QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online October 10, 2008
doi: 10.1242/10.1242/dev.026708

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Yang, S. Articles by Ingram, G. Search for Related Content PubMed PubMed Citation Articles by Yang, S. Articles by Ingram, G. Social Bookmarking                         What's this?

The endosperm-specific ZHOUPI gene of Arabidopsis thaliana regulates endosperm breakdown and embryonic epidermal development

Suxin Yang, Niamh Johnston, Edmund Talideh, Steve Mitchell, Chris Jeffree, Justin Goodrich^*, and Gwyneth Ingram^*,

Institute of Molecular Plant Sciences, School of Biology, University of Edinburgh, Rutherford Building, Mayfield Road, Edinburgh EH9 3JH, UK.

View larger version (16K): [in this window] [in a new window]   Fig. 1. Structure of zou mutant alleles and ZOU expression. (A) Structure of ZOU genomic region and position of T DNA insertions. Exons are indicated by black rectangles; the direction of transcription is indicated by red arrow. The pSKI074 T-DNA insertion in zou1-D is shown as a triangle with black arrowheads indicating the viral enhancer sequences. The origin of the zou2-4 loss of function alleles is described in the Materials and methods. (B) RT-PCR analysis of gene expression in leaves of wild-type (+) and zou-1D/- (z) hemizygotes. Primer sets specific for three different genes were used, as indicated below the gel image. Eukaryotic translation initiation factor 4A (EiF4A) is a control for loading and RNA integrity. (C) Q-PCR analysis of ZOU expression in wild-type rosettes (R), seedlings (Se), unopened flower buds (B), opened flowers (F), siliques containing globular-heart stage embryos (S1) and siliques containing late heart and torpedo stage embryos (S2). Values are relative to expression in S1, which is designated as 1.0. No expression was detected in R, Se or F samples. Values are means of three replicates with standard error of mean.

View larger version (64K): [in this window] [in a new window]   Fig. 2. Phenotype of zou mutant seeds. (A) Seeds from wild-type plant. (B) Shrivelled seeds from self-pollinated zou-2/+ plant (the bulk of seeds were wild-type and non-shrivelled). (C) Dissected silique of self pollinated zou-2/zou-2 ZOU::ZOU-GFP/- primary transformant. The majority of seeds have a wild-type phenotype, owing to complementation by the transgene. Some seeds (asterisk) do not inherit the transgene, owing to segregation, and therefore have zou mutant phenotype. (D) Embryos dissected from mature seeds of wild-type (left) and zou-2 mutants (right). (E-H) Cleared seeds viewed using DIC microscopy. Arrows indicate embryos which are at globular (E), heart (F), late torpedo (G,H) stages. (I,J) Light microscope images of 1µM sections of resin-embedded seeds. Arrow in J indicates the mis-shapen zou embryo, which has adhered to the surrounding cellularized endosperm. (K,L) TEM images of ultra thin sections of osmium stained seeds with torpedo stage embryos. The cuticularized layer (CL) is indicated by arrows. In zou-2 (K), unlike ZOU+ (L), endosperm (en) is appressed to the cuticle of the embryo (em). Scale bars: 200µm in A-H; 100µm in I,J; 500 nm in K,L.

View larger version (66K): [in this window] [in a new window]   Fig. 3. Epidermal phenotypes of zou mutant seedlings. (A,B) Seedlings stained with Toluidine Blue. Arrowheads indicate leaves, arrows indicate cotyledons. Unlike wild type (A), in zou-4 mutant (B) cotyledons and hypocotyls are stained strongly. (C-I) Cryo-SEM images of seedling epidermis. (C)zou-4 mutant seedling. Leaves (arrowhead) are normal, whereas cotyledons (arrow) have large tears in the epidermis. (D) Epidermis of wild-type cotyledon, showing jigsaw-shaped pavement cells and stomata. (E) Epidermis of zou-4 cotyledons. Normal cell types are present, although stomatal spacing is abnormal. (F)zou-2 mutant cotyledon showing large tear, with protruding mesophyll cells. (G)zou-4 mutant cotyledon showing gaps between pavement cells (arrows). The rough cell surfaces are due to adhering endosperm. (H)zou-4 acr4-2 double mutant, showing severely defective cotyledons (arrows). Adhering seed coat is indicated by arrowhead. (I)ale2-1 zou-4 double mutant cotyledons are minute, mis-shapen and partially fused. (J) TEM image of wild-type adaxial cotyledon surface showing cell wall (cw) cuticularized layer (cl) and cuticle proper (cp). (K) TEM image of zou-2 adaxial cotyledon surface showing thickness variability and discontinuity (arrow) of the cp. (L)zou-2 cotyledon surface showing adhering endosperm tissue (arrow) on surface of embryo (e). Scale bars: 1 cm in A,B; 1 mm in C; 300µm in D; 400µm in E; 500µm in F; 50µm in G; 500µm in H; 400µm in I; 100 nm in J,K; 2µm in L.

View larger version (96K): [in this window] [in a new window]   Fig. 4. ZOU expression pattern during seed development. (A-D) Images of in situ hybridizations of digoxigenin-labelled ZOU antisense (A-C) and sense (D) RNA probes to sections of siliques, viewed using DIC microscopy. Signal appears purple; the dark-brown staining of the endothelium (asterisk) does not represent signal. Embryos are indicated with arrowheads, the surrounding endosperm with arrows. (A) Seed with early globular stage embryo. (B) Seed with late heart stage embryo. (C) Seed with torpedo stage embryo. (D) Control hybridization with ZOU sense probe. (E-L) Confocal microscopy images in which autofluorescence appears red and GFP fluorescence green. (E) Seed of transgenic plant carrying ZOU::ZOU-GFP reporter gene fusion. The reporter is expressed in nuclei of the embryo surrounding region (ESR) that surrounds the late heart stage embryo. (F-L) Developing seeds of transgenic plant carrying ZOU::H2B-YFP reporter gene. (F) Unfertilized ovule shows no transgene expression. (G) Seed shortly after fertilization showing strong expression in central cell. (H) Seed with endosperm at the two-nuclei stage, with uniform expression in endosperm. (I) Seed with endosperm at the four-nuclei stage, with uniform expression in endosperm. (J) Seed with endosperm at 12- to 16-nuclei stage; expression is becoming stronger at micropylar pole of endosperm (arrow). (K) Seed with endosperm at 24- to 28-nuclei stage. (L) Seed with endosperm at 44- to 48-cell stage, expression is now largely confined to ESR. Scale bars: 100 µm.

View larger version (74K): [in this window] [in a new window]   Fig. 5. Expression of diverse genes involved in endosperm and embryo development in zou mutant seeds. (A-D) Images of in situ hybridization of digoxigenin-labelled ALE1 antisense RNA probes to sections of siliques, viewed using DIC microscopy. Embryo is indicated by arrowhead, endosperm by arrows. Little or no ALE1 expression was detected in zou-2 mutant seeds (C,D), whereas wild type showed intense signal in ESR (A,B). (E,F) Confocal images of reporter line 9185 in wild-type (E) and zou-4 (F) seeds. (G,H) Images of reporter line ATML1::GFP-ATML1 in wild-type (G) and zou-4 (H) seeds. (I,J) Images of reporter line ACR4::H2B-YFP in wild-type (I) and zou-4 (J) seeds. Scale bar: 100 µm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter