A Regulatory Network for Coordinated Flower Maturation

For self-pollinating plants to reproduce, male and female organ development must be coordinated as flowers mature. The Arabidopsis transcription factors AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 regulate this complex process by promoting petal expansion, stamen filament elongation, anther dehiscence, and gynoecium maturation, thereby ensuring that pollen released from the anthers is deposited on the stigma of a receptive gynoecium. ARF6 and ARF8 induce jasmonate production, which in turn triggers expression of MYB21 and MYB24, encoding R2R3 MYB transcription factors that promote petal and stamen growth. To understand the dynamics of this flower maturation regulatory network, we have characterized morphological, chemical, and global gene expression phenotypes of arf, myb, and jasmonate pathway mutant flowers. We found that MYB21 and MYB24 promoted not only petal and stamen development but also gynoecium growth. As well as regulating reproductive competence, both the ARF and MYB factors promoted nectary development or function and volatile sesquiterpene production, which may attract insect pollinators and/or repel pathogens. Mutants lacking jasmonate synthesis or response had decreased MYB21 expression and stamen and petal growth at the stage when flowers normally open, but had increased MYB21 expression in petals of older flowers, resulting in renewed and persistent petal expansion at later stages. Both auxin response and jasmonate synthesis promoted positive feedbacks that may ensure rapid petal and stamen growth as flowers open. MYB21 also fed back negatively on expression of jasmonate biosynthesis pathway genes to decrease flower jasmonate level, which correlated with termination of growth after flowers have opened. These dynamic feedbacks may promote timely, coordinated, and transient growth of flower organs

Auxin response factors ARF6 and ARF8 promote jasmonic acid production and flower maturation

Pollination in flowering plants requires that anthers release pollen when the gynoecium is competent to support fertilization. We show that i

A Regulatory Network for Coordinated Flower Maturation

For self-pollinating plants to reproduce, male and female organ development must be coordinated as flowers mature. The Arabidopsis transcription factors AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 regulate this complex process by promoting petal expansion, stamen filament elongation, anther dehiscence, and gynoecium maturation, thereby ensuring that pollen released from the anthers is deposited on the stigma of a receptive gynoecium. ARF6 and ARF8 induce jasmonate production, which in turn triggers expression of MYB21 and MYB24, encoding R2R3 MYB transcription factors that promote petal and stamen growth. To understand the dynamics of this flower maturation regulatory network, we have characterized morphological, chemical, and global gene expression phenotypes of arf, myb, and jasmonate pathway mutant flowers. We found that MYB21 and MYB24 promoted not only petal and stamen development but also gynoecium growth. As well as regulating reproductive competence, both the ARF and MYB factors promoted nectary development or function and volatile sesquiterpene production, which may attract insect pollinators and/or repel pathogens. Mutants lacking jasmonate synthesis or response had decreased MYB21 expression and stamen and petal growth at the stage when flowers normally open, but had increased MYB21 expression in petals of older flowers, resulting in renewed and persistent petal expansion at later stages. Both auxin response and jasmonate synthesis promoted positive feedbacks that may ensure rapid petal and stamen growth as flowers open. MYB21 also fed back negatively on expression of jasmonate biosynthesis pathway genes to decrease flower jasmonate level, which correlated with termination of growth after flowers have opened. These dynamic feedbacks may promote timely, coordinated, and transient growth of flower organs

A gain-of-function mutation in IAA18 alters Arabidopsis embryonic apical patterning

Lateral organ emergence in plant embryos and meristems depends on spatially coordinated auxin transport and auxin response. Here, we report the gain-of-function iaa18-1 mutation in Arabidopsis, which stabilizes the Aux/IAA protein IAA18 and causes aberrant cotyledon placement in embryos. IAA18 was expressed in the apical domain of globular stage embryos, and in the shoot apical meristem and adaxial domain of cotyledons of heart stage embryos. Mutant globular embryos had asymmetric PIN1:GFP expression in the apical domain, indicating that IAA18-1 disrupts auxin transport. Genetic interactions among iaa18-1, loss-of-function mutations in ARF (Auxin response factor) genes and ARF-overexpressing constructs suggest that IAA18-1 inhibits activity of MP/ARF5 and other ARF proteins in the apical domain. The iaa18-1 mutation also increased the frequency of rootless seedlings in mutant backgrounds in which auxin regulation of basal pole development was affected. These results indicate that apical patterning requires Aux/IAA protein turnover, and that apical domain auxin response also influences root formation

FIDDLEHEAD, a gene required to suppress epidermal cell interactions in Arabidopsis, encodes a putative lipid biosynthetic enzyme

In plants, the outer epidermal cell wall and cuticle presents a semipermeable barrier that maintains the external integrity of the plant and regulates the passage of various classes of molecules into and out of the organism. During vegetative development, the epidermal cells remain relatively inert, failing to respond to wounding or grafting. During reproductive development and fertilization, however, the epidermis is developmentally more labile and participates in two types of contact-mediated cell interactions: organ fusion and pollen hydration. Here we describe the isolation and characterization of one gene whose product normally functions in blocking both types of epidermal cell interactions during vegetative development: the FIDDLEHEAD gene. As suggested by previous biochemical analyses, the gene encodes a protein that is probably involved in the synthesis of long-chain lipids found in the cuticle and shows similarity to a large class of genes encoding proteins related to β-ketoacyl-CoA synthases and chalcone synthases. In situ hybridization reveals an epidermal pattern of expression consistent with a role for this protein in the synthesis of lipid components that are thought to localize extracellularly and probably modify the properties of the cuticle

Gene expression and jasmonate production in wild-type and mutant flowers.

<p>(A–B) RNA gel blot hybridization using <i>MYB21</i>, <i>MYB24</i>, <i>MYB57</i> and <i>MYB108</i> probes. (A) RNA from wild-type, <i>arf6-2 arf8-3/ARF8</i> and <i>arf6-2 arf8-3</i> inflorescences (left panel), and wild-type stage 1–10, stage 11–12 and stage 13–14 flowers (right panel). (B) RNA from untreated (left panel) or MeJA treated (right panel) wild-type, <i>arf6-2 arf8-3</i>, <i>aos-2</i> and <i>coi1-1</i> inflorescences. (C) RNA gel blot hybridization using <i>ARF6</i>, <i>TPS11</i>, <i>TPS21</i>, <i>MYB108</i> and <i>SAUR63</i> probes. RNA from wild-type, <i>arf6-2 arf8-3</i> and <i>myb21-5 myb24-5</i> inflorescences. (D) RNA gel blot hybridization using <i>SAUR63</i>, <i>IAA2</i>, <i>IAA3</i>, <i>IAA4</i>, <i>IAA7</i>, <i>IAA13</i>, <i>IAA16</i> and <i>IAA19</i> probes. RNA from wild-type, <i>arf6-2 arf8-3</i> and <i>myb21-5 myb24-5</i> stage 12-13 flowers. (E) RNA gel blot hybridization using <i>LOX2</i>, <i>DAD1</i>, and <i>AOS</i> probes. PolyA⁺ RNA from wild-type, <i>arf6-2 arf8-3</i> and <i>myb21-5 myb24-5</i> stage 12–13 flowers. In A–E, numbers beneath each band indicate measured signal level relative to the <i>β-TUBULIN</i> control. (F) cis-JA concentrations in wild-type, <i>arf6-2 arf8-3</i>, <i>myb21-5 myb24-5</i>, and <i>aos-2</i> stage 12-13 flowers. Data are means of two measurements ± SD. n.d., not detected.</p

Global analyses of gene expression in <i>arf6-2 arf8-3</i> and <i>myb21-5 myb24-5</i> stage 12 flowers.

<p>Venn diagram indicates numbers of genes with higher or lower expression in mutant compared to wild-type flowers, based on a t-test (P<0.05) and a two-fold ratio of expression values. Pie charts indicate the proportion of genes in each expression class having highest expression in sepals, petals, stamens, or carpels of wild-type stage 12 flowers <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002506#pgen.1002506-Schmid1" target="_blank">[21]</a>. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002506#pgen.1002506.s010" target="_blank">Table S3</a> lists these genes and provides details of their expression levels.</p

Inflorescence apices and flower phenotypes of <i>myb21</i>, <i>myb24</i>, <i>myb108</i>, and <i>aos</i> mutants.

<p>(A–H) Photographs of inflorescences (left panels) and individual flowers (right panels) of indicated genotypes. Asterisks indicate the position of the first open flower (stage 13) in the inflorescences shown, or the corresponding flower based upon bud size and position compared to a wild-type inflorescence. Individual flowers shown in the right panels are the first open flower (stage 13, A–F) or the fourth open flower (stage 15, G–H). Some sepals and petals have been removed to show inner organs. Scale bar: left panels, 3 mm, right panels, 1 mm. (I, J) Scatter plots showing petal and stamen lengths relative to gynoecium length of individual flowers of indicated genotypes. In I, data from a single experiment are shown. In J, measurements from two experiments were combined. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002506#pgen.1002506.s002" target="_blank">Figure S2</a> shows similar data for additional genotypes.</p

Expression of <i>MYB21</i>, <i>MYB24</i>, and jasmonate pathway genes in wild-type and mutant flowers at stages 12, 13, and 14.

<p>Gene expression was measured by quantitative RT-PCR. Shown are means of two biological replicates each having three technical replicates (± SD). Within each biological replicate, expression levels were normalized to expression in wild-type stage 12 flowers.</p

Expression patterns of <i>MYB21</i> and <i>MYB24</i>.

<p>(A–C) <i>In situ</i> hybridization with a <i>MYB21</i> antisense probe in stage 12 wild-type gynoecia (A,B) or stamen filament (C). (D,E) <i>In situ</i> hybridization with a <i>MYB24</i> antisense probe in stage 12 wild-type nectary (D) and stament filament (E). (F) <i>MYB21 in situ</i> hybridization in a wild-type ovule. (G) <i>MYB21 in situ</i> hybridization in a <i>mARF6</i> ovule. (J–O) X-Gluc staining of <i>P_MYB21:MYB21:GUS</i> flowers. (J) Stage 13 wild-type whole flower. (K) Gynoecium showing ovule funiculi. (L) Gynoecium base showing nectary. (M–O) <i>aos-2 P_MYB21:MYB21:GUS</i> flowers at stage 13 (M), (N) MeJA-treated stage 13, (O) Stage 15 untreated.</p