Antagonistic Gene Activities Determine the Formation of Pattern Elements along the Mediolateral Axis of the <em>Arabidopsis</em> Fruit

<div><p>The <em>Arabidopsis</em> fruit mainly consists of a mature ovary that shows three well defined territories that are pattern elements along the mediolateral axis: the replum, located at the medial plane of the flower, and the valve and the valve margin, both of lateral nature. <em>JAG</em>/<em>FIL</em> activity, which includes the combined functions of <em>JAGGED</em> (<em>JAG</em>), <em>FILAMENTOUS FLOWER</em> (<em>FIL</em>), and <em>YABBY3</em> (<em>YAB3</em>), contributes to the formation of the two lateral pattern elements, whereas the cooperating genes <em>BREVIPEDICELLUS</em> (<em>BP</em>) and <em>REPLUMLESS</em> (<em>RPL</em>) promote replum development. A recent model to explain pattern formation along the mediolateral axis hypothesizes that <em>JAG</em>/<em>FIL</em> activity and <em>BP</em>/<em>RPL</em> function as antagonistic lateral and medial factors, respectively, which tend to repress each other. In this work, we demonstrate the existence of mutual exclusion mechanisms between both kinds of factors, and how this determines the formation and size of the three territories. Medial factors autonomously constrain lateral factors so that they only express outside the replum, and lateral factors negatively regulate the medially expressed <em>BP</em> gene in a non-autonomous fashion to ensure correct replum development. We also have found that <em>ASYMMETRIC LEAVES1</em> (<em>AS1</em>), previously shown to repress <em>BP</em> both in leaves and ovaries, collaborates with <em>JAG</em>/<em>FIL</em> activity, preventing its repression by <em>BP</em> and showing synergistic interactions with <em>JAG</em>/<em>FIL</em> activity genes. Therefore <em>AS</em> gene function (the function of the interacting genes <em>AS1</em> and <em>AS2</em>) has been incorporated in the model as a new lateral factor. Our model of antagonistic factors provides explanation for mutant fruit phenotypes in <em>Arabidopsis</em> and also may help to understand natural variation of fruit shape in Brassicaceae and other species, since subtle changes in gene expression may cause conspicuous changes in the size of the different tissue types.</p> </div

Effect of mutant combinations of loss-of-function alleles of <i>AS1</i> and YAB1 group genes on gene expression.

<p>(A–F) Whole mount staining of the <i>GUS</i> reporter under control of the <i>FUL</i> (A–C) or the <i>SHP2</i> (D–F) promoter in multiple mutant fruits carrying the <i>as1-104</i> alelle. The expression of the <i>ful-1</i> enhancer trap (<i>FUL::GUS</i>) and <i>SHP2::GUS</i> in the <i>as1-104 fil-8</i> mutant appears mostly normal, with exception of some fruits in which the expression of the <i>FUL::GUS</i> declines in both the basal and the apical regions of the ovary (A) where the <i>SHP2::GUS</i> is expressed at higher levels (D). The <i>as1-104 fil-8 YAB3</i>/<i>yab3-2</i> mutant shows reduced valves which express the <i>FUL::GUS</i> (B), whereas the <i>SHP2::GUS</i> expression is detected encompassing the small valves (E). Most <i>as1-104 fil-8 yab3-2</i> triple mutant fruits lack <i>FUL::GUS</i> in the valves (C), and exhibit a line of <i>SHP2::GUS</i> expression running along the basal two-thirds of the valve (F). (G) Relative mRNA levels of <i>BP</i> in stage 10–13 pistils quantified by qRT-PCR. Scale bars: 1 mm.</p

<i>BP</i> misregulation affects fruit morphology and expression of <i>JAG</i>/<i>FIL</i> activity genes.

<p>(A–D) Cross-sections of stage 15 fruits show defects in replum and valve formation in mutants misexpressing <i>BP</i>. Wild type (A), <i>as1-104</i> (B), <i>35S::BP</i> (C), and <i>FIL>>BP</i> (D). (E–K) <i>BP</i> misexpression produces a reduction in the expression of <i>FIL</i>. <i>In situ</i> hybridization of <i>FIL</i> mRNA in cross-sections of stage 8 pistils of the wild type (E), <i>as1-104</i> (F), <i>35S::BP</i> (G), and <i>FIL>>BP</i> (H); and expression of the <i>FIL::GFP</i> reporter in stage 14 gynoecia of wild type (I), <i>as1-104</i> (J), and <i>35S::BP</i> (K). Although <i>FIL</i> mRNA is undetectable by <i>in situ</i> hybridization in <i>35S::BP</i> gynoecia, the <i>FIL::GFP</i> transgene provides a more sensitive detection of <i>FIL</i> promoter expression. (L–M) Whole mount staining of anthesis gynoecia for <i>JAG::GUS</i>, showing higher expression levels in the wild-type (L) than in <i>as1-104</i> (M). (N) Relative mRNA levels of <i>JAG</i>/<i>FIL</i> activity genes in stage 10–13 pistils quantified by qRT-PCR. In A–D, the curved lines indicate replum size. Scale bars: 100 µm (A–D); 50 µm (E–H); 500 µm (I–M).</p

Synergistic interaction between loss-of-function alleles of <i>AS1</i> and <i>JAG</i>.

<p>(A, D) SEM micrographs and (F) cross-section of stage 17 fruits of <i>35S::BP jag-1</i> plants. These fruits show a moderate mutant phenotype (A, F) with the occasional formation of ectopic valve margin at the apical region of valves (white arrowhead in D). (B, C) SEM images and (G) cross-section of stage 17 fruits of <i>as1-104 jag-1</i> plants, showing the synergistic interaction between these two mutant alleles. Although these fruits show a similar appearance to those of <i>ful</i> mutants, with large and twisted repla and small valves (B, G), the presence of ectopic valve margin is only restricted to the apical region of the valves (asterisk in C). (E, H) Whole mount staining in stage 15 <i>as1-104 jag-1</i> fruits for <i>GUS</i> expression driven by the <i>ful-1</i> enhancer trap (E), which is detected in the small valves, and for <i>SHP2::GUS</i> (H), which is detected in the valve margin and in the apical region of valves where ectopic valve margin forms (asterisk). (I) Relative mRNA levels of <i>CUC1</i>, <i>CUC2</i> and <i>AtMYB117</i>/<i>LOF1</i> in stage 10–13 pistils quantified by qRT-PCR. r, replum; v, valve. Scale bars: 100 µm (A, C, D, F–H); 1 mm (B, E).</p

Simplified model for patterning along the mediolateral axis of the <i>Arabidopsis</i> fruit.

<p>The antagonistic activities of lateral factors (<i>JAG</i>/<i>FIL</i> activity and <i>AS1</i>/<i>2</i> genes) and medial factors (<i>BP</i> and <i>RPL</i>) are responsible for the formation and size of the three pattern elements in the mediolateral axis: valve, valve margin (VM) and replum. Lateral factors form a gradient of activity to determine valve and valve margin development. High activity of lateral factors promotes <i>FUL</i> expression in valves whereas lower levels of this activity induce the expression of valve margin identity genes (VM genes) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003020#pgen.1003020-Dinneny3" target="_blank">[35]</a>. Medial factors specify replum development <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003020#pgen.1003020-AlonsoCantabrana1" target="_blank">[19]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003020#pgen.1003020-Ripoll1" target="_blank">[36]</a>. Lateral factors negatively regulate the expression of medial factors in a non-autonomous fashion and medial factors impede in an autonomous way the expression of lateral factors in the replum. The whole of genetic interactions along the mediolateral axis indicates that genes in all three tissue types negatively regulate the genes expressed in the other territories <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003020#pgen.1003020-Roeder2" target="_blank">[13]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003020#pgen.1003020-Liljegren2" target="_blank">[16]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003020#pgen.1003020-AlonsoCantabrana1" target="_blank">[19]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003020#pgen.1003020-Dinneny3" target="_blank">[35]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003020#pgen.1003020-Ripoll1" target="_blank">[36]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003020#pgen.1003020-Girin2" target="_blank">[88]</a>.</p

<i>BP</i> misexpression enhances the fruit defects of mutants with impaired <i>JAG</i>/<i>FIL</i> activity.

<p>(A–D) SEM micrographs of stage 17 fruits of <i>35S::BP fil-8</i> and <i>35S::BP yab3-2</i> plants. Similar to <i>fil yab3</i> fruits, in <i>35S::BP fil-8</i> (A, B) and <i>35S::BP yab3-2</i> (C, D) fruits, the apical regions lack valve margin whereas the basal regions show ectopic valve margin tissue. (E–I) SEM micrographs and (J–L) cross-sections of stage 17 fruits of several combinations of <i>as1-104</i> with mutant alleles in YAB1 group genes. In panels E–H, insets indicate the magnified area shown in the image below. The apical regions of the ovaries in <i>as1-104 fil-8</i> (E, J) and <i>as1-104 yab3-2</i> (F) fruits show ectopic valve margin, which is reminiscent of <i>fil YAB3</i>/<i>yab3</i> fruits. The increase in the mutant phenotype is evident in <i>as1-104 fil-8 YAB3</i>/<i>yab3-2</i> fruits (G), resembling <i>ful</i> mutants, although unlike these the valves of the multiple mutant show a few interspersed stomata. The fruit of the <i>as1-104 fil-8 yab3-2</i> triple mutant exhibits an extreme phenotype, which implies the complete absence of valves and the presence of two very huge repla separated by valve margin tissue in the basal region of the ovary (H, L), whereas this tissue is absent in its apical region (H, K). Fruits of <i>35S::BP fil-8 yab3-2</i> show in all their lengths the same phenotype exhibited in the apical region of triple mutant ovaries (I). evm, ectopic valve margin; r, replum; v, valve; vm, valve margin. Scale bars: 100 µm (A–D, insets in E–H, J–L,); 1 mm (upper images in E–H, I).</p

Main pattern elements along the mediolateral axis of the <i>Arabidopsis</i> wild-type fruit.

<p>Scanning Electron Microscope (SEM) micrograph of a stage 17 fruit artificially colored to highlight the main pattern elements along the mediolateral axis: valves in green, replum in blue and valve margins in purple (A) and a higher magnification of the same fruit (B). Cross-section of a fruit at the level of the ovary with the same color code as in A (C). gy, gynophore; nc, nectaries; ov, ovary; r, replum; sg, stigma; st, style; v, valve; vm, valve margin. Scale bars: 1 mm (A), 100 µm (B, C).</p

The loss of <i>HUA-PEP</i> activity is epistatic over the <i>ful</i> phenotype.

<p>A) SEM images of the top portion of a <i>ful</i> fruit. The typical long style and wide zig-zag replum were suppressed in <i>ful hua1 hua2</i> and <i>ful hua1 hua2 pep/+</i> pistils, and sepaloid giant cells were observed on the valve surface. Simple or branched trichomes (white and yellow arrows, respectively) were occasionally observed on the surface of <i>ful hua1 hua2 pep/+</i> pistils. B) SEM images of the abaxial ovary surface in wild-type (WT) and different mutant backgrounds. Observe interspersed stomata (arrows) in a <i>ful hua1 hua2 pep/+</i> panel. C-F) <i>GUS</i> reporter whole-mount staining (<i>ful-1</i>) in <i>ful</i> (C), <i>ful hua1 hua2</i> (D), <i>ful hua1 hua2 pep/+</i> (E) pistils and wild-type sepal (F). Observe long gynophores and full petaloid conversion of stamens in (E). Scale bars: 100 μm (A, B), except 10 μm in the last B panel (<i>ful hua1 hua2 pep/+</i> genotype), and 1 mm (C-F). r, replum; v, valve.</p

Loss of <i>FLK</i> dramatically enhances the floral phenotypes of <i>hua1 hua2</i> plants.

<p>A) <i>flk hua1 hua2</i> flower with all stamens converted into petals. B) Gynoecium with a long gynophore. A sepaloid valve was manually removed to better observe a new flower developing inside. C) SEM image of a sepaloid carpel with giant cells and epicuticular wax ridges. D) SEM magnification of the inner flower shown in (B). E) Close-up view of the sepaloid organ shown in (D). F, G) qPCR relative expression levels of <i>AG</i> mRNA (F), and <i>AG</i> transcripts including intron 2 sequences (G) in wild type (WT) and mutant backgrounds. Error bars, SD. Asterisks indicate statistically significant differences from <i>hua1 hua2</i> plants (**P < 0.01). Scale bars: 1 mm (A, B), 20 μm (C) 500 μm (D) and 50 μm (E).</p

Detection of the AP1-GFP protein in <i>hua1 pep</i> gynoecia.

<p>A) Apical region of a mildly affected gynoecium with recognizable pistil morphology. Specific AP1-GFP signal is detected in some style cells. B) Fourth whorl organs of a pre-anthesis flower displaying a severe sepaloid phenotype. C) Adaxial (inner) view of a manually open pistil with severe sepaloid transformations, but containing some developing ovules (do). D) Detail of a fourth whorl organ from panel D showing nuclear-localized AP1-GFP. A cell has been outlined with a dotted line and the nucleus marked with an arrow. Scale bars: 25 μm (A), 50 μm (B and C) and 10 μm (D).</p