The ZmBBM2-BBIP1-RCC1-4 module enhances callus growth and genetic transformation efficiency in maize

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BABY BOOM (BBM) encodes an AP2 transcription factor belonging to the AINTEGUMENTA-LIKE (AIL) subfamily and is widely recognized as a master regulator of embryogenesis (Boutilier et al. 2002; Deng et al. 2009; El Ouakfaoui et al. 2010). In maize, ectopic expression of ZmBBM markedly improves genetic transformation efficiency across multiple inbred lines (Lowe et al. 2016). Similarly, overexpression of another maize BBM homolog, ZmBBM2, promotes rapid callus proliferation and significantly enhances transformation efficiency in elite germplasm (Du et al. 2019).

At the molecular level, BBM acts as a central transcriptional hub that initiates embryogenic programs through extensive downstream gene regulation. BBM and other AINTEGUMENTA-LIKE (AIL) proteins interact with L1-expressed HOMEODOMAIN GLABROUS (HDG) transcription factors and co-regulate key embryogenesis-related targets (Horstman et al. 2015), while BBM also directly activates the core embryonic network genes LEC1, LEC2, FUS3, and ABI3 (Horstman et al. 2017). Genome-wide ChIP analyses further identified chromatin-associated factors such as AHL15/19/20 as direct BBM targets (Karami et al. 2021). In parallel, BBM-mediated somatic embryogenesis is tightly linked to auxin biosynthesis, as BBM activates YUCCA family genes in both Arabidopsis and rice, thereby elevating endogenous auxin levels and promoting embryogenic reprogramming (Li et al. 2022; Khanday et al. 2023). Despite these advances, the molecular mechanisms by which BBM proteins orchestrate embryogenic reprogramming in maize remain poorly understood, particularly with respect to their interacting protein partners and the downstream regulatory modules that mediate BBM-driven transcriptional programs.

In this study, we integrate ChIP-seq, protein–protein interaction, and functional genetic analyses to identify a BBM-centered regulatory module that directly connects transcriptional control to somatic embryogenesis and callus growth. ZmBBM2 exhibits high expression in embryo, with its protein localized to the nucleus (Figure S1). To elucidate the gene regulatory network underlying ZmBBM2-mediated embryogenic development, we performed ChIP-seq using transgenic maize calli overexpressing ZmBBM2 (Figure S2). Across two biological replicates, 88,741 overlapping peaks were identified and considered high-confidence ZmBBM2-binding sites. These binding events were distributed across multiple genomic regions, with a substantial proportion located in intergenic regions (Fig. 1a). Motif analysis using the MEME suite identified a novel ZmBBM2-binding E-box motif (CACGT) (Fig. 1b). We further performed a genome-wide analysis to profile the distribution of this motif across all ZmBBM2 ChIP-seq peaks. Among the total 27,145 binding peaks, 62.24% (16,895) contained at least 1 CACGT motif with a score >5 and P < 0.05. To identify putative direct targets of ZmBBM2, we focused on ChIP-seq peaks located within 2 kb upstream of transcription start sites, yielding 27,146 annotated genes as candidate targets. Gene Ontology (GO) enrichment analysis revealed significant enrichment in molecular functions related to DNA binding and transmembrane transporter activity, as well as biological processes including DNA conformation change, membrane-bound organelle organization, regulation of nucleic acid–templated transcription, and RNA biosynthetic processes (Fig. 1c). To identify target genes, we further performed transcriptome analysis using ZmBBM2 calli, and 4,163 and 5,673 genes were significantly upregulated and downregulated, respectively (Data S1). Based on GO terms, KEGG pathway, gene annotations, and literature evidence, we retrieved 40 candidate downstream genes associated with callus formation and cell proliferation, including RCC1-4, RMV1, LRR-RLK, and KRP3 (Table S1). Direct binding of ZmBBM2 to the promoter regions of RMV1, LRR-RLK, and KRP3 was confirmed by electrophoretic mobility shift assays (EMSAs) (Figure S3).

Figure 1.

ZmBBM2-BBIP1-RCC1-4 module enhances callus growth and transformation efficiency in maize. a) Genome-wide identification of ZmBBM2-binding regions in maize by ChIP-seq. b) Motif enrichment analysis of ZmBBM2-bound regions performed using HOMER, revealing core cis-elements associated with ZmBBM2 binding. c) GO enrichment analysis of differentially expressed genes (DEGs) in ZmBBM2-positive callus tissues. d) ChIP–qPCR validation of ZmBBM2 binding to the ZmRCC1-4 promoter in vivo. Callus tissues from 3×Flag and ZmBBM2–3×Flag transgenic lines were used. Protein–chromatin complexes were immunoprecipitated with anti-Flag antibodies. Error bars represent the standard deviation of three biological replicates. Data are presented as mean ± SD (n = 6). Asterisks indicate statistically significant differences (t-test, P < 0.001). e) Y1H assay demonstrating the binding of ZmBBM2 to the ZmRCC1-4 promoter. f) EMSA showing direct binding of ZmBBM2 to the ZmRCC1-4 promoter. The black arrow indicates the shifted DNA–protein complex. g) Dual-luciferase reporter assay showing relative LUC activity driven by the ZmRCC1-4 promoter in Nicotiana benthamiana leaves and maize protoplasts. Data are presented as mean ± SD (n = 3). Different letters indicate statistically significant differences. Schematic representation shows 35S-driven ZmBBM2 as the effector and the ZmRCC1-4 promoter (−1 to −2,000 bp) fused to the LUC reporter gene. h) Subcellular localization of ZmRCC1-4 in the nucleus. YFP, yellow fluorescent protein; RFP, red fluorescent protein. Scale bar = 50 μm. i) Y2H assay showing the interaction between ZmBBM2 and BBIP1. Positive controls were pGADT7-T (AD-T) and pGBKT7-53 (BD-53); negative controls were pAD-T and pGBKT7-Lam (BD-Lam). Y2H Gold strains carrying the indicated constructs were grown on SD−Trp−Leu (DDO) and SD−Trp−Leu−His−Ade (QDO) media. j) Pull-down assay confirming the interaction between ZmBBM2 and BBIP1. Recombinant proteins were immunoprecipitated using MBP beads and detected by immunoblotting with anti-His antibodies. k) BiFC assay showing the interaction between ZmBBM2 and BBIP1. Scale bar = 50 μm. l) Representative callus phenotypes obtained after two rounds of glufosinate-ammonium selection in maize transformed with the empty vector (control) or ZmRCC1-4 overexpression construct. Scale bar = 1 cm. m) Quantification of transformation efficiency. Data are presented as mean ± SD (n = 3). Asterisks indicate statistically significant differences (t-test, P < 0.01). n) Overexpression of ZmRCC1-4 increases the frequency of callus formation from maize embryos. Data are presented as mean ± SD. Asterisks indicate statistically significant differences (t-test, P < 0.01). o) Proposed working model of the ZmBBM2-BBIP1-ZmRCC1-4 regulatory module during maize embryogenic reprogramming.

Among the identified targets, ZmRCC1-4 (Zm00001eb367810), encoding a Regulator of Chromatin Condensation 1 (RCC1) family protein, emerged as a prominent ZmBBM2 target (Figure S3g). RCC1 controls RanGTP gradient formation; its overexpression increases RanGTP, which in turn impacts the cell cycle and DNA damage response (Bischoff and Ponstingl 1991; Cekan et al. 2016). ChIP–quantitative PCR (qPCR) analysis demonstrated that ZmBBM2 specifically binds to a promoter region of ZmRCC1-4 containing the CACGT motif, but not to a region lacking this motif (Fig. 1d). Yeast one-hybrid (Y1H) and EMSA assays further confirmed direct binding of ZmBBM2 to this promoter region in vitro (Fig. 1e and 1f). Transient expression assays in Nicotiana benthamiana leaves and maize protoplast showed that ZmBBM2 significantly activated the pRCC1-4:LUC reporter (Fig. 1g). Consistent with the known function of RCC1 as a guanine nucleotide exchange factor for Ran, confocal microscopy revealed nuclear localization of ZmRCC1-4–YFP fusion protein (Fig. 1h).

To identify proteins interacting with ZmBBM2, we performed yeast two-hybrid (Y2H) screening using a maize cDNA library. This analysis yielded several candidate interactors (Table S2), including a 14-3-3 protein that we designated BBIP1 (Baby Boom–Interacting Protein 1). Targeted Y2H assays confirmed the interaction between ZmBBM2 and BBIP1 (Fig. 1i), which was further validated by in vitro pull-down assays (Fig. 1j) and in vivo bimolecular fluorescence complementation (BiFC) analysis (Fig. 1k). The BiFC analysis revealed that the interaction between BBIP1 and ZmBBM2 occurred within the nucleus, consistent with the localization of ZmBBM2 in the nucleus. Notably, dual-luciferase reporter assays demonstrated that co-expression of ZmBBM2 and BBIP1 in Nicotiana benthamiana leaves and maize protoplast significantly enhanced pRCC1-4::LUC activity compared with ZmBBM2 alone (Fig. 1g), indicating a synergistic effect of this interaction on downstream gene activation.

To evaluate the functional role of ZmRCC1-4 in maize transformation, a pUBI::ZmRCC1-4 vector was constructed, in which ZmRCC1-4 is driven by the maize ubiquitin promoter. All the transformation experiments were carried out using the maize CAL inbred line. Transformation efficiency with pUBI::ZmRCC1-4 vector was significantly increased to 7.8%, compared with the control vector (2.1%) (Fig. 1l and 1m). T₁ plants overexpressing ZmRCC1-4 were grown, which did not show any developmental abnormalities, and immature kernels from T₂ ears were utilized for callus induction. PCR-positive calli overexpressing ZmRCC1-4 exhibited a significantly higher growth rate compared to nontransgenic calli over a 2-wk period (Fig. 1n).

In summary, our study extends the current understanding of BBM function beyond its established role as a general inducer of somatic embryogenesis. We demonstrate that ZmBBM2 directly engages a chromatin-associated regulatory pathway by transcriptionally activating ZmRCC1-4. Specifically, as a Guanine Nucleotide Exchange Factor for Ran, ZmRCC1-4 is expected to generate a local RanGTP gradient on chromatin, thereby influencing chromatin condensation and mitotic progression, which provides a possible mechanistic link between the transcriptional activation of ZmRCC1-4 and the chromatin structure. Importantly, this transcriptional activation is potentiated through the physical interaction between ZmBBM2 and the 14-3-3 protein BBIP1, revealing an additional regulatory layer that fine tunes BBM activity. Functional analyses further establish ZmRCC1-4 as a downstream effector capable of independently enhancing callus proliferation and genetic transformation efficiency in maize. Together, these findings define a BBM-centered transcription factor–protein partner module that explains how embryogenic transcriptional programs drive callus growth, providing a mechanistic basis for improved maize regeneration and transformation efficiency (Fig. 1o).

Author contributions

Y.L. and Y.Y. designed the project and wrote the manuscript. Q.F., Q.W., and C.Q. performed the experiments. A.Z. and X.Y. analyzed the data.

Supplementary material

Supplementary material is available at Plant Physiology online.

Funding

This work was supported by Biological Breeding-National Science and Technology Major Project (2024ZD04077), the National Natural Sciences Foundation of China (82325049), the Key Project at Central Government Level: the Ability Establishment of Sustainable Use for Valuable Chinese Medicine Resources (2060302), and the Agricultural Science and Technology Innovation Program of CAAS.

Data availability

The data supporting the fndings of this study are available within the article and/or its supplementary materials.