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First published online August 4, 2003
doi: 10.1242/10.1242/dev.00634

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The Arabidopsis ACR4 gene plays a role in cell layer organisation during ovule integument and sepal margin development

Institute of Cell and Molecular Biology, Kings Buildings, University of Edinburgh, Edinburgh EH9 3JR, UK

View larger version (120K): [in a new window]   Fig. 1. Expression of ACR4 during development. (A,C) Fluorescence images of two- to four-cell (A) and eight-cell (C) embryos (arrows). (B,D) In situ hybridisations of the same sections with ACR4 antisense probe. Expression is detected as light brown coloration in the eight-cell embryos but not in two- to four-cell embryos. (E-H) ACR4 expression in the L1 (outer) cell layer of developing embryo, inflorescence (im) and floral meristems (fm). In situ hybridisation (F) and confocal images (E,G,H) of H2B::YFP expression (green) in pACR4 transactivation lines. (I) In mature ovules expression is detected in the outer cell layer of the funiculus (f), the outer integument (oi) inner integument (ii) and endothelium (en). (J) In the root tip expression occurs in at least four columella (c) cells layers, the lateral root cap (LRC) and the quiescent centre (QC) but not in the epithelial cell file (e). (K) Expression in the root epithelial cell file (e) initiates as epithelial cells emerge from the LRC. c, cotyledon primordia; rp, embryonic root pole; sam, embryonic shoot apical meristem; fg, female gametophyte; m, micropyle. Scale bars: 25 µm except for K (10 µm).

View larger version (76K): [in a new window]   Fig. 2. acr4 mutant seed phenotype. (A,B) Opened siliques showing differences in seed size and texture between a wild-type (A) and homozygous (B) plant at comparable stages (12 days after pollination). Aborted ovules (arrowhead), retarded seeds (star) and seeds with epidermal outgrowths (arrow) are frequent in mutant siliques. (C,D) Comparison between mature wild-type (C) and mutant (D) seeds observed by SEM. Scale bars: 100 µm.

View larger version (98K): [in a new window]   Fig. 3. The internal structure of acr4 mutant ovules. Wild-type (A) compared with mutant (B,C,D) ovules. Micropyles are indicated by arrowheads. Female gametophytes (fg) and densely staining endothelial cells (e) are labelled where present. (A) In wild type neatly organised cell layers are visible, with the female gametophyte (fg) surrounded by an orderly endothelium. (B) Mutant ovule with weak phenotype. Fg is visible but outer integument is disrupted (to left of star). Endothelial layer is visible. (C) Mutant ovule with intermediate phenotype showing disorganised cell layers and replacement of fg with divided cells. (D) Two fused (star) mutant ovules with extreme phenotypes. Both show cell layer disorganisation but one has distinguishable (probably abnormal) fg. Scale bar: 25 µm.

View larger version (138K): [in a new window]   Fig. 4. Phenotypic analysis of wild-type and acr4 mutant plants. (A,B) Ovule primordia immediately prior to integument initiation in wild-type (A) and mutant (B) plants. (C,D) The initiation of the inner (arrowheads) and outer (arrows) integuments in wild-type and mutant ovules respectively. In the mutant, the irregular initiation of integument outgrowth is visible, with at least two outgrowths observed in one region of outer integument initiation (asterisks), whereas other regions have no outgrowths. (E,F) Ongoing integument outgrowth. In wild-type ovules (E) the leading edges of the integuments are smooth, whereas in mutants (F) they are ragged and often retarded. (The nucellus has snapped off in the right hand ovule of F.) (G) A mature wild-type ovule at anthesis. The outer integument has overgrown the inner integument and nucellus to give a narrow micropyle (m) facing the funiculus (f). (H-J) A weak, a medium and a severe mature mutant ovule phenotype, respectively. In H, the retardation of outer integument (arrow) growth has left an open micropyle within which the inner integument (arrowhead) and nucellus are visible. In I the inner integument (arrowhead) looks relatively normal whereas the outer integument (arrow) has failed to elongate correctly. (J) The inner integument (arrowhead) has failed to grow out leaving the nucellus almost completely exposed. (K) Wild-type sepal margin (arrowhead) showing well organised border cells covered in cuticular decoration. (L) Mutant sepal margin (arrowhead) showing typical irregularities in cell organisation, `lumpy' appearance and regions devoid of cuticular decoration. Scale bars: 20 µm.

View larger version (123K): [in a new window]   Fig. 5. ACR4 kinase activity. Coomassie Blue-stained gel (top) showing products of kinase assay on the wild-type ACR4 kinase domain (K+) and kinase null variant (negative control, K-). Autoradiography of this gel (below) shows that the native kinase domain has kinase activity whereas the kinase null variant does not.

View larger version (90K): [in a new window]   Fig. 6. Protein localisation in lines expressing an ACR4::GFP protein fusion. (A) localisation of fusion proteins (gren) in the plasma membranes of the ovule epidermis. (B,C) Expression patterns of fusion proteins (green) in embryos (B) and meristems (floral meristem, fm) (C). Plasma membrane localisation can be observed (arrow in B). (D-F) Protein localisation in root meristems. (D) Lateral root tip, columella and LRC expression are visible. No protein is seen in the epidermis (e) before emergence from the LRC. (E) Main root tip with columella cell layers indicated by stars. (F) surface view when mounted in 0.8 M mannitol. Cell wall area is clear of fluorescence. (G,H) Protein localisation in comparable untreated and 2-hour BFA-treated root samples, respectively, showing relative decrease in plasma membrane localisation and the appearance of bright perinuclear bodies. (I) mature ovule with fluorescence seen in outer integument (oi) (where little protein is detected in the outermost cell plasma membrane; arrowhead), inner integument (ii) and outer cell layer of funiculus (f). rp, embryonic root pole; c, cotyledon primordia; s, sepal primordia; m, micropyle; fg, female gametophyte. Scale bars: 25 µm.