In rice, ectopic expression of the BABY BOOM1 transcription factor (OsBBM1) induces somatic embryogenesis without exogenous hormones by directly activating YUCCA (OsYUC) auxin biosynthesis genes. The preprint that established this module (Khanday et al. 2020) and its published version (New Phytologist, 2023) left several mechanistic questions open. The originally posed question of conservation between somatic and zygotic BBM-YUCCA pathways has been substantially resolved (the same OsBBM-OsYUC module operates in both, with zygotic OsYUC targets expressed only from the maternal genome), but a coherent open core remains: the role of the LAFL (LEC1-LEC2-FUS3-ABI3) embryonic network in rice somatic embryogenesis, the mechanism by which OsBBM1 represses cytokinin response genes and how cytokinin supplementation overcomes the shoot-regeneration block, the mechanistic basis for why endogenous OsYUC-mediated auxin biosynthesis cannot be replaced by exogenous auxin, and whether OsBBM2/OsBBM3 use the same auxin-biosynthesis-based mechanism. These questions jointly ask for the downstream regulatory architecture through which a single embryogenic trigger executes embryo initiation.

Background

Somatic embryogenesis (SE) is the process by which differentiated vegetative (somatic) plant cells are reprogrammed to form embryos without fertilization; it underpins clonal propagation and is the regeneration step required for genetic transformation. Many agronomically important cereal crops are recalcitrant to SE and transformation except from embryonic tissues. BABY BOOM (BBM) proteins are members of the AP2/ERF family of transcription factors (defined by the APETALA2/ETHYLENE RESPONSE FACTOR DNA-binding domain), originally identified in Brassica napus microspore-derived embryos (embryos formed in culture from microspores, the immature precursor cells of pollen grains); ectopic BBM expression is sufficient to induce SE, including in cereals. In rice, OsBBM1 is expressed in the male gamete and delivered to the zygote at fertilization, where it acts as an embryogenic trigger; its ectopic expression in somatic tissues induces SE without exogenous auxin. The mechanistic basis was established by Khanday et al. (2020, published 2023): transient OsBBM1 induction directly upregulates auxin biosynthesis genes OsYUCCA6, OsYUCCA7 and OsYUCCA9. YUCCA (YUC) proteins are flavin monooxygenases that catalyze a rate-limiting, tryptophan-dependent step (the indole-3-pyruvic acid pathway) in producing indole-3-acetic acid (IAA), the natural plant auxin. Genetic analysis showed that OsYUC6/7/9 are required for normal rice development and that yuc triple mutants are strongly compromised in SE even when exogenous auxin is supplied; conversely, SE induction by exogenous auxin itself requires functional BBM genes. The published version added that the OsBBM1-direct OsYUC targets are the same zygote-expressed genes that act in zygotic embryogenesis, that in the zygote these OsYUC genes are expressed only from the maternal genome while the functional OsBBM1 is provided by the paternal genome, and that exogenous auxin acts upstream by inducing OsBBM1 expression, which then activates endogenous OsYUC genes. In the eudicot Arabidopsis, BBM similarly regulates YUC3 and YUC8 during SE (Li et al. 2021), and BBM activates the LEC1-ABI3-FUS3-LEC2 (LAFL) seed-regulatory network (Horstman et al. 2017), but with only partial YUC requirement, in contrast to the near-complete block in rice yuc mutants. Several mechanistic issues left open by the rice study remain unaddressed. The LAFL network comprises LEAFY COTYLEDON1 (LEC1, an NF-YB/HAP3 transcription factor), LEAFY COTYLEDON2 (LEC2) and FUSCA3 (FUS3) (B3-domain transcription factors), and ABA INSENSITIVE3 (ABI3); in Arabidopsis these govern embryo identity and seed maturation and are direct targets of BBM in somatic embryos, but their role in rice SE is undetermined: no detectable expression of OsLEC1A, OsLEC1B or OsLEC2-related genes was observed in leaf samples 6 h after OsBBM1 induction, whereas OsLEC1A and OsLEC1B are expressed in somatic embryos established subsequent to OsBBM1 induction; promoter occupancy by OsBBM1 was assayed only at OsYUC6/OsYUC7/OsYUC9, so direct regulation of LAFL genes at other stages or in other tissues has not been tested, and the source offers only the hypothesis that LEC genes may matter during later stages rather than at initiation. Cytokinin signaling in rice proceeds through two-component signaling whose negative-feedback type-A response regulators (OsRR genes, e.g. OsRR4, OsRR9/10) mark cytokinin responsiveness; OsBBM1 induction downregulates these genes, which may partially account for the requirement of cytokinin to complete shoot formation in OsBBM1-induced somatic embryos. Their downregulation is cycloheximide-insensitive (the OsRR genes fall in the source's set of genes "directly regulated by OsBBM1-GR" in the DEX+CYC treatment), and TOPLESS-related (TPR) corepressor-mediated repression is hypothesized by analogy with Arabidopsis BBM, but no promoter-occupancy or corepressor experiment has been reported; how OsBBM1 represses them and how cytokinin supplementation overcomes the shoot-regeneration block therefore remains unknown. Zygote-electrofusion experiments with bbms triple mutants (disrupting OsBBM1, OsBBM2, OsBBM3) showed that all three paralogs act paternally with overlapping effects on chromatin- and cell-cycle-related genes in early zygotic development (Akter et al. 2025), but whether OsBBM2/OsBBM3 trigger SE through the same OsYUC activation as OsBBM1 was not tested; the source infers their involvement only presumptively, and frames extension of the pathway to other cereals as a prospective application rather than as a posed mechanistic question, even as BBM genes are deployed as morphogenic transgenes (transgenes expressing developmental-regulator genes to confer or enhance regeneration capacity) across crops.

Problem Statement

Building on the established OsBBM1-OsYUC auxin biosynthesis module in rice, determine the remaining downstream regulatory architecture of OsBBM1-mediated somatic embryogenesis. Specifically: (1) define the role of the LAFL network (LEC1A, LEC1B and related LEC2/FUS3/ABI3-family genes) in rice somatic embryogenesis, including whether these genes act downstream of OsBBM1 and are required for somatic embryo initiation or for later stages and maturation, given that no detectable transcript induction of OsLEC1A/OsLEC1B or OsLEC2-related genes was observed in leaf tissue 6 h after OsBBM1 induction while OsLEC1A/OsLEC1B are expressed in established OsBBM1-induced somatic embryos, and whether they are directly regulated by OsBBM1 at stages or in tissues not yet assayed; (2) identify the mechanism by which OsBBM1 represses cytokinin response genes such as OsRR4, OsRR9 and OsRR10, whose downregulation is cycloheximide-insensitive and for which TOPLESS-related corepressor-mediated repression is hypothesized but untested by promoter occupancy, determining whether repression involves direct promoter occupancy and corepressor recruitment or indirect action, and explain how cytokinin supplementation overcomes the shoot-regeneration block in OsBBM1-induced tissue; (3) establish the mechanistic basis for why endogenous auxin biosynthesis through OsYUC6, OsYUC7 and OsYUC9 is required for somatic embryogenesis and cannot be substituted by application of exogenous auxin; and (4) determine whether OsBBM2 and OsBBM3 induce somatic embryogenesis through the same direct OsYUC-activation mechanism as OsBBM1, which the source infers only presumptively and did not test. All four sub-questions are jointly required, and each is resolved only when every claim it contains is settled; a partial answer resolves none of them. The original question of whether BBM-YUCCA pathways are conserved between somatic and zygotic embryogenesis is excluded as substantially resolved by the published 2023 version, which demonstrated the shared OsBBM-OsYUC module and the maternal-genome expression of zygotic OsYUC targets.

References

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