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. 2009 Aug;21(8):2269-83.
doi: 10.1105/tpc.109.065862. Epub 2009 Aug 28.

Differential Recruitment of WOX Transcription Factors for Lateral Development and Organ Fusion in Petunia and Arabidopsis

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Free PMC article

Differential Recruitment of WOX Transcription Factors for Lateral Development and Organ Fusion in Petunia and Arabidopsis

Michiel Vandenbussche et al. Plant Cell. .
Free PMC article

Abstract

Petal fusion in petunia (Petunia x hybrida) results from lateral expansion of the five initially separate petal primordia, forming a ring-like primordium that determines further development. Here, we show that MAEWEST (MAW) and CHORIPETALA SUZANNE (CHSU) are required for petal and carpel fusion, as well as for lateral outgrowth of the leaf blade. Morphological and molecular analysis of maw and maw chsu double mutants suggest that polarity defects along the adaxial/abaxial axis contribute to the observed reduced lateral outgrowth of organ primordia. We show that MAW encodes a member of the WOX (WUSCHEL-related homeobox) transcription factor family and that a partly similar function is redundantly encoded by WOX1 and PRESSED FLOWER (PRS) in Arabidopsis thaliana, indicating a conserved role for MAW/WOX1/PRS genes in regulating lateral organ development. Comparison of petunia maw and Arabidopsis wox1 prs phenotypes suggests differential recruitment of WOX gene function depending on organ type and species. Our comparative data together with previous reports on WOX gene function in different species identify the WOX gene family as highly dynamic and, therefore, an attractive subject for future evo-devo studies.

Figures

Figure 1.
Figure 1.
Phenotypic Analysis of Petunia maw Mutants. (A) to (F) Vegetative phenotypes of petunia wild type and maw mutants at comparable stages of development. (A) and (B) Seedlings. (C) Top view of 7-week-old wild-type and maw plants with the first flower opening. (D) Close-up of the leaf margins; note the thickened margins in maw leaves. (E) and (F) Scanning electron microscopy images of freeze-fractured cross sections through wild-type and maw leaves. Adaxial palisade parenchyma tissue has been artificially colored in green. (G) to (P) Flower phenotypes of petunia wild type and maw mutants at comparable stages of development. (G), (H), (L), and (M) Scanning electron microscopy images of flower primordia. Sepals have been removed to reveal inner organization. Petal primordia are artificially colored in red and carpels in green. (G) and (L) Stage in which carpel primordia start to emerge. Petal primordia are already flattened in the wild type and merge at their base. In maw mutants, lateral expansion of the petal primordia is reduced. (H) and (M) Later stage showing the fusion of the two carpels in the wild type and the development of abaxial trichomes on the petal main veins. In maw mutants, carpels fail to fuse completely, while the distal ends of the petals are radial rather than flattened structures. Note the trichomes in these regions extending toward the adaxial side. (I) and (N) Top view of fully developed flowers. (J) and (O) Close-up of the pistil showing stigma. (K) and (P) Entire pistils showing ovary, style, and stigma. (O) A maw pistil with partially unfused carpels. (P) A maw pistil with carpels unfused all the way down to the base of the ovary. Bars = 2 mm in (A) and (B), 1 cm in (C), (N), and (I), 1 mm in (D), (H), (K), (M), and (P), 0.5 mm in (J) and (O), and 100 μm in (E) to (G) and (L).
Figure 2.
Figure 2.
Phenotypic Analysis of Petunia chsu and maw chsu Double Mutants. Genotypes are indicated in italics. (A) Fully grown leaves of 5-week-old plants. (B) and (C) Scanning electron microscopy images of freeze-fractured cross sections through fully grown leaves. Adaxial palisade parenchyma tissue has been artificially colored in green. Note the further reduction and disorganization of palisade parenchyma in (C). (D) Entire pistils showing fusion defects in mutant backgrounds and exposure of the ovules in maw chsu pistils (arrow). (E) Rosette of a 4-week-old maw chsu mutant showing an almost completely radialized leaf (arrow). (F) Scanning electron microscopy image of a maw chsu flower. Four of the five sepals have been removed to reveal inner organization. Petal primordia are artificially colored in red, and abaxial trichomes develop on all sides. (G) to (J) Side view of fully grown flowers. Bars = 1 cm in (A), 100 μm in (B) and (C), 5 mm in (D) and (G) to (J), and 1 mm in (F).
Figure 3.
Figure 3.
Cloning and Molecular Characterization of Petunia MAW. (A) Genomic structure of the petunia MAW gene. Exons are represented by green boxes, introns by a single line, and dTph1 transposon insertions by red triangles. Allele names are indicated with insert positions in superscript as number of base pairs downstream of the ATG start codon in the genomic sequence. Blue box marked with HD indicates the homeodomain. (B) to (E) In situ localization of petunia MAEWEST transcripts in developing leaves and flower buds. Sections were hybridized with a digoxigenin-labeled antisense MAW RNA probe (red staining). (B) and (C) MAW expression in vegetative organs (leaves and bracts). (B) Longitudinal section through a meristem with an emerging bract primordium. Expression is localized to the adaxial side at the base of the emerging bract (asterisk) and, more distally, confined to a narrow stripe corresponding to the provascular tissue (arrow). (C) Cross-section through developing leaves, including a young still round-shaped leaf stage and a further expanded leaf. Sections are oriented with the adaxial side above. MAW is detected in a central stripe in the youngest leaf cross section (arrows), and this pattern is maintained during later stages. (D) and (E) MAW expression in developing flowers. (D) Longitudinal section through a young flower bud with just emerging carpel primordia. At this stage, MAW transcripts are found central at the distal ends of emerging petal primordia (arrow) and more uniformly distributed in the developing stamens and emerging carpel primordia. (E) Cross section through an almost fully developed flower bud sectioned at the height of the stigma. Strongest expression remains at the adaxial side of the carpels where fusion occurs, in the stamen loculi, and the margins of the petals. Identity of the floral organs in (D) and (E) are indicated as follows: s, sepal; p, petal; st, stamen; and ca, carpel. Bars = 200 μm in (C) to (E) and 100 μm in (B). (F) Subcellular localization of the 35S:GFP-MAW construct in tobacco BY cells analyzed by confocal laser microscopy. Left, nuclear-localized GFP signal; middle, differential interference contrast image; right, merged image of GFP and differential interference contrast images.
Figure 4.
Figure 4.
Phylogenetic and Structural Analysis of the WOX Gene Family. For the neighbor-joining tree, 1000 bootstrap samples were generated to assess support for the inferred relationships. Local bootstrap probabilities are indicated near the branching points. Species names precede protein names and are abbreviated as follows: Vv, Vitis vinifera; At, Arabidopsis thaliana; Am, Antirrhinum majus; Ph, Petunia hybrida; Pt, Populus trichocarpa; Os, Oryza sativa; Sb, Sorghum bicolor; Zm, Zea mays. Accession codes are provided in Supplemental Table 2 online. An asterisk after the gene name indicates a deviating gene model compared with automatic predictions in the database. Short conserved peptide motifs are shown right from the tree and are named after their position relative to the homeodomain: 5′, upstream; 3′int, downstream internal; 3′c, C-terminal, combined with the name of subfamily(ies) for which they are diagnostic. Asterisks after C-terminal motifs represent stopcodons; in all other cases, the numbers after motifs indicate the number of remaining nonconserved amino acid residues before the stop codon is encountered. Exon/intron structures for members of the WUS/WOX1-7 subclass are shown at the right. Exons are represented by green boxes and introns by black lines. HD, homeodomain region. Yellow and purple boxes indicate position of the 5′ WOX1/4 box and the 3′int WUS box, respectively.
Figure 5.
Figure 5.
Leaf and Flower Phenotypes of Arabidopsis Wild-Type and wox1 prs Mutants. Genotypes are indicated in italics below the images. (A) Seedling stage. (B) Rosette at the beginning of flowering. (C) Flowering plants. (D) Inflorescence top view. (E) Side view of individual flowers. (F) to (I) Scanning electron microscopy images. (F) and (G) Freeze-fractured cross sections through wild-type and wox1 prs leaves. Adaxial palisade parenchyma tissue has been artificially colored in green. (H) Epidermis of wild-type petals showing the typical conical petal cells at the adaxial side. (I) wox1 prs petals showing the flattened abaxial petal epidermis cells extending to the adaxial side. A small group of normal conical cells remains in the middle (arrow). Bars = 0.25 cm in (A) and (D), 1 cm in (B) and (C), 500 μm in (E), and 100 μm in (F) to (I).
Figure 6.
Figure 6.
Organ Polarity Gene Expression Analysis in the Wild Type and Mutants by Quantitative RT-PCR. The y axis shows x-fold expression with respect to the lowest encountered value equal to one. Error bars represent sd calculated from two technical replicates done on two biological replicates. Gene names of measured transcript levels are indicated below the bars. (A) Expression levels in shoot samples, including leaf primordia of 2-week-old Arabidopsis wild type (white bars) and wox1 prs mutants (black bars). (B) Expression levels in shoot samples, including leaf primordia of 30-d-old petunia wild type (white bars), maw mutants (light-gray bars), chsu mutants (dark-gray bars), and maw chsu mutants (black bars). AB, abaxial determinants; AD, adaxial determinants. Differential expression values between mutant and wild-type samples were tested for statistical significance (see Methods). Differences with a P < 0.05 (one asterisk), and P < 0.001 (two asterisks) resulting from a one-way analysis of variance test are indicated.

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