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. 2014 Jun 11;5:235. doi: 10.3389/fpls.2014.00235

Figure 2.

Figure 2

Live-imaging of hPED initiation and extension. Top right, time after first imaging time point (t = 0; A–E,G–K) or DAG (F,L). Bottom left, reproducibility quotient (Table 2). Scale bars: (A–E) 20 μm; (F,L) 1 mm; (G–K,N–Q) 10 μm. (A–F) PIN1:GFP expression in a first leaf from 3 to 4.5 DAG. Note the appearance of freely-ending hPEDs in (B) (white arrowheads) and in (E) (yellow arrowhead) Matching higher order veins in leaf at 14 DAG marked by arrowheads in (F) and boxed area enlarged in (L). (G–L) Detail of PIN1:GFP expression and xylem pattern in the boxed areas shown in (A–E,F), respectively. Note weak PIN1 expression in cells adjacent to the first loop (white arrow in G) initiating higher-order vein. Upregulated PIN1 in the neighbours of one of these cells (white arrow in H). PIN1 localization is directed toward the center of the hPED (“central”) and/or directed toward the lower order loop (“basal”; turquoise-boxed inset in H). The hPED is extended through the upregulation of PIN1 in additional cells at the free end of the hPED (J,K). The first recruited cells in the “proximal” part of the hPED (nearest to the connecting lower order vein) further upregulate PIN1 (yellow arrow in I), and elongate and/or divide along the predicted axis of auxin flow, becoming procambium (red arrowheads in J,K). The final vein pattern of the same leaf area is shown in (L). (M–R) Gradient of cell length (N) and PIN1 expression (P, intensity monitored in Q) along the proximal-distal axis of the hPED (labeled with a red arrow in N,P,Q). Schematic gradient in M green shading reflects PIN1 expression levels, red arrowheads PIN1 polarity. (O) graph of average normalized cell length of the 2, 3, and 4th cells of the hPED (n = 42). Cell length values were normalized against the cell length value of the 4th cell for each hPED. (R) graph of average normalized PIN1 intensity of the first four cells of the hPED (n = 16). PIN1 intensity values were normalized against the PIN1 intensity value of the 4th cell for each hPED. Error bars in (O,R) show the standard error. (See Methods for details).