Ectopic expression of BABY BOOM triggers a conversion from vegetative to embryonic growth

Abstract

The molecular mechanisms underlying the initiation and maintenance of the embryonic pathway in plants are largely unknown. To obtain more insight into these processes, we used subtractive hybridization to identify genes that are upregulated during the in vitro induction of embryo development from immature pollen grains of Brassica napus (microspore embryogenesis). One of the genes identified, BABY BOOM (BBM), shows similarity to the AP2/ERF family of transcription factors and is expressed preferentially in developing embryos and seeds. Ectopic expression of BBM in Arabidopsis and Brassica led to the spontaneous formation of somatic embryos and cotyledon-like structures on seedlings. Ectopic BBM expression induced additional pleiotropic phenotypes, including neoplastic growth, hormone-free regeneration of explants, and alterations in leaf and flower morphology. The expression pattern of BBM in developing seeds combined with the BBM overexpression phenotype suggests a role for this gene in promoting cell proliferation and morphogenesis during embryogenesis.

Figure 1.

Sequence Comparison of the BBM Proteins and Related AP2 Domain–Containing Proteins. The amino acid sequences of the first AP2/ERF domain repeat (Repeat 1), the second AP2/ERF domain repeat (Repeat 2), and the linker region lying between the two repeats (Linker) of the Brassica and Arabidopsis BBM proteins were aligned with the amino acid sequences of related AP2/ERF domain–containing proteins. The shaded boxes indicate the percentage of sequences at that position with the same amino acid identity (dark gray, 100%; medium gray, 80%; light gray, 60%). Protein names or accession numbers are indicated at the left. BnBBM1/2, Brassica BBM1 and BBM2 (the BnBBM1 and BnBBM2 protein sequences are identical in the AP2/ERF domain and linker regions); AtBBM, Arabidopsis BBM; ANT, Arabidopsis AINTEGUMENTA; ZMMHCF1, maize ZMMHCF1; GL15, maize Glossy15; AP2, Arabidopsis APETALA2. NP196613, NP20135, NP175530, NP188720, NP200549, and NP177401 are accession numbers for hypothetical Arabidopsis proteins.

Figure 2.

BBM Gene Expression in Brassica. (A) RNA gel blot analysis of BBM gene expression in Brassica microspore cultures. Total RNA was isolated from microspores at the start of culture (MIC 0d), after 4 days in culture at 32.5°C (EMB 4d), after 4 days in culture at 25°C (MIC 4d), after 1 day of culture at 25°C followed by 3 days of culture at 32.5°C (NE 4d), and from purified microspore-derived embryos at the globular (EMB 10d) and cotyledon (EMB 21d) stages of development. RNA samples (15 μg) were blotted and hybridized to a BBM1/BBM2-specific probe. Ethidium bromide staining of RNA was used to compare sample loading. EMB, embryogenic microspore culture; MIC, microspores and pollen; NE, nonembryogenic microspore culture. (B) RT-PCR analysis of BBM transcripts in developing seeds. RNA samples for RT-PCR analysis were isolated from whole seeds collected at specific days after pollination (DAP). These developmental time points correspond approximately to the globular (7 DAP), heart/torpedo (14 DAP), early cotyledon (21 DAP), mid cotyledon (28 DAP), and mid to late cotyledon (35 DAP) stages of development. RT-PCR analysis of torpedo-stage microspore-derived embryos was included as a positive control (CON). Ethidium bromide staining of RT-PCR–amplified CyP cyclophilin cDNA (CYC) from the same reverse-transcribed RNAs was used as a control for cDNA synthesis.

Figure 3.

In Situ Localization of BBM mRNA in Embryos and Seeds. Sections of Brassica microspore-derived embryos and Arabidopsis seeds were hybridized with antisense (AtBBM, [A] to [E]; BnBBM, [H] and [I]) or sense (AtBBM, [F] and [G]; BnBBM, [J]) digoxigenin-UTP–labeled probes. The transcript hybridization signal is purple-brown. (A) to (G) Longitudinal sections through developing Arabidopsis seeds. (H) Globular-stage microspore embryo (arrow) and undeveloped microspores from an 8-day-old Brassica microspore embryo culture. (I) and (J) Fourteen-day-old Brassica microspore-derived embryo culture. Bars = 25 μm in (A) and (F), 50 μm in (B) to (E) and (G), 100 μm in (H), 350 μm in (I), and 200 μm in (J).

Figure 4.

Ectopic Expression of BBM Induces Somatic Embryo Formation in Arabidopsis and Brassica. All images correspond to transgenic plants. (A) Somatic embryos (arrows) formed on the cotyledon margins of a 35S::BBM Arabidopsis (C24) seedling. (B) Scanning electron microscopy image of a 35S::BBM Arabidopsis (Columbia) seedling showing somatic embryos (arrows) on the cotyledons and leaves. Note the absence of trichomes on the somatic embryos. (C) Somatic embryos (arrows) formed on the petiole and shoot apex of a UBI::BBM Arabidopsis (C24) plant. (D) Formation of somatic embryos on the shoot apex and activation of cell division (arrow) along the cotyledon margin of a 35S::BBM Arabidopsis (C24) seedling. (E) Somatic embryos (arrow) formed on the leaf margin of a 35S::BBM Brassica plant. (F) Recurrent embryogenesis along the cotyledon margin and petiole of a 35S::BBM Arabidopsis (C24) seedling. The leaves of this seedling have been replaced by cotyledon-like organs (arrow). (G) Longitudinal semithin section through the point of attachment of a single 35S::BBM (C24) somatic embryo (se) to the underlying cotyledon (cot). (H) Longitudinal semithin section through one of the cotyledons and attached embryos of the seedling shown in (A). The somatic embryo attached to the seedling cotyledon (cot) is bipolar and contains all of the organs and tissue types seen in zygotic embryos. Secondary somatic embryos (arrows) initiate from the cotyledons of the primary somatic embryo. gp, ground parenchyma; prot, protoderm; pv, provascular tissue; rm, root meristem; sm, shoot meristem. (I) A 35S::BBM (C24) transgenic seedling showing recurrent embryogenesis from the shoot apex and callus formation on the root/hypocotyl. (J) Recurrent somatic embryogenesis from the shoot apex of a 35S::BBM (C24) seedling. (K) Recurrent embryogenesis and cell proliferation in a 35S::BBM Arabidopsis (C24) seedling. The photograph shows one of 10 such dishes obtained after 3 months of biweekly subculture of a single 35S::BBM seedling on minimal tissue culture medium. Bars = 1 mm in (A), (C), (D), (F), (I), and (J), 0.16 mm in (B), 20 mm in (E), 0.03 mm in (G), 0.2 mm in (H), and 5 mm in (K).

Figure 5.

Ectopic Overexpression of BBM Induces Pleiotropic Phenotypes in Arabidopsis. (A) UBI::BBM seedling (C24) showing lobed cotyledons and leaves, and increased shoot production at the apex. (B) 35S::BBM seedling (C24) showing ectopic shoots on the cotyledon edge and short roots. (C) Wild-type (C24) seedling. (D) Homozygous, single-locus 35S::BBM line (C24) showing the class I phenotype of rounded leaves and decreased size. (E) 35S::BBM (C24) plant with serrated leaves. (F) 35S::BBM plant (Columbia) with rumpled leaves. (G) Wild-type (C24) plants. (H) 35S::BBM (C24) flowers showing alterations in floral organ length. (I) Wild-type C24 flowers. Bars = 1 mm in (A), 1.5 mm in (B) and (C), 10 mm in (D), 8 mm in (E) and (F), 17 mm in (G), 0.05 mm in (H), and 1.5 mm in (I).

Figure 6.

Regenerative Capacity of Wild-Type and Transgenic UBI::BBM Arabidopsis Plants. Leaf and hypocotyl explants from wild-type C24 ([A] and [C]) and transgenic UBI::BBM seedlings ([B] and [D]) were placed successively on medium containing callus-inducing growth regulators followed by shoot-inducing growth regulators ([A] and [B]) or medium lacking growth regulators ([C] and [D]). UBI::BBM explants regenerate on hormone-free medium and exhibit enhanced regeneration in the presence of growth regulators.