Figure 1.
Vein patterning functions of Arabidopsis PIN genes.
(A,B) Vein pattern of WT mature first leaf. In (A), dark grey, midvein; grey, loops; light grey, minor veins. (B–H) Dark-field illumination of cleared mature first leaves illustrating phenotype classes: unbranched, narrow midvein and scalloped vein-network outline (B); bifurcated midvein and scalloped vein-network outline (C); fused leaves with scalloped vein-network outline (D); conspicuous marginal vein (E); fused leaves with conspicuous marginal vein (F); wide midvein (G); fused leaves with wide midvein (H). (I) Percentages of leaves in phenotype classes. Difference between pin1 and WT, between pin1;6 and pin1, and between UBQ10::amiPIN6;pin1 and pin1 was significant at P<0.001 (***) by Kruskal-Wallis and Mann-Whitney test with Bonferroni correction. Sample population sizes: WT, 65; pin2, 68; pin3, 68; pin4, 68; pin5, 68; pin6, 68; UBQ10::amiPIN6-5, 65; UBQ10::amiPIN6-10, 65; pin7, 68; pin8, 68; pin1, 71; pin1;2, 71; pin1;3, 77; pin1;4, 69; pin1;5, 72; pin1;6, 65; UBQ10::amiPIN6-5;pin1, 65; UBQ10::amiPIN6-10;pin1, 67; pin1;7, 77; pin1;8, 68. Bars: (B–F) 1.5 mm; (G,H) 0.75 mm.
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.
Figure 3.
Genetic interaction between PIN1, PIN5, PIN6, and PIN8 in vein patterning.
Percentages of leaves in phenotype classes (defined in Figure 1). Difference between pin1;6;8 and pin1;6, and between pin1;5;6;8 and pin1;6;8 was significant at P<0.05 (*) or P<0.001 (***) by Kruskal-Wallis and Mann-Whitney test with Bonferroni correction. Sample population sizes: pin1;6, 114; pin1;5;6, 92; pin1;6;8, 95; pin1;5;6;8, 114.
Figure 4.
PIN8 expression in leaf development.
(A–F,H–K) Top right: genotype, leaf age in days after germination (DAG). Bottom left: reproducibility index. Confocal laser scanning microscopy without (A–F) or with (H–K) transmitted light; first leaves. (A–E) Green: PIN8::PIN8:GFP expression; magenta: chlorophyll. (F) Expression at 3.25 DAG of PIN8::PIN8:GFP (left), staining by ER-Tracker Red (centre) and their overlay displayed with a dual-channel LUT (defined in Figure 2) (right). (G) 5-DAG first leaf illustrating positions of close-ups in (D) and (K). (H–K) PIN6::YFPnuc expression. Bars: (A,H) 10 µm; (B–E,I–K) 50 µm; (F) 2 µm.
Figure 5.
Necessity of PIN5, PIN6, and PIN8 for vein network formation.
(A) Vein network complexity as mean ± SE number of vein branching points per first-leaf area unit in mm2 [28]. Difference between pin6;8 and all other genotypes was significant at P<0.01 (**) by one-way ANOVA and Tukey's test. Sample population sizes: WT, 30; pin5, 30; pin6, 30; pin8, 27; pin5;6, 28; pin5;8, 28; pin6;8, 28; pin5;6;8, 28. (B–N) Top right: genotype, markers and leaf age in days after germination (DAG). Bottom left: reproducibility index. (B–D) Dark-field illumination of cleared mature first leaves. (E–N) Confocal laser scanning microscopy; first leaves. LUT (in Figure 2K) visualizes expression levels. (E–G) DR5rev::YFPnuc expression, 4 DAG. Dotted line: leaf outline. (H–N) PIN1::PIN1:YFP expression. (O) 4-DAG first leaf illustrating positions of close-ups in (E–G) and (H–N). Bars: (B–D) 1.5 mm; (E–G,L–N) 50 µm; (H–K) 25 µm.
Figure 6.
Sufficiency of PIN5, PIN6, and PIN8 for vein network formation.
(A–G,J–P) Top right: genotype, markers and leaf age in days after germination (DAG). Bottom left: reproducibility index. (A–G) Confocal laser scanning microscopy; first leaves. LUT (in Figure 2K) visualizes expression levels. (J–P) Dark-field (J–L,N,P) or differential-interference-contrast (M,O) illumination of cleared mature first leaves. (A,B) DR5rev::YFPnuc expression. (C,D) ATHB8::CFPnuc expression. Magenta line connects nuclei in the first loop (l1). Dotted line: leaf outline. (E–G) PIN1::PIN1:YFP expression. (H) 4-DAG first leaf illustrating positions of close-ups in (A–D), (E–G) and (M,O). (I) Percentage of first leaves with 0, 1, 2 or ≥3 open loops. Difference between MP::PIN6 and WT, and between MP::PIN8 and WT was significant at P<0.05 (*) or P<0.001 (***) by Kruskal-Wallis and Mann-Whitney test with Bonferroni correction. Sample population sizes: WT, 43; MP::PIN6-38, 40; MP::PIN6-26, 42; MP::PIN8-10, 40; MP::PIN8-4, 40. (Q) Vein network complexity as mean ± SE number of vein branching points per first-leaf area unit in mm2 [28]. Difference between MP::PIN5 and WT was significant at P<0.001 (***) by unpaired, two-tailed t-test. Sample population sizes: WT, 28; MP::PIN5, 28. l1, first loop; l2, second loop; l3, third loop. Bars: (A,B) 25 µm; (C–G) 50 µm; (J–L,N,P) 1.5 mm; (M,O) 0.2 mm.
Figure 7.
Control of PIN1-dependent vein patterning by intracellular auxin levels.
(A) Percentages of leaves in phenotype classes (defined in Figure 1). Difference between pin1 and WT, between pin1;6 and pin1, and between PIN6::iaaL;pin1 and pin1 was significant at P<0.001 (***) by Kruskal-Wallis and Mann-Whitney test with Bonferroni correction. Sample population sizes: WT, 50; PIN6::iaaL-15, 50; PIN6::iaaL-12, 50; pin1, 61; pin1;6, 61; PIN6::iaaL-15;pin1, 60; PIN6::iaaL-12;pin1, 62. (B,C) Dark-field illumination of cleared mature first leaves. Top right: genotype. Bottom left: phenotype class. Bars: (B,C) 2 mm.
Figure 8.
(A,B) Two potential, non-mutually-exclusive regulatory circuits for PIN1 and PIN5/PIN6/PIN8 in vein patterning. Arrows indicate positive regulation; blunt-ended lines indicate negative regulation. Distinct functions of PIN1-mediated intercellular auxin transport and of PIN5/PIN6/PIN8-mediated intracellular auxin transport converge on vein patterning. Additionally, PIN1-mediated intercellular auxin transport could control vein patterning through regulation of PIN6 expression, and PIN5/PIN6/PIN8-mediated intracellular auxin transport could control vein patterning through regulation of PIN1 expression (A). Alternatively, vein patterning could feed back on expression of PIN1 and PIN6 (B). PIN1-independent functions of PIN5/PIN6/PIN8-mediated intracellular auxin transport in vein patterning could include intercellular auxin transport by a mechanism nonhomologous to that used by PM-localized PIN proteins [63] (grey arrows). (C,D) Localization of PIN1 (blue) at the basal plasma membrane and of PIN5 (orange), PIN6 (green) and PIN8 (yellow) at the endoplasmic reticulum/nuclear envelope (ER/NE) of vascular cells. For simplicity, PIN5, PIN6 and PIN8 are shown to be expressed in the same cell, only PIN6 is shown to be also localized at the NE, and localization of AUX/LAX [30], ABCB/PGP/MDR [32] and PILS [31] auxin transporters is not shown. Arrows indicate presumed directions of auxin transport. Antagonism between pin5 and pin6;8 in vein patterning could reflect opposite directions in which PIN5 and PIN6/PIN8 transport auxin (C), different abilities of PIN5 and PIN6/PIN8 to transport different auxins or auxin conjugates (orange, green and yellow arrows) (D; for simplicity, the mirror-image scenario, i.e. transport to the ER/NE lumen, is not shown), or varied combinations of the two. See text for additional details.