BABY BOOM Genes Research Articles

Cloning, Characterization, and Expression Pattern Analysis of the BBM Gene in Tree Peony (Paeonia ostii)

BABY BOOM (BBM) is one of the members of the plant-specific APETALA2/ethylene-responsive factor (AP2/ERF) transcription factor superfamily. It acts as a key regulator of plant cell pluripotency, playing a significant role in promoting somatic embryogenesis. In this study, a BBM gene named PoBBM was screened, cloned, and identified from the third-generation full-length transcriptome data of Paeonia ostii. Its open reading frame was 2136 bp, encoding 711 amino acids. Sequence feature analysis revealed that it possessed two AP2 conserved domains and eight motifs, including bbm-1. The phylogenetic tree indicated that PoBBM clusters with AtBBM in the euANT group of the Arabidopsis AP2 family, which is most closely related to grape VvBBM and may have the same ancestry as grape. Subcellular localization demonstrated that the PoBBM protein was localized in the nucleus. Semi-quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to assess the PoBBM transcript levels during ten developmental stages of somatic embryos and in five tissue types of peonies. The results indicate that PoBBM was highly expressed in the early stages of peony somatic embryo development. The expression on 0–15 d was the highest and decreased gradually with somatic embryogenesis. The gene is almost not expressed after 40 d since somatic embryo formation. PoBBM was expressed in roots, stems, leaves, seeds, and calli, with the highest levels in seeds, followed by leaves and calli. The PoBBM protein displayed transcriptional self-activation activity, which may facilitate further research on its relationships with other proteins. The above results provide a key gene PoBBM for somatic embryogenesis in peonies, which is significant for advancing the establishment of a stable and efficient regeneration and genetic transformation system for peonies.

GmBBM7 promotes callus and root growth during somatic embryogenesis of soybean (Glycine max)

The BABY BOOM (BBM) subfamily of the AP2/ERF transcription factor family is the main regulator of totipotency in plant cells and plays an important role in regulating cell proliferation and plant growth and development. Previously, we identified GmBBM7, a key gene of the soybean BBM gene subfamily (GmBBM family). In this study, we aimed to analyze its molecular characteristics and role in somatic embryogenesis in soybean. First, we identified 17 members of GmBBM family. Phylogenetic and collinear analyses revealed that the GmBBM family genes mainly exhibited a strong genetic relationship with the those of PvBBM family, the BBM family of common bean (Phaseolus vulgaris). Analysis of the promoters revealed that GmBBM family genes were mainly regulated by plant hormones. The expression of GmBBM7 varied in various tissues of soybean. Particularly, it was higher in the roots, immature embryos, grains and callus. Subcellular localization analysis indicated that GmBBM7 encodes a nuclear protein. Furthermore, overexpressed GmBBM7 could significantly enhance callus formation by regulating the levels of gibberellin A1, A3 and A7; abscisic acid; and salicylic acid and could promote root growth and development. In summary, GmBBM7 is an important regulatory gene for somatic embryogenesis and root elongation in soybean. GmBBM7 overexpression in soybean could increase the callus formation rate and density through hormonal pathways and could increase root growth. This study provided theoretical basis for efficient breeding of soybean via gene editing, transgenic and other techniques.

Effect of BBM gene on callus growth and ginsenoside content in Panax quinquefolius

Baby Boom(BBM) gene is a key regulatory factor in embryonic development and regeneration, cell proliferation, callus growth, and differentiation promotion. Since the genetic transformation system of Panax quinquefolius is unstable with low efficiency and long period, this study attempted to transfer BBM gene of Zea mays to P. quinquefolius callus by gene gunship to investigate its effect on the callus growth and ginsenoside content, laying a foundation for establishing efficient genetic transformation system of P. quinquefolius. Four transgenic callus of P. quinquefolius with different transformation events were obtained by screening for glufosinate ammonium resistance and molecular identification by PCR. The growth state and growth rate of wild-type and transgenic callus were compared in the same growth period. The content of ginsenoside in transgenic callus was determined by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry(UPLC-MS/MS). The results showed that transgenic callus growth rate was significantly higher than that of wild-type callus. In addition, the content of ginsenoside Rb_1, Rg_1, Ro, and Re was significantly higher than that in wild-type callus. The paper preliminarily proved the function of BBM gene in promoting growth rate and increasing ginsenoside content, which provided a scientific basis to establish a stable and efficient genetic transformation system for Panax plants in the future.

BABY BOOM regulates early embryo and endosperm development.

The BABY BOOM (BBM) AINTEGUMENTA-LIKE (AIL) AP2/ERF domain transcription factor is a major regulator of plant cell totipotency, as it induces asexual embryo formation when ectopically expressed. Surprisingly, only limited information is available on the role of BBM during zygotic embryogenesis. Here we reexamined BBM expression and function in the model plant Arabidopsis thaliana (Arabidopsis) using reporter analysis and newly developed CRISPR mutants. BBM was expressed in the embryo from the zygote stage and also in the maternal (nucellus) and filial (endosperm) seed tissues. Analysis of CRISPR mutant alleles for BBM (bbm-cr) and the redundantly acting AIL gene PLETHORA2 (PLT2) (plt2-cr) uncovered individual roles for these genes in the timing of embryo progression. We also identified redundant roles for BBM and PLT2 in endosperm proliferation and cellularization and the maintenance of zygotic embryo development. Finally, we show that ectopic BBM expression in the egg cell of Arabidopsis and the dicot crops Brassica napus and Solanum lycopersicon is sufficient to bypass the fertilization requirement for embryo development. Together these results highlight roles for BBM and PLT2 in seed development and demonstrate the utility of BBM genes for engineering asexual embryo development in dicot species.

BABY BOOM (BBM): a candidate transcription factor gene in plant biotechnology.

Plants have evolved a number of transcription factors, many of which are implicated in signaling pathways as well as regulating diverse cellular functions. BABY BOOM (BBM), transcription factors of the AP2/ERF family are key regulators of plant cell totipotency. Ectopic expression of the BBM gene, originally identified in Brassica napus, has diverse functions in plant cell proliferation, growth and development without exogenous growth regulators. The BBM gene has been implicated to play an important role as a gene marker in multiple signaling developmental pathways in plant development. This review focuses on recent advances in our understanding of a member of the AP2 family of transcription factor BBM in plant biotechnology including plant embryogenesis, cell proliferation, regeneration, plant transformation and apogamy. Recent discoveries about the BBM gene will inevitably help to unlock the long-standing mysteries of different biological mechanisms of plant cells.

A fern AINTEGUMENTA gene mirrors BABY BOOM in promoting apogamy in Ceratopteris richardii.

Asexual reproduction is widespread in land plants, including ferns where 10% of all species are obligate asexuals. In these ferns, apogamous sporophytes are generated directly from gametophytes, bypassing fertilization. In the model fern Ceratopteris richardii, a sexual species, apogamy can be induced by culture on high sugar media. BABY BOOM (BBM) genes in angiosperms are known to promote somatic embryogenesis, which like apogamy produce sporophytes without fertilization. Here, a Brassica napus BBM (BnBBM) was used to investigate genetic similarity between apogamy in ferns and somatic embryogenesis in angiosperms. A C.richardii transcriptome was constructed from which one AINTEGUMENTA-LIKE unigene, CrANT, was identified. Whole mount insitu hybridization showed that CrANT is expressed in sperm and fertilized eggs. Phylogenetic analysis grouped CrANT with other non-seed-plant ANT genes to the euANT clade but in a branch separate from BBM genes. Overexpression of CrANT or BnBBM promotes apogamy in C.richardii without sugar supplement. CrANT knockdown gametophytes responded weakly to sugar for apogamy promotion. Theses results suggest some genetic conservation between apogamy and somatic embryogenesis and that such asexual reproduction may be ancient.

Micropropagation and Androgenesis in coconut: an assessment of Sri Lankan implication

The coconut palm is an important cash crop mostly in the sub-tropical countries which provides almost all the necessities of life to the mankind. Genetic improvement for increased productivity is a prioritized research area of coconut palm. In vitro cloning of elite palms via somatic embryogenesis seems to be a promising method due to its potential for massive propagation. Based on the Sri Lankan experience, this paper summarizes the notable achievements towards developing a reliable protocol for clonal propagation of coconut and also dihaploid plant production. The newel explants described by Coconut Research Institute Sri Lanka (CRISL), unfertilized ovaries dissected from immature inflorescences collected from -4 stage (inflorescence to be opened in 4 months time), is a somatic tissue which causes minimal damage to the mother palm while collecting. Limited number of female flowers in an inflorescence which limits large scale culturing could overcome by callus multiplication phase to produce higher number of plants. Somatic embryogenesis and germination of somatic embryos was achieved by reduction of auxin concentration in the culture medium followed by an addition of cytokinin. AINTEGUMENTA-LIKE (ANT-like) gene isolated from coconut genome which shows higher conserved domain sequence similarities with BABYBOOM gene will assist development of suitable molecular markers to assess the morphogenic potential of in vitro cultured coconut tissues and callus. Successful plant regeneration from cultured anthers was reported by culturing anthers at three weeks before splitting (3-WBS) inflorescence stage which contains late uninucleate microspores. Heat pretreatment of anthers for six days at 38°C induced androgenesis in coconut upon culturing in 2, 4-D and 1-naphthyl acetic acid (NAA) added medium. Current clonal propagation protocol is efficient when responsive genotype is in use.Further improvement to current protocol will enable commercially viable protocol for coconut micropropagation.

Molecular cloning, characterization, and functional analysis of CcBBM gene from camphor tree (Cinnamomum camphora L.)

Cloning and functional analysis of CcBBM gene. Baby boom (BBM), as a transcription factor of AP2/ERF family, has great potence in promoting somatic embryogenesis. In this study, a BBM gene from somatic embryos of camphor tree (Cinnamomum camphora L.), named CcBBM, was cloned and characterized. A partial fragment of 560 bp of CcBBM was amplified by degenerate PCR based on the two conserved AP2 domains of other annotated BBMs. Full-length cDNA of CcBBM was obtained by a cloning strategy combing degenerate PCR and RACE technique. The open-reading frame of CcBBM is 2169 bp in length, which encodes 722 amino acids. A comparison of the deduced amino acid sequence of CcBBM with BBM from other species showed high similarities, ranging from 51 to 80 %. The mRNA levels of CcBBM in different tissues were detected by semi-quantitative reverse transcription PCR (RT-PCR). CcBBM was expressed in most tissues, with highest expression level in somatic embryos, followed by roots, young fruits and flowers. Ectopic expression of CcBBM gene in Arabidopsis exhibited a variety of deformed and variegated leaves and short roots. No spontaneous somatic embryogenesis was observed in 35S::CcBBM transgenic plants. These results will help us understand the role of CcBBM in somatic embryogenesis in camphor tree.

Enhanced somatic embryogenesis in Theobroma cacao using the homologous BABY BOOM transcription factor.

BackgroundTheobroma cacao, the chocolate tree, is an important economic crop in East Africa, South East Asia, and South and Central America. Propagation of elite varieties has been achieved through somatic embryogenesis (SE) but low efficiencies and genotype dependence still presents a significant limitation for its propagation at commercial scales. Manipulation of transcription factors has been used to enhance the formation of SEs in several other plant species. This work describes the use of the transcription factor Baby Boom (BBM) to promote the transition of somatic cacao cells from the vegetative to embryonic state.ResultsAn ortholog of the Arabidopsis thaliana BBM gene (AtBBM) was characterized in T. cacao (TcBBM). TcBBM expression was observed throughout embryo development and was expressed at higher levels during SE as compared to zygotic embryogenesis (ZE). TcBBM overexpression in A. thaliana and T. cacao led to phenotypes associated with SE that did not require exogenous hormones. While transient ectopic expression of TcBBM provided only moderate enhancements in embryogenic potential, constitutive overexpression dramatically increased SE proliferation but also appeared to inhibit subsequent development.ConclusionOur work provides validation that TcBBM is an ortholog to AtBBM and has a specific role in both somatic and zygotic embryogenesis. Furthermore, our studies revealed that TcBBM transcript levels could serve as a biomarker for embryogenesis in cacao tissue. Results from transient expression of TcBBM provide confirmation that transcription factors can be used to enhance SE without compromising plant development and avoiding GMO plant production. This strategy could compliment a hormone-based method of reprogramming somatic cells and lead to more precise manipulation of SE at the regulatory level of transcription factors. The technology would benefit the propagation of elite varieties with low regeneration potential as well as the production of transgenic plants, which similarly requires somatic cell reprogramming.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0479-4) contains supplementary material, which is available to authorized users.

A putative BABY BOOM-like gene (CaBBM) is expressed in embryogenic calli and embryogenic cell suspension culture of Coffea arabica L

The acquisition of embryogenic cell suspension (ECS) cultures has been one of the main objectives to maximize clonal propagation of the coffee plant. However, the majority of somatic embryogenesis induction requirements are genotype-dependent. Therefore, molecular markers linked to the embryogenic transition events may be useful. The BABY BOOM (BBM) gene can be considered as one of those markers, as it is related to the embryogenic process and to cell proliferation. BBM homologous sequences were obtained from Expressed Sequence Tags (ESTs) in a databank generated by the Brazilian Coffee Genome Project. We selected EST-contigs that showed similarities with BBM sequence from different species. Two EST-contigs (C2 and C9) were expressed in silico in cellular suspension libraries and embryogenic calli of coffee. Contig C9, defined as BBM-like (CaBBM), presented similarity with BBM genes and showed 2-fold change in expression in ECS relative to embryogenic calli (EC). Contig C2, on the other hand, was related to the ERF-like family. It showed basal expression in non-embryogenic calli (NEC) and approximately 66- and 311-fold less in ECS and EC compared with CaBBM in the same samples, respectively. These data suggest that CaBBM is likely to be a BBM ortholog in Coffea arabica, which has potential for use as a molecular marker to further increase the methodological efficiency of in vitro culture of coffee.

Expression of the BBM gene during somatic embryogenesis of Arabidopsis thaliana

The relationship between somatic embryogenesis (SE) and the expression of the BABY BOOM (BBM) gene was studied in cultured immature zygotic embryos (IZEs) using a transgenic line of Arabidopsis thaliana containing a BBMPro::GUS construct. Results showed spatio-temporal differences in BBM expression in explants during culture. BBM promoter activity was observed in freshly isolated IZEs except distal parts of cotyledons. At the beginning of culture, considerable increase of GUS staining intensity was observed in all parts of explants, which maintained at high level over next few days and coincide with cell divisions. Gradual decrease of GUS distribution in explants was observed at about the 5th day of culture. BBM promoter activity became largely restricted to dividing cells, then to developing somatic embryos, shoot-like structures and callus. In parts of explants not involved in morphogenesis BBM promoter activity was absent or hardly seen. Thus the in vitro expression of BBM coincides with cell proliferation and morphogenesis.

Expression of the BBM gene during somatic embryogenesis of Arabidopsis thaliana

The relationship between somatic embryogenesis (SE) and the expression of the BABY BOOM (BBM) gene was studied in cultured immature zygotic embryos (IZEs) using a transgenic line of Arabidopsis thaliana containing a BBMPro::GUS construct. Results showed spatio-temporal differences in BBM expression in explants during culture. BBM promoter activity was observed in freshly isolated IZEs except distal parts of cotyledons. At the beginning of culture, considerable increase of GUS staining intensity was observed in all parts of explants, which maintained at high level over next few days and coincide with cell divisions. Gradual decrease of GUS distribution in explants was observed at about the 5th day of culture. BBM promoter activity became largely restricted to dividing cells, then to developing somatic embryos, shoot-like structures and callus. In parts of explants not involved in morphogenesis BBM promoter activity was absent or hardly seen. Thus the in vitro expression of BBM coincides with cell proliferation and morphogenesis.

A novel method for induction of plant regeneration via somatic embryogenesis

To induce plant regeneration via somatic embryogenesis, a binary expression vector system was constructed containing a heat shock-inducible FRT/FLP system. In this system, the BABY BOOM ( BBM) gene, which shows similarity to the AP2/ERF family, was under the control of cauliflower mosaic virus 35S (CaMV 35S) promoter and the gene coding for FLP site-specific recombinase was under the control of a heat shock-inducible promoter HSP18.2 from Arabidopsis thaliana. The promoters and genes were flanked by two FRT recombination sites in direct orientation. BBM overexpression induced formation of somatic embryos from calli of Chinese white poplar ( Populus tomentosa Carr.). Six somatic embryos germinated to form transgenic plants with phenotypic alterations. The stem cuttings of the transgenic plants were treated with heat shock and they developed into phenotypically normal plants. Southern blot analysis further confirmed that the BBM gene was excised from the genomic DNA of the transgenic plants with normal phenotype. The system reported here can be used to obtain transgenic plants without the use of antibiotic or herbicide resistance genes and to provide a novel strategy for plant regeneration via somatic embryogenesis.

Heterologous expression of the BABY BOOM AP2/ERF transcription factor enhances the regeneration capacity of tobacco (Nicotiana tabacum L.)

Gain-of-function studies have shown that ectopic expression of the BABY BOOM (BBM) AP2/ERF domain transcription factor is sufficient to induce spontaneous somatic embryogenesis in Arabidopsis (Arabidopsis thaliana (L.) Heynh) and Brassica napus (B. napus L.) seedlings. Here we examined the effect of ectopic BBM expression on the development and regenerative capacity of tobacco (Nicotiana tabacum L.) through heterologous expression of Arabidopsis and B. napus BBM genes. 35S::BBM tobacco lines exhibited a number of the phenotypes previously observed in 35S::BBM Arabidopsis and B. napus transgenics, including callus formation, leaf rumpling, and sterility, but they did not undergo spontaneous somatic embryogenesis. 35S::BBM plants with severe ectopic expression phenotypes could not be assessed for enhanced regeneration at the seedling stage due to complete male and female sterility of the primary transformants, therefore fertile BBM ectopic expression lines with strong misexpression phenotypes were generated by expressing a steroid-inducible, post-translationally controlled BBM fusion protein (BBM:GR) under the control of a 35S promoter. These lines exhibited spontaneous shoot and root formation, while somatic embryogenesis could be induced from in-vitro germinated seedling hypocotyls cultured on media supplemented with cytokinin. Together these results suggest that ectopic BBM expression in transgenic tobacco also activates cell proliferation pathways, but differences exist between Arabidopsis/B. napus and N. tabacum with respect to their competence to respond to the BBM signalling molecule.