakly in GM cells in the distal half of the developing primordium, consistently elevated along the prospective first loops of 2^nd order procambial strands (F). At 5 DAG, GT5211-GUS expression had become strong in those loops (G), which are procambial at this stage. At 5.5 and 6 DAG, strong GUS expression also marked the formation of second loops of 2^nd order procambial strands (H,I). At 6.5 DAG, weak GT5211-GUS expression in the first and second IA became more pronounced along the routes of 3^rd order veins (J,K), followed by strong expression in higher-order veins at 7 DAG (L). As illustrated in higher-order vein formation in M,N, expression typically progressed from a more diffused to a more vein-associated pattern in the formation of higher-order veins. With the emergence of fully differentiated xylem in various vein orders, two types of GT5211-GUS expression became apparent. In thick, lower-order veins, GT5211-GUS expression persisted, while in higher-order veins expression subsided with the appearance of differentiated xylem (O,P). In summary, high levels of GT5211-GUS expression were tightly associated with procambium formation. At a distinctly lower level, GT5211-GUS expression was initially more broadly distributed in the GM and then became elevated in preprocambial domains. The marker does not seem to be expressed in any type of mature vascular tissue as it disappears completely from mature higher-order veins, which consists exclusively of fully differentiated cells.

• Supplemental Figure 2 -

  Fig. S2. Expression of 553-643-GUS during development of the first leaf. (A,C) Lateral view, (B,D-L) abaxial view. (A-G,I,J,L) DIC microscopy, (H,K) dark-field optics. Arrow in G indicates transient expression in GM cell at the tip of 2.5 to 6.5 primordia. Upper right: primordium age in DAG. Lower left: fraction of primordia showing nearly identical features. Scale bars: (A-F) 25 mm (G,H) 50 mm (I-K) 100 mm (L) 1 mm. Expression of 553-643-GUS. Expression of GUS in line 553-643 preceded the formation of the primary procambial strand in the 2.5 DAG primordium (B). This expression did not change in intensity or distribution when cells of the 1^st order strand had adopted procambial identity (3 and 3.5 DAG; C,D). Expression of 553-643-GUS was particularly strong at the leaf tip, where not all cells expressing the marker later acquired procambial identity. As the lamina became wider in the 4 DAG primordium, 553-643-GUS was expressed in GM cells in domains that precisely foreshadowed the emergence of the first loops of 2^nd order procambial strands (E). Subsequently, at 5 DAG, 553-643-GUS expression preceded the formation of second loops of 2^nd order procambial strands (F). Loops of 553-643-GUS expression in 2^nd order veins invariably emerged as pairs on both sides of the 1^st order vein. At 6 DAG, 553-643-GUS expression foreshadowed the formation of 3^rd order veins in the first IAs, followed by a similar expression pattern in the second IAs at 6.5 DAG (G-I). Expression domains presaging the formation of third loops of 2^nd order procambial strands typically appeared at 7 DAG, concurrent with expression outlining the higher-order vein network in the third IAs (J). Unlike all other markers in this study, 553-643-GUS expression increased upon further maturation in all vein orders. At 6 DAG in the 1^st order vein (G,H) and at 7 DAG in the first loops of 2^nd order veins (J,K), GUS expression increased with overt xylem differentiation, and in mature leaves at 14 DAG, strong GUS expression was observed in all vein classes (L). In conclusion, 553-643-GUS was clearly expressed in GM cells at stages preceding procambium formation as well as transiently in some unrelated GM cells at the tip of the early primordium (G). In contrast to all other markers in our study, 553-643-GUS expression became stronger in maturating veins, presumably because of expression in mature phloem, which has been reported (Mattsson et al., 1999).

• Supplemental Figure 3 -

  Fig. S3. Vascular-specific GUS expression during first leaf development. (A-N) Abaxial view, DIC microscopy. Upper right: marker line identity (see Table S2). Lower left: stage in DAG. GUS expression in each line was monitored in leaves with primarily procambial (3-5 DAG) and mature vasculature (14 DAG). (A,I,K,M) Little or no procambial GUS expression. (B,F,H) GUS expression in mature veins of all orders. (C) Strong GUS expression in the primary, and very weak GUS expression in first loops of 2^nd order procambial strands. (D,J,N) GUS expression is restricted to 1^st order and most proximal section of the 2^nd order veins, clearly weaker in J and N. (E,O) GUS expression restricted to GM cells at primordium tip and 1^st order procambial strand. (G) Weak and diffuse GUS expression around, but somewhat elevated in first loops of 2^nd order procambial strands and second loops of 2^nd order preprocambial strands. (L) Weak GUS expression in veins of all orders, but expression is discontinuous within individual differentiated strands. (P-R) Heterogeneous GUS expression in the emerging veins of the 2^nd and 3^rd order. Reduction or absence of expression in the already differentiating vein of the 1^st order. Growth condition A and histochemical localization of GUS activity as described in the Materials and methods, except for incubation for 2 (DR5-GUS) or 16 hours (all other markers) in staining buffer containing 0.5 (AtP5K1-1-GUS), 1 (GT5978, GT6224 and GT6644), 2 (ET3845 and GT525) or 5 (GT2845 and DR5-GUS) mM each of potassium ferrocyanide and potassium ferricyanide. Scale bars: (A,C,E,G,I,K,M,O-R) 25 mm; (B,D,F,H,J,L,N) 1 mm.