Ectopic CLE41 expression results in disrupted vascular bundles. (A-D) Diagrammatic representation of vascular cell divisions specified by CLE41 and PXY in wild-type and transgenic lines. Native CLE41 is represented by black dots, CLE41 from overexpression in red. In wild type (A), CLE41 is expressed in the phloem and signals to PXY in the procambium, thus providing positional information to the dividing cell, which sets the appropriate division plane (dotted line). In 35S::CLE41 (B) and IRX3::CLE41 (C) lines, the polar nature of the CLE41 signal is disrupted, resulting in loss of oriented cell division. SUC2::CLE41 (D) lines have more ligand than do wild-type lines, but it is expressed in a polar manner such that positional information is maintained and the cell division is oriented. (E-H) Col stems in transverse section (E) compared with 35S::CLE41 (F), IRX3::CLE41 (G) and SUC2::CLE41 (H) lines. Phloem sieve plates fluoresce green in Analine Blue stain (top panel) and are found in a discrete arc in wild-type (E) and SUC2::CLE41 (H) lines, but are displaced towards the centre of the stem in 35S::CLE41 and IRX3::CLE41 lines (F,G, white arrows). Procambial cells divide in a highly ordered process (middle panel) in wild type (E) and SUC2::CLE41 (H) lines compared with 35S::CLE41 (F) and IRX3::CLE41 (G) lines, where some cell divisions are present at altered orientations (black arrows). A large number of undifferentiated cells are present in 35S::CLE41 and SUC2::CLE41 lines (F,H, bottom panel, black arrows) compared with wild-type lines (E). Pc, procambium; Ph, phloem; X, xylem. Scale bars: 50 μm in E-H top and bottom panels; 20 μm in E-H middle panels.

PXY-CLE41 signalling acts early in vascular development. (A,B) Transverse sections through wild-type (A) and 35S::CLE41 (B) vascular tissue in recently initiated vascular bundles 5 mm below the shoot apex of 4-week inflorescence stems. (C,D) In situ hybridisation showing that both PXY (C) and CLE41 (D) expression is absent from the apical meristem (top panels, black arrow), appearing just below it as vascular development is initiated (middle panel, arrowheads), and is apparent in several vascular bundles in older tissue (lower panel). Scale bars: 20 μm in A,B; 50 μm in C,D.

Ectopic CLE41 expression results in intercalation of xylem and phloem in hypocotyls. (A-D) Transverse sections of Toluidine Blue-stained hypocotyls (upper panels) demonstrate that in wild-type (A) and SUC2::CLE41 (D) hypocotyls, the vascular cylinder is highly ordered with a continuous ring of xylem. Conversely, 35S::CLE41 (B) and IRX3::CLE41 (C) hypocotyls are disorganised, with cell types interspersed. In sections stained with Aniline Blue (lower panels), wild-type (A) and SUC2::CLE41 (D) hypocotyls have phloem (fluorescing green) on the outside of the stem surrounding xylem on the inside. In 35S::CLE41 (B) and IRX3::CLE41 (C) hypocotyls, phloem cells develop towards the centre of the stem (arrowheads) surrounded by xylem. (E-G) Toluidine Blue-stained close-up images show that in wild-type (E) and SUC2::CLE41 (H) hypocotyls, all xylem cells are running parallel (arrowheads) into the plane of section; however, in 35S::CLE41 (F) and IRX3::CLE41 (G) hypocotyls, xylem vessels are in a variety of orientations (arrowheads). Xylem cells running parallel to the plane of section demonstrate a lack of order along the apical-basal axis and this is clearly demonstrated in longitudinal section (lower panels). C, cambium; Ph, phloem; X, xylem. Scale bars: 100 μm in A-D; 20 μm in E-H upper panels; 50 μm in E-H lower panels.

The diameter of plant hypocotyls at senescence. Error bars are standard error. t-tests show that pxy is significantly smaller than Col (P<0.005) and that 35S::CLE41 and 35S::CLE42 are significantly larger than Col (P<0.0001). 35S::CLE41 pxy and 35S::CLE42 pxy were not significantly different from pxy.

Changes in gene expression in 35S::CLE41 compared with wild type, normalised to 18s rRNA. (A-C) Quantitative RT-PCR was used to assay IRX3 (A), ATHB8 (B) and PXY (C) expression in stems and hypocotyls of wild-type plants compared with 35S::CLE41. Error bars are standard error.