Differential expression and co-localization of transcriptional factors during callus transition to differentiation for shoot organogenesis in the water fern Ceratopteris richardii

Abstract

Background and aims: In flowering plants, regeneration can be achieved by a variety of approaches, and different sets of transcriptional factors are involved in these processes. However, regeneration in taxa other than flowering plants remains a mystery. Ceratopteris richardii is a representative fern capable of both direct and indirect organogenesis, and we aimed to investigate the genetics underlying the transition from callus proliferation to differentiation.

Methods: Morphological and histological analyses were used to determine the type of regeneration involved. RNA sequencing and differential gene expression were used to investigate how the callus switches from proliferation to differentiation. Phylogenetic analysis and RNA in situ hybridization were used to understand whether transcriptional factors are involved in this transition.

Key results: The callus formed on nascent leaves and subsequently developed the shoot pro-meristem and shoot meristem, thus completing indirect de novo shoot organogenesis in C. richardii. Genes were differentially expressed during the callus transition from proliferation to differentiation, indicating a role for photosynthesis, stimulus response and transmembrane signalling in this transition and the involvement of almost all cell layers that make up the callus. Transcriptional factors were either downregulated or upregulated, which were generally in many-to-many orthology with genes known to be involved in callus development in flowering plants, suggesting that the genetics of fern callus development are both conserved and divergent. Among them, an STM-like, a PLT-like and an ethylene- and salt-inducible ERF gene3-like gene were expressed simultaneously in the vasculature but not in the other parts of the callus, indicating that the vasculature played a role in the callus transition from proliferation to differentiation.

Conclusions: Indirect de novo shoot organogenesis could occur in ferns, and the callus transition from proliferation to differentiation required physiological changes, differential expression of transcriptional factors and involvement of the vasculature.

Keywords: Ceratopteris richardii; in situ hybridization; Transcriptome; callus; ferns; organogenesis; pluripotency; regeneration.

Fig. 1.

Callus morphology at different stages. (A) In the BA-containing medium. (B) In the BA-lacking medium for 4 days. (C) In the BA-lacking medium for 8 days. (D) In the BA-lacking medium for 12 days. (E) In the BA-lacking medium for 19 days. (F) In the BA-lacking medium for 26 days. Arrows indicate rod-like structures covered with white trichomes. Scale bars: 1 mm.

Fig. 2.

Histology of the callus at different stages. (A) On the explant. Arrows indicate individual callus on the aerial part and in the embedded roots of a young leaf. (B) In the BA-containing medium. Arrows indicate protrusions, and the arrowhead indicates the protoderm. (C) In the BA-lacking medium for 4 days. Arrows indicate protrusions, the arrowhead indicates the protoderm, and the ellipse indicates dynamic cell divisions. (D) In the BA-lacking medium for 8 days. Arrows indicate shoot pro-meristems, and the inset shows the apex and the cell arrangement of shoot pro-meristems. (E) In the BA-lacking medium for 12 days. The arrow indicates the shoot meristem, and the inset shows the dome shape of the shoot meristems. (F) In the BA-lacking medium for 26 days. The arrow indicates a shoot meristem, the arrowhead indicates one leaf, and the asterisk indicates the deteriorated leakages within the callus. Scale bars: 100 μm.

Fig. 3.

Clustering of enriched GO and KEGG terms. Five groups are indicated by black vertical bars and letters. The number of genes in the terms is shown in parentheses. Terms are classified by colour, with biological process terms in red, cellular component terms in orange, molecular function terms in blue and significant KEGG terms in green, while insignificant terms are in black.

Fig. 4.

Genes and their expression levels in four functional groups. (A) Group I centred on stimulus response. (B) Group II centred on photosynthetic light reaction. (C) Group III centred on photosynthetic dark reaction. (D) Group IV centred on transmembrane signalling. Genes have been described with reference to the best hit of BLAST against Arabidopsis thaliana.

Fig. 5.

Cell type analysis and RT-qPCR analysis. (A) Distribution of differentially expressed genes (DEGs) across cell types. The horizontal axis indicates c0–c9 cell types, and the vertical axis indicates the proportion of hits within DEGs to hits within both DEGs and non-DEGs in each cell type. This analysis was performed separately using the best hit, top two hits and top three hits. (B) RT-qPCR of the STM-like 04G064800 and the LBD16-like 38G014600.

Fig. 6.

Phylogenetic analysis of multiple transcriptional factors. (A) 04G064800 in many-to-many orthology with STM. (B) 31G036600 in a different lineage from PLTs. (C) 19G009000 in many-to-many orthology with WOX11. (D) 25G000100 and 38G014600 in a different lineage from LBD16. Support values along branches are hidden if <50. Branches leading to fern genes are in green, with the Ceratopteris genes under investigation also indicated by names in green. Branches leading to seed plants are in red, to lycophytes in green, to bryophytes in blue, and to algae in purple.

Fig. 7.

In situ hybridization. (A–D) Expression pattern of the STM-like 04G064800. (A) The proliferating callus. (B) Callus differentiated for 4 days. (C) Callus differentiated for 8 days. (D) Callus differentiated for 12 days. (E–H) Expression pattern of the ANT-like 31G036600. (I–L) Expression pattern of the ESE3-like 11G046500. Scale bars: 100 μm.