Morpholinos for splice modificatio

Morpholinos for splice modification


Auxin-dependent regulation of lateral root positioning in the basal meristem of Arabidopsis
Ive De Smet, Takuya Tetsumura, Bert De Rybel, Nicolas Frei dit Frey, Laurent Laplaze, Ilda Casimiro, Ranjan Swarup, Mirande Naudts, Steffen Vanneste, Dominique Audenaert, Dirk Inzé, Malcolm J. Bennett, Tom Beeckman
  1. Fig. 1.

    Correlation between Arabidopsis root waving and lateral root positioning. (A) Vertically grown Col-0 seedling with wavy growth pattern of the root. (B) Detail of boxed area in A showing the left (L) - right (R) alternating lateral roots.?, wave where lateral root is not apparent. (C) Root grown at 45° to enhance waving. Positions of lateral root primordia are marked by the CYCB1;1::GUS activity. (D) Schematic illustrating the method to determine lateral root positioning/root wave relationship. a, amplitude of the wave; b, root length between the curve tops; the red line marks the top of the wave; +, lateral roots perfectly on top of the wave; -, lateral root at the side of a wave. (E) Quantification of left-right alternation of lateral roots in vertically grown seedlings with an equal and dissimilar percentage for wild type (Col-0) and aux1-7, respectively. *, statistically significant difference for right side values as determined by Student's t-test (P<0.001). Error bars, s.e.m. (F) Vertically grown agravitropic aux1-7 seedling root with curled main root. The left (L) position of lateral roots is indicated.

  2. Fig. 2.

    Role of targeted AUX1 expression in lateral root formation. (A) Root tip of a GAL4-GFP-expressing line (J0951) used for targeted expression of AUX1 to the lateral root cap and epidermis. Arrowheads mark the end of the root cap. (B,C) Analysis of lateral root initiation and positioning in wild type (Col-0), aux1-22, both parental lines (UAS:AUX1,aux1-22 and J0951,aux1-22), and transactivation line (J0951≫AUX1,aux1-22). (B) Lateral root density (seedlings grown at 45°). *, statistically significant differences for values compared with wild type as determined by Student's t-test (P<0.001). (C) Quantification of left-right alternation of lateral roots in vertically grown seedlings. *, statistically significant difference for right side values compared with wild type as determined by Student's t-test (P<0.001). Error bars, s.e.m.

  3. Fig. 3.

    Responsiveness of the basal meristem to auxin and specificity of DR5 auxin reporter maximum. (A) Transfer from MS medium to 10 μM NAA: induction of lateral roots all over the root (*, visualized using CYCB1;1::GUS) and emergence of a big cluster in the basal meristem (arrowhead). (B) Quiescent center marker line (QC184) demonstrating differentiation in fused lateral root primordia (arrowheads). (C-E) DR5::GUS expression in apical part of root grown on MS medium for 40 HAG (C); transferred from MS medium to 10 μM NAA for 6 hours (D); and grown for 72 HAG on NPA (E). Scale bar: 100 μm. The boxed area in C indicates DR5::GUS expression in the basal meristem. Arrowheads mark the end of the root cap and start of the basal meristem. (F-H) Transverse sections through the basal meristem of seedling roots expressing DR5::GUS (F) and IAA2::GUS (G,H) grown on MS for 40 HAG (F,G) and on NPA for 72 HAG (H). *, protoxylem cells; p, pericycle layer.

  4. Fig. 4.

    Regular lateral root spacing and oscillation in DR5::GUS activity in the basal meristem. (A) DR5::GUS-activity in a 7.5-hour time course in Col-0 (gray) and aux1-7 (white) expressed as percentage of seedlings with strong GUS staining at each time point. The green line highlights the oscillation in DR5 activity in Col-0. Labels a-d (green circles) mark the time points with high DR5 activity. ND, not determined. (B) Schematic representation of toner label experiment. CYCB1;1::GUS seedlings were labeled with toner particles (left) and after 30 hours stained for GUS (right). (C) Detail of seedling root with toner particles attached on top of the lateral root initiation site (*). Arrowhead, adventitious root. (D) Percentage of seedlings with toner label on top of lateral root initiation site. Labels a-d (green circles) mark the time points with higher percentage of seedlings having toner label on top of lateral root initiation site. (E) Percentage of seedlings with 0-3 lateral root primordia at 30, 45 and 60 HAG. Labels a-c (blue circles) indicate time points at which the next lateral root appears at the earliest. (F) Timeline indicating the intervals between four successive maxima of DR5 activity (green circles) and the periodicity of initiation of the first three lateral roots after germination (blue circles). The 15-hour periodicity in the DR5::GUS expression matches the timing of lateral root initiation.

  5. Fig. 5.

    AXR3 dependency for asymmetric cell division in the xylem pole pericycle. (A) J0121 with specific expression in the xylem pole pericycle starting from the basal meristem onward. (B-E) Analysis of lateral root formation in wild-type control (Col-0), the parental lines (J0121 and UAS:axr3-1), and the transactivation line (J0121≫UAS:axr3-1). (B) Lateral root densities. *, statistically significant differences for values compared with wild type as determined by Student's t-test (P<0.001). (C) Lateral root stages [total number of detected primordia from ten individual roots at each developmental stage according to Malamy and Benfey (Malamy and Benfey, 1997)]. (D) Differential interference contrast image of adjacent pericycle cells with migrated nuclei in J0121≫UAS:axr3-1. *, nuclei; arrowhead, cell wall. (E) Average number of lateral roots per seedling at stage I-VIII (gray) and arrested at the stage of displaced nuclei (white). *, statistically significant difference for values for displaced nuclei as determined by Student's t-test (P<0.02). Error bars, s.e.m.

  6. Fig. 6.

    Involvement of IAA14/SLR in lateral root initiation above the basal meristem. (A) IAA14::GUS expression in the root tip limited to the root cap. Arrowheads point to the edge of the root cap. (B,C) Analysis of pIAA14:mIAA14-GR seedlings upon transfer from media with Dex to media with (B) and without (C) Dex. Above the black line, lateral roots are formed in the region previously subjected to Dex after transfer to Dex-free medium (C), whereas this is not the case after transfer to Dex medium (B). The region `a' in C between solid and broken line is the distal part of the root that can form lateral roots after transfer to Dex-free medium.

  7. Fig. 7.

    Hypothetical model for auxin signaling in the basal meristem and subsequent lateral root initiation. (A,B) Possible presence of an auxin (response) gradient based on the staining patterns of auxin reporters. In accordance with the auxin gradient in the quiescent center and surrounding initials, the lateral root initiation `stem cells' are triggered by the neighboring xylem cell showing high auxin concentration and/or response (A). When this gradient disappears (as is the case of auxin transport inhibition), auxin is distributed equally (even into the pericycle) and no lateral root initiation can take place (B). Light to dark blue shades reflect low to high auxin content. Xylem pole pericycle cells that will initiate a lateral root are in red. (C) Scheme of two adjacent pericycle cells on the same cell file undergoing early developmental steps prior to lateral root initiation: from priming by auxin in the basal meristem (associated with migration of nuclei as indicated with the dotted arrows) to the auxin response required for asymmetric cell division. (D) Hypothetical scheme showing the longitudinal progression of pericycle cells in time and space in the main root with the indication of the major developmental steps toward lateral root initiation. First, auxin (blue arrows) is targeted to the basal meristem (BM) from the root apical meristem (RAM) via AUX1-mediated transport in the lateral root cap (dark gray). Subsequently, reflux (PIN-mediated) is presumably involved in generating the auxin maximum in the protoxylem cells (blue strands) adjacent to the pericycle cells (green). Later, in the differentiation zone, the primed pericycle cells (light blue) are exposed again to auxin response and signaling mechanisms (Aux/IAAs, for instance IAA14/SLR) before CYCB1;1 becomes expressed and the actual division occurs (light-blue cells with dark-blue center)