A regulatory network for coordinated flower maturation

Abstract

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.

Figure 1. Gene expression and jasmonate production in wild-type and mutant flowers.

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

Figure 2. Expression patterns of MYB21 and MYB24.

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

Figure 3. Inflorescence apices and flower phenotypes of myb21, myb24, myb108, and aos mutants.

(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. Figure S2 shows similar data for additional genotypes.

Figure 4. Expression of MYB21, MYB24, and jasmonate pathway genes in wild-type and mutant flowers at stages 12, 13, and 14.

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.

Figure 5. Global analyses of gene expression in arf6-2 arf8-3 and myb21-5 myb24-5 stage 12 flowers.

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 . Table S3 lists these genes and provides details of their expression levels.

Figure 6. Phenotypes related to insect attraction.

(A–D) Base of gynoecia of indicated genotypes. Arrows indicate nectaries. Scale bar, 0.1 mm. (E) Comparative quantitative analyses of floral volatile sesquiterpene emissions from wild-type, myb21-5, myb24-5, and myb21-5 myb24-5 mutants. Emitted compounds were collected for 7 h from 40 detached inflorescences by a closed-loop stripping procedure. Emission was determined in ng h⁻¹ per 40 inflorescences. Values are averages and standard deviations of three independent collections. Only emissions of (E)-β-caryophyllene, the product of TPS21, and thujopsene, the product of TPS11, are shown. Different letters indicate significant differences in emissions of each compound between genotypes ( p≤0.001). (F) GC-MS analyses of sesquiterpene hydrocarbons collected via SPME from 20 inflorescences of wild-type, myb21-5 and arf6-2 arf8-3 mutants. Peaks marked with circles represent sesquiterpenes produced by the terpene synthase TPS21. Compounds not labeled with circles are products of the terpene synthase TPS11, with the exception of α-farnesene (α-farn). 1, (E)-β-caryophyllene; 2, thujopsene; 3, α-humulene; 4, β-chamigrene. Peaks marked with asterisks are other sesquiterpene products of TPS11 or TPS21 .

Figure 7. Genetic model of Arabidopsis flower maturation.

(A) Diagram of principal regulatory pathways. Arrows indicate regulatory events established in this work or by previous studies. Both gibberellins and ARF6 and ARF8 auxin response factors promote jasmonate biosynthesis at flower stage 12. Auxin presumably enables ARF activity, and this may also be regulated by the circadian rhythm. Jasmonates in turn activate expression of genes for jasmonate biosynthesis, in a positive feedback loop requiring the JA-Ile receptor COI1. The underexpression of potential direct ARF6- and ARF8-targets in myb21 myb24 flowers suggests that MYB21 and MYB24 may also participate in an additional positive feedback loop that promotes ARF6 and ARF8 activity, possibly through effects on auxin level (shown as dashed arrows). MYB21 represses jasmonate biosynthesis, and after the flower has opened (stage 13 and later), this negative feedback arrests flower maturation functions. In the absence of jasmonate signaling, ARF6 and ARF8 also contribute to MYB21 expression in late-stage petals. (B) Illustration of flower developmental events regulated by the network between flower stage 12 (left) and stage 13 (right). The network induces downstream effectors that promote multiple events including petal and stamen filament elongation (regulated by ARF16 and by SAUR proteins), anther dehiscence (regulated by MYB108), volatile compound production (by TPS11 and TPS21 terpene synthases), nectary growth and development (regulated by CRC), and gynoecium growth and maturation. These and other effector genes may be activated directly or indirectly by MYB21 and MYB24, or by ARF6 and ARF8 independently of the MYB proteins. N, nectary.