Figure 1.
Four canonical long PIN clades are found in the angiosperms.
(A) Bayesian phylogram of angiosperm long PIN sequences. Basal plant groups (Physcomitrella patens and Selaginella moellendorffii) are not visible, see Figure S1 in supplementary material for complete phylogeny. All nodes without asterisks (*) have at least 0.95 posterior probability. Arabidopsis sequences are marked with stars. Brachypodium SoPIN1, PIN1a, and PIN1b are marked with arrows. Major clades are labeled at right. Plant families at major phylogenetic nodes are colored according to (B). Each sequence name is followed by a family abbreviation also defined in (B). (B) Summary tree showing the relationships amongst the angiosperm families sampled in (A), monocot and eudicot groups are labeled [57]. (C) Summary of inferred phylogenetic relationships between Arabidopsis PIN1 and Brachypodium SoPIN1, PIN1a, and PIN1b. The “X” indicates the loss of Arabidopsis SoPIN1. Scale: 0.08 substitutions per site.
Figure 2.
SoPIN1, PIN1a, PIN1b, and DR5 expression in the Brachypodium spikelet meristem.
(A) Organization of the Brachypodium spikelet meristem. Lemma primordia are colored red, floral meristems blue. Lemma primordia are labeled, I2, I1, P1, P2, P3 (see text). The spikelet meristem (SM), a single floral meristem (FM) and the P2 lemma (L) are labeled. Dashed box indicates area of detail images in Figure 3A–F. (B) Right, mature inflorescence with one lateral and one terminal spikelet, in brackets. Middle, a spikelet broken into the 2 bracts and 10 individual florets. Left, a single floret lemma, not to scale. (B Inset) Look-up-table scales for all fluorescence images. (C–H) Confocal maximum projections of PIN-Citrine and DR5-RFP expression in staged spikelet meristems. See supplemental Video S1. (C) SoPIN1 maximum projection including epidermal sections. (D) Internal SoPIN1 sections in same sample as (C), the spikelet meristem, floral meristems and lemma primordia are outlined in white for clarity. (E,G,H) Internal confocal sections of PIN1a and DR5 from a single plant expressing PIN1a and DR5. (E) PIN1a channel only, (G) DR5 only, and (H) DR5 and PIN1a. (F) Complete maximum projection showing PIN1b. Asterisks in (A), (C), and (D) show lemma midvein convergence points. Circles in (A) and (C) show lemma lateral vein convergence points on one side of meristem, see also Video S1. Ratios below each figure label indicate the number of times each phenomenon discussed in the text was observed out of the total imaged. Scale bars: 1 cm in (B), 25 µm in (C–H).
Figure 3.
SoPIN1, PIN1a, and PIN1b localization during lemma initiation.
Internal confocal maximum projections showing cellular localization of SoPIN1, PIN1a, PIN1b and DR5 in I2-P1 lemma primordia. (A) SoPIN1. Inset shows an alternate confocal section of the I1 primordium with an epidermal (L1) periclinal cell division. (B) SoPIN1 and DR5 localization in an alternate sample at an earlier stage than (A). (C) PIN1a and DR5. (D–E) PIN1b, (D) at earlier stage than (E). Double arrow shows the forming I1 midvein trace. (F), same dataset as (C), DR5 channel only. Large arrow shows region of lowered DR5 expression along the P1 midvein spanned by PIN1a, see (C). (G–H) I1 details of SoPIN1 and DR5 localization in two different samples, (H) at a later stage than (G). (I) PIN1a in I2 at similar stage as (H). Note polarization towards the center of the meristem, surrounding the L2 periclinal division. (J–L) DR5 expression is reduced in areas of high PIN1b expression. (J) PIN1b, (K) DR5, and (L) merged images of a P1 lemma primordium. Large arrow in (K) shows region of reduced DR5 in the PIN1b midvein domain. Double arrow in (J) and (L) shows beginning of PIN1b expression during formation of the I1 midvein. (M) Detail of SoPIN1 and DR5 in P1 midvein. (N) Detail of PIN1a and DR5 in P1 midvein. Images in A, C, E, F are details of same samples as Figure 2C–H. Detail area for (A–F) is indicated by the dashed box in Figure 1A. See Figures S5, S6, S7, S8, S9, S10 for split DR5 and PIN channel images of (B), (C), (G), (H), (M), and (N). Ratios below each figure label indicate the number of times a given phenomena discussed in the text was observed out of the total imaged. Primordia I2, I1 and P1 are labeled. Asterisks show SoPIN1 convergence points in (A), (B), (G), (H), and (M). Small arrows indicate inferred polarity of PIN. Scale Bars: 10 µm.
Figure 4.
Basic features of the proposed model of polar auxin transport.
(A) The layout of cells in the tissue model used to simulate convergence point and vascular strand patterning. Cells in the L1 are colored green, those in the L2 blue, and corpus cells are colored red. Cells assumed to export auxin outside the explicitly modeled part of the shoot are stippled. In addition, a sink is located at the base of the tissue (black cell). Faded cells represent parts of the tissue outside the scope of the model. (B) Schematic view of a cell and its neighbors that illustrates the factors determining PIN allocation to segments of the cell membrane. PIN1a is shown in white, PIN1b in blue and SoPIN1 in yellow. Exocytosis is represented by green arrows, endocytosis by red arrows.
Figure 5.
Simulated dynamics of SoPIN1, PIN1b, and PIN1a during the initiation of a single convergence point and vein segment.
Columns (1–5) correspond to progress of time (5960, 6100, 6430, 6610, 6850 simulation time units). Row (A) SoPIN1, (B) PIN1b, (C) PIN1a, and (D) combined. The concentration of each PIN in the cell membrane is indicated by the width of the colored wedges along each cell (increasing with concentration). The concentrations of each PIN on the membrane, and auxin in each cell, are also indicated by colors, as shown by the scale bars on the right. In rows (B) and (D), the direction and magnitude of PIN1b polarization is further highlighted by a blue arrow in each cell. Cells in the region outlined in white in Panel 2B exhibit increased polarization following the formation of a new convergence point (compare to Panel 1B). The region outlined in white in Panel 3B is the triangular fan of cells with increased polarization at the rootward end of the developing vein. The white arrow in Panel 3C points to a gap in auxin concentration along an emerging vein. The white arrow in Panel 4C points to two cells exhibiting a transient increase in auxin concentration.
Figure 6.
The state of the simulation after the successive emergence of five convergence points.
The concentration of auxin in each cell, PIN on each cell membrane and PIN1b polarities are represented as in Figure 5. (A–C) The expression of each PIN protein: (A) SoPIN1, (B) PIN1b, and (C) PIN1a. (D) The combined expression of the three PINs.
Figure 7.
SoPIN1, PIN1a and PIN1b act in concert during leaf initiation.
(A) SoPIN1 acts up-the-gradient to create an auxin maximum, which is directed to the sink by PIN1b (B), and refined by PIN1a (C), both acting with-the-flux. In (C) PIN1b begins to polarize the adjoining tissue, converting the newly formed trace into a sink for new organs. Auxin concentration as inferred by DR5 is colored red, SoPIN1 green, PIN1b blue and PIN1a red. Arrows show the direction of transport and the relative polarity of the tissue.