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First published online 11 July 2007
doi: 10.1242/dev.006296

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Regulation of the Arabidopsis root vascular initial population by LONESOME HIGHWAY

Kyoko Ohashi-Ito^* and Dominique C. Bergmann

Stanford University, Department of Biological Sciences, Stanford, CA 94305, USA.

View larger version (98K): [in this window] [in a new window]   Fig. 1. Phenotype of LONESOME HIGHWAY mutants. (A) Cross-section diagram of a mature Arabidopsis root; tissues are arranged radially from outside in: epidermis (white), cortex (purple), endodermis (dark blue) and stele [which consists of a ring of pericycle cells (light blue) that surrounds xylem (yellow) and phloem (red) arranged in bilateral symmetry]. (B,C) Confocal images of wild type (WT, B) and lhw-1 (C) expressing the xylem-associated pericycle marker J0121::GFP (green). Roots are counterstained with propidium iodide (PI, red) to visualize the outlines of cells. (D) Bright-field image of a lhw-1 root, which has all lateral roots (black arrowheads) emerging from a single side of the primary root. (E,F) Confocal image of basic fuchsin staining of xylem in wild type (E) and lhw-1 (F). (G,H) Confocal images of wild type (G) and lhw-1 (H) expressing the phloem marker APLpro::APL-GFP (green). (I,J) Whole-plant phenotypes of wild-type Col (I) and lhw SALK_079402 (J). (K) Root growth of wild type (left) and lhw-1 (right) on agar plates at 20 dpg. Notice the root-waving and short-root phenotypes in lhw-1. (L,M) Vascular pattern in mature (13 dpg) wild-type (L) and lhw-1 (M) cotyledons. (N,O) Higher-magnification images of xylem from images L and M, respectively. The black arrow points to the end of mature xylem elements; to the right, elongated cells typical of procambium are still seen. For each marker, the wild-type and lhw image pair are at the same magnification.

View larger version (113K): [in this window] [in a new window]   Fig. 2. Cross sections of lhw roots and hypocotyls. (A) Schematic of stele in cross sections from wild type (WT), lhw and wol-1; pericycle is in blue and xylem in yellow; wild type and lhw were traced from 2F and 2G, respectively. (B-G) Bright-field images of wild-type and lhw root sections taken at increasing distance from the tip of the root; for each, the same pericycle cell is marked with a blue star for orientation: (B) wild type, 30 µm; (C) lhw-2, 30 µm; (D) wild type, 60 µm; (E) lhw-2, 60 µm; (F) wild type, 120 µm; (G) lhw-2, 120 µm. (H,I) Toluidine-blue staining of wild type (H) and lhw-2 (I) in the mature zone. (J,K) Section through the lower third of the hypocotyl in wild type (J) and lhw-2 (K). (L) Center of a wol-1 root, 66 µm from the tip. (M) Center of a wol-1;lhw-1 root, 66 µm from the tip. (N,O) DIC (N) and toluidine-blue (O) images of wol-1;lhw-1 mature roots, showing the very reduced stele filled with xylem elements. Each image pair is at the same magnification. Scale bars: 20 µm.

View larger version (55K): [in this window] [in a new window]   Fig. 3. Measures of auxin response in lhw. (A-C) Confocal images of wild type (WT, A) and lhw-1 (B,C) expressing J0121::GFP (green). (A,B) After treatment with 20 µM NPA for 7 days. (C) After treatment with 30 nM 2,4D for 7 days. (D,E) DIC images of roots treated with 20 µM NPA for 21 days. Black arrows point to xylem elements. (F-I) Bright-field images of 7-dpg seedling root tips; (F,G) DR5::GUS expression; (H,I) PIN4::GUS expression. Each image pair (and A-C) is at the same magnification. Scale bars: 20 µm.

View larger version (27K): [in this window] [in a new window]   Fig. 4. LHW gene and protein structure, and its behavior in a two-hybrid screen. (A) LHW gene structure, with exons represented as boxes and introns as lines. The LHW coding region is in black. The location and nature of lhw mutant alleles is indicated above the exons. (B) LHW protein structure. Two domains that are conserved with other plant proteins are indicated as grey boxes. Part of the C-terminal conserved region resembles the bHLH domain of transcriptional regulators. A sequence alignment of the putative bHLH domain (boxed) is diagrammed for LHW and for related proteins from Arabidopsis and rice. (C) Graphical representation of LHW protein fragments used in the yeast two-hybrid assay and their ability to dimerize with full-length LHW. (D) GAL4 transcriptional activation activity of LHW variants; pACT is included as a negative control. GUS measurements are based on four replicates/sample. Error bars ±s.e.m. NT, not tested.

View larger version (74K): [in this window] [in a new window]   Fig. 5. LHW expression. (A,B) Confocal images of lhw-2 roots expressing 35S::LHW-GFP in their nuclei (green). Expression of this transgene is sufficient to rescue the lhw-2 xylem phenotype (B). (C-E) LHWpro::GUS reporter expression in root tips after staining for 1, 4 or 8 hours, respectively. (F) RT-PCR of LHW and ACTIN (ACT) expression. S, 14-dpg seedling; L, expanded rosette leaf; RT, 5 mm of 14-dpg root tip; R, whole 14-dpg root; SAM, transition SAM and youngest leaves; F, flower stage 8-15; 79402, 14-dpg seedling of SALK_79402.

View larger version (104K): [in this window] [in a new window]   Fig. 6. The effects of LHW on RM establishment and maintenance. (A-P) Expression of meristem markers (green) in wild-type (WT) and lhw-1 7-dpg seedlings. (A,B) SCR::GFP expression; (C) SCR::GFP expression in a torpedo-stage lhw-1 embryo; (D,E) columella differentiation marker Q1630::GFP expression; (F,G) CYCB1;2::GUS expression; (H-P) QC25::GUS (blue) and starch granules (purple) mark the degeneration of the lhw meristem over time (H-I, 7 dpg; J-L, 13 dpg; M-P, 18 dpg). (L) Higher-magnification image of K; star indicates starch-granule-containing cells adjacent to QC25-marked cells. (N) Higher-magnification image of M. For each marker, the wild-type and lhw image pair are at the same magnification. Arrows point to quiescent center (QC) cells. (Q) Graph of wild-type (dark grey) and lhw-1 (light grey) root growth over time. Error bars ±s.e.m. (R) Model for LHW action in generating vascular pattern. LHW is required to establish the radial extent of the root vascular tissues in the embryo and promotes postembryonic divisions in these tissues. LHW therefore acts as a meristem size-control protein for the center of the root. LHW and WOL are both required for these cell divisions, but appear to act at least somewhat independently. We propose that the eventual slowing down of longitudinal growth in lhw mutant roots is not due to a direct requirement for LHW in meristem maintenance, but because LHW is required to create the tissue that normally produces SHR. Without adequate levels of SHR, SCR is not maintained in the QC and meristems eventually terminate. Scale bars: 30 µm.