The BABY BOOM Transcription Factor Activates the LEC1-ABI3-FUS3-LEC2 Network to Induce Somatic Embryogenesis

Abstract

Somatic embryogenesis is an example of induced cellular totipotency, where embryos develop from vegetative cells rather than from gamete fusion. Somatic embryogenesis can be induced in vitro by exposing explants to growth regulators and/or stress treatments. The BABY BOOM (BBM) and LEAFY COTYLEDON1 (LEC1) and LEC2 transcription factors are key regulators of plant cell totipotency, as ectopic overexpression of either transcription factor induces somatic embryo formation from Arabidopsis (Arabidopsis thaliana) seedlings without exogenous growth regulators or stress treatments. Although LEC and BBM proteins regulate the same developmental process, it is not known whether they function in the same molecular pathway. We show that BBM transcriptionally regulates LEC1 and LEC2, as well as the two other LAFL genes, FUSCA3 (FUS3) and ABSCISIC ACIDINSENSITIVE3 (ABI3). LEC2 and ABI3 quantitatively regulate BBM-mediated somatic embryogenesis, while FUS3 and LEC1 are essential for this process. BBM-mediated somatic embryogenesis is dose and context dependent, and the context-dependent phenotypes are associated with differential LAFL expression. We also uncover functional redundancy for somatic embryogenesis among other Arabidopsis BBM-like proteins and show that one of these proteins, PLETHORA2, also regulates LAFL gene expression. Our data place BBM upstream of other major regulators of plant embryo identity and totipotency.

Figure 1.

BBM binds and transcriptionally regulates LAFL genes. A, ChIP-seq BBM-binding profiles for embryo-expressed genes: 35S:BBM-GFP (top profiles) and BBM:BBM-YFP (bottom profiles). x axis, Nucleotide positions (TAIR 10 annotation; black bars indicate exons, and lines indicate introns); y axis, ChIP-seq score (fold enrichment of the BBM-GFP/YFP ChIP to the control ChIP), as calculated by the CSAR package in Bioconductor; <<, direction of gene transcription; *, peaks with scores that are considered statistically significant (false discovery rate < 0.05). B and C, Transcriptional regulation of LAFL genes. Relative expression was determined by quantitative real-time reverse transcription-PCR (qPCR) in 35S:BBM-GR and Columbia-0 (Col-0) seeds 1 d after seed plating. Samples were treated for 3 h with dexamethasone (DEX) and cycloheximide (CHX; both at 10 µm). B, Error bars indicate se values of the three biological replicates. Statistically significant differences (asterisks) between DEX+CHX-treated 35S:BBM-GR and DEX+CHX-treated Col-0 were determined using Student’s t test (P < 0.01). C, LEC1:LEC1-GFP regulation by BBM. Samples were treated with 10 µm DEX 1 d after plating and imaged on subsequent days as indicated. The images show the adaxial sides of cotyledons, unless indicated (ab, abaxial side). The green signal in Col-0 and LEC1:LEC1-GFP cotyledon tips is autofluorescence. Bars = 250 µm.

Figure 2.

BBM-induced embryogenesis is modulated by LAFL genes. The percentage of primary embryogenic transformants obtained is shown after transformation of the 35S:BBM-GR construct to the wild-type Wassilewskija (Ws) or Col-0 background or the indicated mutant lines. Statistically significant differences (asterisks) in the number of embryogenic transgenic lines between the mutant and the corresponding wild-type line were determined using Pearson’s χ² test (P < 0.05). The total number of transformants per line is indicated above each bar. Somatic embryo formation was not observed in any of the individual mutant backgrounds alone or in mutant + 35S:BBM-GR backgrounds in the absence of BBM-GR activation.

Figure 3.

BBM overexpression phenotypes are dose dependent. Phenotypes are shown for 35S:BBM-GR seedlings grown for 2 weeks (A–E and H) or 3 weeks (F and G) on the DEX concentration (µm) indicated in each image. A, A phenotypically normal seedling. B, A small seedling with epinastic leaves and cotyledons. C, A small, epinastic seedling with a trichome-bearing ectopic leaf (arrow) on the cotyledon petiole. D, A seedling with ectopic leaves on the petioles of both cotyledons (arrows). E, A magnified view of the ectopic leaf in D. F, A 35S:BBM-GR seedling with an ectopic root (asterisk). G, A magnified view of the ectopic root in F. H, A seedling with somatic embryos on the cotyledon margins (arrowheads). Bars = 2.5 mm.

Figure 4.

Quantification of BBM dose-dependent phenotypes. A, BBM-GFP-GR nuclear localization increases with increasing DEX concentration. The effect of DEX on BBM localization in roots of 35S:BBM-GFP-GR seedlings grown for 7 d in medium containing the indicated DEX concentration is shown. Non-DEX-treated (Col-0) roots are shown as a GFP-negative control. Subcellular BBM-GFP-GR localization was quantified for each DEX concentration by calculating the average nuclear-cytoplasmic GFP ratio (63–133 cells of five to eight roots per DEX concentration), which is indicated on the bottom of the images (±se). The average ratios are significantly different from each other (P < 0.01, Student’s t test). Green, GFP; magenta, propidium iodide. B, Frequency of phenotypes observed in 35S:BBM-GR seedlings from two independent transgenic lines (solid and hatched bars) grown for 2 weeks on medium containing different DEX concentrations (n = 100–350 seedlings). Leaf, Ectopic leaves; SE, somatic embryos. Seedlings that showed both ectopic shoots and somatic embryos were scored as SE. SE refers to both embryogenic tissue (smooth, swollen, bright green in color, and lacking trichomes) and cotyledons as well as histodifferentiated embryos. C, A relatively low BBM dose induces smaller and less-lobed leaf pavement cells compared with the control. The abaxial sides of cleared first leaves of 9-d-old 35:BBM-GR seedlings grown on medium without DEX (left) or with 0.1 μm DEX (right) are shown. Bars = 25 μm. D, Stomatal differentiation in DEX-treated 35:BBM-GR seedlings is reduced compared with untreated seedlings. In DEX-treated 35S::BBM-GR seedlings, fewer cells are committed to stomatal development, as reflected by the lower stomatal lineage index (SLI). Also, fewer mature stomata were found in leaves of DEX-treated seedlings (stomatal index [SI]), while the number of stomatal meristemoids was increased (meristemoid index [MI]). Error bars indicate se. Asterisks indicate statistically significant differences compared with the non-DEX-treated control (*, P < 0.05, Student’s t test).

Figure 5.

BBM-induced embryogenesis is context dependent. A, 35S:BBM-GR plants were cultured with 10 µm DEX starting at the dry seed stage (d0) or after germination (d4). The culture time after DEX application is indicated on the bottom right of each image. Arrowheads, Callus; arrows, somatic embryos/embryogenic tissue; le, callused leaf tissue. The bottom image is a magnification of the boxed region in the d4 +14 image. B, LEC1:LEC1-GFP regulation by BBM. Seedlings were treated with 10 µm DEX after germination (d4) and imaged on subsequent days as indicated. The images show the adaxial sides of cotyledons. Arrowhead, Callus on the distal end of the cotyledon blade; arrows, GFP-positive embryo clusters. The outline of the cotyledon margins is shown with dashed lines. Autofluorescence (magenta) was used to delineate the tissue. Bars = 250 μm.