Figure 7.

Co-expression of PIN1:GFP and Athb8::YFPer during vein formation. PIN1:GFP (green) and Athb8::YFPer (red). Top left, time after first imaging (t = 0; F,H,K,L,N,P,Q,S,U,W,Y,AC,AD) or at leaf age in DAG (A,D,E,J,AD). Bottom left, reproducibility quotient (Table 2). Scale bars: (A,F–H,L–N,Q–AB) 10 μm; (D,E) 1 mm; (J,AD) 0.5 mm; (K,P) 20 μm. (A–E) Area enclosed by the third loop. Co- (A) and separate (B,C) expression of PIN1:GFP and Athb8::YFPer. Note Athb8 confinement to central, high PIN1 expressing, elongated cells (yellow arrowhead in A) and absence in lower PIN1 expressing cells (white arrowheads in A). This pattern is reflected in the final vein pattern (E), enlarged in (D). (F–J) Area enclosed by the first loop.Co- (F) and separate (G, upper, lower) expression of PIN1:GFP and Athb8::YFPer. Initial (F), Athb8 expression proximal, nearest to attachment to lower order PED in elongated cells with higher PIN1 expression (yellow arrowhead in F), and absent from polygonal cells with low PIN1 expression at the edges and free ends of hPEDs (white arrowheads in H). Co- (H) and separate (I, left, right) expression of PIN1:GFP and Athb8::YFPer. Note that Athb8 is now expressed in some terminal cells, as these have upregulated PIN1 and elongated slightly (yellow asterisk in H; same cell labeled with yellow asterisk in F), but not yet in the newly added cell at the free end (white arrowhead in H). Vein corresponding to hPED in F–I in mature leaf (J). (K–M) Leaf at 4 DAG (0 h; K). Blue-boxed area in (K) enlarged in (L), and separated for PIN1:GFP and Athb8::YFPer expression in (M) (top, bottom). Expression of Athb8 in elongated, PIN1 expressing upper cells in first loop PED (red arrowhead in L), but absence from lower cells of the first loop PED that are polygonal with diminished PIN1 expression (white arrowhead in L). (N–P) 24 h later visualization of leaf in (K). Yellow-boxed area in (P) enlarged in (N) and split in separate expression channels in the top and bottom panels of (O). Note persistence of Athb8 expression in upper cells of the first loop (red arrowhead in N), while the lower cells of the first loop PED no longer express PIN1 (white arrowhead in N). (P–T) Red-boxed area in (P) enlarged in (Q) and expression separated in the top and bottom panels of (R). The central cells of this a newly emerging hPED with highest PIN1 expression express Athb8 (yellow asterisk in Q), while edge cells do not (white arrowhead in Q). (S,T) Same hPED at 48 h (S) and expression separated in left and right panels of (T). Central Athb8 expressing cells (yellow asterisk in Q) have persisted (yellow asterisk in S), while the majority of the edge cells without Athb8 expression (white arrowhead in Q) have also lost PIN1 expression (white arrowhead in S). By contrast, a cell at the free end of the hPED, which maintained PIN1 expression and elongated now expresses also Athb8 (blue asterisks in Q,S). (U–Y) Leaf at 4 DAG (0 h) with hPED extending from the first loop (U). Expression channels separated in the left and right panels of (V). Expression of Athb8 in central, proximal cells (yellow asterisk in U). (W) Same leaf as in (U) and expression separated in the left and right panels of (X). More distal cells have elongated, upregulated PIN1 and express Athb8 (white arrowhead in W). [Same cell marked by yellow asterisk in (U,W) as positional reference). A new freely-ending hPED (blue arrowhead in W); the cells of this new hPED does not express Athb8. (Y) Same hPED as in (W) at 48 h. Note that the upper hPED expressing Athb8 has persisted (white arrowhead in Y), while the lower without Athb8 expression (blue arrowhead in W) has also lost PIN1 expression at 48 h (blue arrowhead in Y). (Z–AD) Leaf at 5 DAG (0 h) with branched, freely-ending hPED from the first loop (Z). Expression displayed separately in the left and right panels of (AA). No Athb8 expression in the hPED at 0 h (Z,AA). (AB) Same sample at 24 h and separately in (AC) (left/right). Cells of upper branch (yellow arrowhead) elongated and express Athb8, those of lower branch have also lost PIN1 expression. A corresponding vein appears in the mature leaf (AD).