Patterning of Leaf Vein Networks by Convergent Auxin Transport Pathways
Figure 2
PIN6 expression in leaf development.
(A–O,Q–S) Top right: leaf age in days after germination (DAG), marker and genotype. Bottom left: reproducibility index. First leaves. (A–D) Midvein, loops and minor veins differentiate progressively later in the same region of the developing leaf, and loops and minor veins differentiate in a tip-to-base sequence during leaf development [6], [7], [57]; purple, green and magenta: successive stages of vein differentiation. Box in (D) illustrates position of close-ups in (I–L). (E–O,Q–S) Confocal laser scanning microscopy with (E–H,L) or without (I–K,M–O,Q–S) transmitted light. (E–H) PIN6::PIN6:GFP expression. (I) Expression of PIN6::YFPnuc and PIN1:PIN1:CFP at 4 DAG; blue: chlorophyll. (J–L) PIN6::PIN6:GFP expression in WT (J) and pin1 (K,L) at 4 DAG. LUT (in K) visualizes expression levels. Dotted line: leaf outline. (M–O,Q–S) Expression of PIN6::PIN6:GFP (M,Q), 35S::YFPer (N), 35S::LTI6B:YFP (R) and respective overlays displayed with a dual-channel LUT [66] (O,S): prevalence of cyan over colocalized magenta signal is shown in green, opposite in red, and colocalized cyan and magenta signals of equal intensity in yellow. (P,T) Quantification of colocalized GFP and YFP signals (as mean ± SE of Manders' coefficient ‘r’) in populations (n = 11) of positive controls: J1721::GFPer;35S::YFPer (P), 35S::YFPpm;FM4-64 (T); negative controls: ATHB8::GFPnuc;35S::YFPer (P), J1721::GFPer; 35S::YFPpm (T); and samples: PIN6::PIN6:GFP;35S::YFPer (P), PIN6::PIN6:GFP; 35S::YFPpm (T). (P) Difference between negative control and positive control, and between negative control and sample was significant at P<0.01 (**) by one-way ANOVA and Tukey's test. (T) Difference between positive control and negative control, and between positive control and sample was significant at P<0.01 (**) by one-way ANOVA and Tukey's test. e, epidermis; hv, minor vein; l1, first loop; l2, second loop; mv, midvein. Bars: (E,I,L) 10 µm; (F,G) 20 µm; (H,J,K) 50 µm; (M–O) 5 µm; (Q–S) 2.5 µm.