Genome-wide analysis of MpBHLH12, a IIIf basic helix-loop-helix transcription factor of Marchantia polymorpha

Abstract

The evolution of plants on land required adaptation to UV radiation and dry environments, and involved the appearance and/or rewiring of genetic connections, known as gene regulatory networks (GRNs), which consist of one or more transcription factors (TFs). The liverwort, Marchantia polymorpha, is a basal land plant, with a recently sequenced genome. The number of genes encoding basic helix-loop-helix (bHLH) family members is considerably higher in M. polymorpha than in charophyte green algae, suggesting the contribution of bHLH proteins to the evolution of GRNs associated with the adaptation of plants to land. Although an understanding of the evolutionary aspects of GRNs is fundamental for elucidating the mechanisms of environmental adaptation, the evolution of GRNs that led to land adaptation in plants remains poorly understood. In this study, we isolated a single gene encoding a IIIf bHLH TF from M. polymorpha, MpBHLH12. Transgenic M. polymorpha constitutively overexpressing MpBHLH12 showed smaller and fewer gemma cups than wild type, suggesting that MpBHLH12 is involved in the regulation of morphological development. Transcriptomic analysis of MpBHLH12 overexpressor (MpBHLH12ox) lines revealed an overlap with the GRN of MpMYB14, which regulates the biosynthesis of anthocyanins and phenolic compounds. However, MpBHLH12ox did not show anthocyanin accumulation. Results of the transient reporter assay suggest that MpBHLH12 could function in repression rather than activation. Our findings suggest that although the IIIf bHLH MpBHLH12 shows highest amino acid similarity with IIIf bHLH clade and is involved in developmental process and partly biosynthesis of phenolic compounds in M. polymorpha like Arabidopsis IIIf bHLH, the GRN involving MpBHLH12 would be distinct one from those of the IIIf bHLH TFs of seed plants.

Keywords: Arabidopsis thaliana; Gene regulatory network; Marchantia polymorpha; bHLH transcription factor.

Fig. 1

Phylogenic analysis of basic helix-loop-helix (bHLH) proteins in Marchantia polymorpha and Arabidopsis thaliana. The phylogenetic tree was drawn using the amino acid sequence of the bHLH transcription factors. Green and blue boxes indicate the bHLH proteins of A. thaliana and M. polymorpha, respectively. Gene names belonging to clade III are shown

Fig. 2

Morphological features of MpBHLH12 overexpressor (MpBHLH12ox) lines. a Phenotypes of the thallus of wild-type Tak-1 (left), MpBHLH12ox#2 (middle), and MpBHLH12ox#3 (right) grown on 1/2 Gamborg’s B5 agar medium for 3 weeks (scale bar = 1 cm). b Images of gemma cups of 3-week-old wild-type Tak-1 (left) and MpBHLH12ox#2 (right) captured under a digital microscope (scale bar = 500 µm). c Area of gemma cups of 3-week-old Tak-1 and MpBHLH12ox#2. The area of gemma cups was approximately fourfold lower in MpBHLH12ox#2 than in wild-type Tak-1 (**p < 0.01; n = 10). d Quantitative real-time PCR (qRT-PCR) analysis of MpBHLH12 expression in the gemma cups and thalli of MpBHLH12ox#2 (**p < 0.01; n = 4)

Fig. 3

Measurement of anthocyanin content in wild-type Tak-1 and MpBHLH12ox lines. Tak-1 and MpBHLH12ox#8 were grown on 1/2 Gamborg’s B5 culture medium lacking phosphorus for 1 week. a Images of thalli of Tak-1 (left) and MpBHLH12ox#8 (right) captured under a digital microscope (scale bar = 1 mm). In both Tak-1 and MpBHLH12ox#8, a part of the thallus showed brown discoloration. b Quantification of anthocyanin content in the thalli of 1-week-old Tak-1 and MpBHLH12ox#8 (**p < 0.01; n = 3). Anthocyanin content was measured in an arbitrary unit. MpBHLH12ox#2 and #3 were shown similar phenotypes as well

Fig. 4

Analysis of the transcriptome of MpBHLH12ox lines. a Scatter plot of MpBHLH12ox#2 and #3. X- and Y-axes indicate log₂ (fold-change) in gene expression in MpBHLH12ox#2 and #3, respectively, relative to that in Tak-1 A total of 1,205 genes were differentially expressed in both overexpressor lines. b, c Gene ontology (GO) analyses of the up-regulated (b) and down-regulated (c) genes. All significantly enriched GO terms (p < 0.01) are shown

Fig. 5

Heat map showing the comparison of shikimate and flavonoid pathway genes among MpBHLH12ox lines (this study) and transgenic M. polymorpha lines overexpressing MpMYB14 and MpMYB02 (Albert et al. 2017; Kubo et al. 2018). Color indicates log₂ (fold-change) in gene expression in overexpressor lines compared with that in wild-type Tak-1; degree of difference is shown from blue to red. Differentially expressed genes (DEGs) identified in MpBHLH12ox lines are shown in a black box. Genes with no expression data available are indicated with diagonal lines

Fig. 6

Transient reporter assays performed using Arabidopsis protoplasts. a Plasmid expressing luciferase (LUC) under the control of the AtLDOX promoter was used as a reporter. The transcription factor (TF) used in each effector plasmid is shown as (+) under the boxplots. Plasmid encoding a vesicle-associated membrane protein (VAMP) was used as a negative control. Statistically significant data are indicated with asterisks (*p < 0.05; **p < 0.01; n = 4). b Plasmid expressing LUC under the control of a GAL4-binding sequence (UAS) was used as a reporter (n = 8). GAL4DB is bound to UAS. Activity of the protein added as an effector was measured