Abstract Liverworts, a section of the bryophyte plants which pioneered the colonization of terrestrial habitats, produce cyclic bisbibenzyls as secondary metabolites. These compounds are generated via the phenylpropanoid pathway, similar to flavonoid biosynthesis, for which basic helix–loop–helix (bHLH) transcription factors have been identified as one of the important regulators in higher plants. Here, a bHLH gene homolog (PabHLH) was isolated from the liverwort species Plagiochasma appendiculatum and its contribution to bisbibenzyl biosynthesis was explored. Variation in the abundance of PabHLH transcript mirrored that of tissue bisbibenzyl content in three different liverwort tissues. A phylogenetic analysis based on the bHLH domain sequence suggested that the gene encodes a member of bHLH subgroup IIIf, which clusters proteins involved in flavonoid synthesis. The gene’s transient expression in onion epidermal cells implied that its product localized to the nucleus, and a transactivation assays in yeast showed that it was able to activate transcription. In both callus and thallus, the overexpression of PabHLH boosted bisbibenzyl accumulation, while also up-regulating PaPAL, Pa4CL1, PaSTCS1 and two genes encoding P450 cytochromes, and its RNA interference (RNAi)-induced suppression down-regulated the same set of genes and reduced the accumulation of bisbibenzyls. The abundance of PaCHS and PaFNSI transcript was related to flavonoid accumulation in transgenic thallus. PabHLH represents a candidate for the metabolic engineering of bisbibenzyl content. Introduction The bryophytes (liverworts, mosses and hornworts) are phylogenetically placed between the algae and the vascular plants (Kennck and Crane 1997). They synthesize a range of metabolites bearing aromatic side chains, such as the bisbibenzyls (Asakawa 1995). The macrocyclic bisbibenzyls, which comprise two bibenzyl moieties linked by either two ether bridges or two biaryl bonds, or one of each (Keserü and Nogradi 1995, Asakawa et al. 2000), are rather specific to the liverworts; these compounds display a broad spectrum of biological activity, including toxicity to the bacteria Pseudomonas aeruginosa and Staphylococcus aureus (Asakawa 1990), the fungi Aspergillus fumigatus and Candida albicans (Asakawa 1994), and the virus HIV-1 (Asakawa 2007). They also can be used as muscle relaxants (Taira et al. 1994) and exhibit cardiotonic activity (Asakawa 1990). Based on a labeled precursor feeding experiment, the synthesis of the marchantin-type bisbibenzyl is thought to be initialized from phenylalanine, which is converted to cinnamic acid in a reaction catalyzed by phenylalanine ammonia lyase (PAL), followed by a hydroxylation to form p-coumaric acid through the action of cinnamic acid 4-hydroxylase (C4H) and a reduction to dihydro-p-coumaric acid. Thereafter 4-coumarate:coenzyme A ligase (4CL) catalyzes an acylation reaction to form dihydro-p-coumaroyl CoA, which is condensed with three malonyl-CoA molecules to yield lunularic acid with the help of stilbene carboxylate synthase (STCS) (Eckermann et al. 2003). Finally, a reaction catalyzed by CytP450 generates the bisbibenzyls (Friederich et al. 1999a, Friederich et al. 1999b). The biosynthesis of bisbibenzyls follows the same pathway as that utilized by higher plants to produce flavonoids (Gao et al. 2015, Yu et al. 2015) (Fig. 1). Fig. 1 View largeDownload slide The phenylpropanoid pathway in liverworts. (A) The flavonoid pathway and proposed bisbibenzyl pathway. PAL, phenylalanine ammonia lyase; C4H, cinnamic acid 4-hydroxylase; 4CL, 4-coumarate: coenzyme A ligase; STCS, stilbene carboxylate synthase; CHS, chalcone synthase; P450, CytP450 enzymes. (B) Several of the bisbibenzyls synthesized by liverworts. Fig. 1 View largeDownload slide The phenylpropanoid pathway in liverworts. (A) The flavonoid pathway and proposed bisbibenzyl pathway. PAL, phenylalanine ammonia lyase; C4H, cinnamic acid 4-hydroxylase; 4CL, 4-coumarate: coenzyme A ligase; STCS, stilbene carboxylate synthase; CHS, chalcone synthase; P450, CytP450 enzymes. (B) Several of the bisbibenzyls synthesized by liverworts. Basic helix–loop–helix (bHLH) proteins form a large, dispersed family of transcription factors (TFs). Their characteristic approximately 60 residue bHLH domain is predicted to form two amphipathic α-helices connected by a loop of variable length (Jones 2004). The basic region functions as the binding motif, while the HLH region forms homodimers or heterodimers between bHLH proteins (Mullen et al. 1994). Although the bHLH domain sequence is strongly conserved, their distinction was considered to make their function discrepant (Littlewood and Evan 1995, Seo et al. 2011). Plant bHLH TFs have been documented to regulate the response to light and hormones (Huang et al. 2013, Liu et al. 2013), the development of floral organs (Groszmann et al. 2008), the transport of symbiotic ammonium (Kaiser et al. 1998), the stress response (Feng et al. 2013) and the synthesis of flavonoids (Matus et al. 2010). Flavonoids occur widely in the plant kingdom; their synthesis starts from phenylalanine, which is converted via p-coumaroyl-CoA to the precursor compound naringenin chalcone (Nesi et al. 2000). It has been established that the regulation of the structural genes involved in flavonoid biosynthesis is controlled by bHLH, R2R3-MYB and WD40 proteins, functioning as complexes or working alone (Koes et al. 2005). The first member of the plant bHLH TF factor family was identified in maize in 1989, and was shown to regulate anthocyanin biosynthesis (Toledo-Ortiz et al. 2003). The bHLH TF MYC-RP from Perillafrutescens control anthocyanin accumulation in leaves and stem (Gong et al. 1999). According to Heim, the 133 Arabidopsis thaliana bHLHs fall into into 12 subgroups, one of which (subgroup IIIf) houses members involved in the regulation of flavonoid synthesis (Heim et al. 2003, Hichri et al. 2010). Little is known concerning the function of the bHLH TFs encoded by liverwort genomes. Although the synthetic pathways used to produce bisbibenzyls and flavonoids are very similar, the regulatory machinery controlling bisbibenzyl production has not been revealed in any detail to date. Here, the isolation of a bHLH sequence from the liverwort species Plagiochasma appendiculatum (a member of the Marchantiales, see Singh et al. 2006) is described, along with a functional analysis based on its overexpression and RNA interference (RNAi)-based suppression. Results Pattern of PabHLH in different tissues and its response to phytohormone treatment The bisbibenzyl contents in three different P. appendiculatum tissues (Fig. 2A) were quantified. The bisbibenzyl content of thallus growing in the greenhouse was higher than that of either axenically cultured thallus or callus in medium (Fig. 2A, B). At the same time, a gene referred to as PabHLH from a sequenced normalized cDNA library (Cheng et al. 2013) of P. appendiculatum thallus, containing a highly conserved bHLH domain in the C-terminal region, was found showing a consistent tendency to be expressed in the three different liverwort tissues according to the bisbibenzyl contents (Fig. 2C). In brief, the bisbibenzyl content variation mirrored the expression profile of PabHLH. These results indicate that PabHLH plays a positive role in bisbibenzyl accumulation. Fig. 2 View largeDownload slide The bisbibenzyl content and the abundance of PabHLH transcript in P. appendiculatum. (A) Three different tissues of P. appendiculatum: 1, axenic callus; 2, axenic thallus; 3, thallus growing in a greenhouse. (B) Total bisbibenzyl content in three tissues. NMA, neo-marchantin A; IMC, iso-marchantin C; PE, perrottetin E; RC, riccardin C; RD, riccardin D. (C) PabHLH transcript abundance by qRT-PCR in the three tissue types referred to in (B). Data are shown in the form mean ± SD (n = 3). **Means differ significantly from the level of sample 1 at P < 0.01. Fig. 2 View largeDownload slide The bisbibenzyl content and the abundance of PabHLH transcript in P. appendiculatum. (A) Three different tissues of P. appendiculatum: 1, axenic callus; 2, axenic thallus; 3, thallus growing in a greenhouse. (B) Total bisbibenzyl content in three tissues. NMA, neo-marchantin A; IMC, iso-marchantin C; PE, perrottetin E; RC, riccardin C; RD, riccardin D. (C) PabHLH transcript abundance by qRT-PCR in the three tissue types referred to in (B). Data are shown in the form mean ± SD (n = 3). **Means differ significantly from the level of sample 1 at P < 0.01. The plant hormones salicylic acid (SA) and jasmonic acid (JA) play key roles in responses to stresses (Zhu et al. 2012, Shimizu et al. 2013), and we detected the presence of JA (0.75 ng g−1 FW) (Supplementary Figs. S3, S4) and SA (4.77 ng g−1 FW) (Supplementary Figs. S5, S6) in P. appendiculatum thallus. The abundance of PabHLH transcript in thallus was increased to a low extent after a 6 h exposure to either SA or methyl jasmonate (MeJA), peaking after 36 h; the peak abundance induced by SA was >5-fold the background level, while that induced by MeJA treatment was >8-fold the background level. The level of PabHLH transcription began to decline at 60 h (Fig. 3). In conclusion, both MeJA and SA treatment could cause strong induction of the PabHLH transcript level. A previous investigation indicated that stress treatment could induce the accumulation of bisbibenzyls and the expression level of the genes encoding the key enzymes in the biosynthesis of bisbibenzyls (Yu et al. 2014). The expression of PabHLH responded to SA and MeJA treatment, indicating that it might be involved in the biosynthesis of the bisbibenzyls. Fig. 3 View largeDownload slide Expression patterns of PabHLH in response to SA and MeJA at different time points (0, 6, 12, 24, 36, 48, 60 and 72 h). Data are shown in the form mean ± SD (n = 3). *,**Means differ significantly from the level of sample t = 0 h at P < 0.05 and <0.01, respectively. Fig. 3 View largeDownload slide Expression patterns of PabHLH in response to SA and MeJA at different time points (0, 6, 12, 24, 36, 48, 60 and 72 h). Data are shown in the form mean ± SD (n = 3). *,**Means differ significantly from the level of sample t = 0 h at P < 0.05 and <0.01, respectively. The PabHLH sequence and its phylogeny A 3' fragment of a coding region sequence from the sequenced normalized P. appendiculatum thallus cDNA library was selected because of its highest similarity to genes of bHLH subgroup IIIf. The full-length cDNA, as recovered by RACE (rapid amplification of cDNA ends) PCR, included a 1,566 nucleotide (nt) open reading frame (ORF) predicted to encode a 521 residue polypeptide of molecular mass 57.9 kDa and pI 9.20. Since a BlastX search indicated it as a member of the bHLH-MYC-N superfamily, the gene was designated PabHLH (GenBank accession No. MF983804). Its genomic sequence of length 2,055 nt was separated into three exons (exon#1, 86 nt; #2, 1,339 nt; #3, 141 nt) interrupted by introns #1 (308 nt) and #2 (181 nt) (Fig. 4A). The bHLH domain was not interrupted by intronic sequence. Alignments based on the conserved bHLH domain sequences revealed that it shared a relatively high level of similarity with sequences from various higher plants (Fig. 4B). The deduced full PabHLH peptide sequence, however, exhibited only 17.81, 11.52, 18.59 and 15.47% identity with VvMYC1, AtTT8, PhAN1 and IpIVS, respectively (data not shown). A phylogenetic analysis based on the bHLH sequence showed that PabHLH resembled most closely the sequence of various regulators of flavonoid synthesis, in particular the subgroup IIIf protein AtTT8 (Fig. 5). Its peripheral position in the cluster suggested that PabHLH represents an ancestral form of the higher plant subgroup of IIIf bHLH TFs. Fig. 4 View largeDownload slide The structure of PabHLH. (A) The 2,055 nt genomic sequence is separated into three exons. The full-length cDNA harbors a 1,566 nt ORF. (B) Alignment of the bHLH domains of PabHLH and other plant bHLHs. Identical and similar residues are shown in black and gray, respectively. Fig. 4 View largeDownload slide The structure of PabHLH. (A) The 2,055 nt genomic sequence is separated into three exons. The full-length cDNA harbors a 1,566 nt ORF. (B) Alignment of the bHLH domains of PabHLH and other plant bHLHs. Identical and similar residues are shown in black and gray, respectively. Fig. 5 View largeDownload slide The phylogeny of plant bHLH domains. The numbers associated with each node indicate the percentage support provided by a bootstrap analysis (1,000 replicates). Fig. 5 View largeDownload slide The phylogeny of plant bHLH domains. The numbers associated with each node indicate the percentage support provided by a bootstrap analysis (1,000 replicates). Subcellular localization and transactivation activity Transient expression of the p35S::PabHLH-GFP (green fluorescent protein) transgene in onion epidermal cells was used to determine in which subcellular compartment the PabHLH product was deposited. While the control the p35S::GFP transgene generated signal throughout the cell, that generated by p35S::PabHLH-GFP was restricted to the nucleus (Fig. 6A). The transactivation assay carried out in yeast was based on either the full or certain truncated versions of the PabHLH coding sequence fused in-frame with the GAL4 DNA-binding domain. All transformants grew well on the selective SD-Trp medium, whereas those carrying the full-length PabHLH sequence exhibited weak growth on SD-Trp-His-Ade medium. Cells harboring either an empty vector or encoding an N-terminal (amino acids 1–160) or C-terminal (amino acids 274–521) truncated form of PabHLH were unable to survive on the SD-Trp-Ade-His medium. However cells harboring the PabHLH fragment encoding residues 1–273 or 161–521 were able to grow on the selective medium (Fig. 6B). The conclusion was that the transactivation activity of PabHLH relied on the sequence lying between amino acids 16 and 273, and not on its bHLH domain. Fig. 6 View largeDownload slide Subcellular localization and transactivation assays of PabHLH. (A) Patterns of expression (GFP signal) derived from the transient expression in onion epidermal cells of the transgenes p35S::GFP or p35S::PabHLH-GFP. (B) Transactivation by PabHLH in yeast. Either the full-length or four different truncated versions of PabHLH were transformed into yeast, and the transgenic cells were cultured on either SD-Trp or SD-Trp-His-Ade medium. Fig. 6 View largeDownload slide Subcellular localization and transactivation assays of PabHLH. (A) Patterns of expression (GFP signal) derived from the transient expression in onion epidermal cells of the transgenes p35S::GFP or p35S::PabHLH-GFP. (B) Transactivation by PabHLH in yeast. Either the full-length or four different truncated versions of PabHLH were transformed into yeast, and the transgenic cells were cultured on either SD-Trp or SD-Trp-His-Ade medium. The accumulation of bisbibenzyls in transgenic P. appendiculatum callus Transgenic P. appendiculatum callus was generated in which PabHLH was either overexpressed (OE) or RNAi-suppressed (RNAi). The OE and RNAi constructs were successfully transformed into P. appendiculatum using Agrobacterium-mediated genetic transformation (Fig. 7A). The transgenic status of the material was confirmed by a genomic DNA (gDNA)-based PCR assay (Supplementary Fig. S1) based on a primer pair amplifying hptII, and three independent transformants per construct were chosen for further analysis. There was no obvious morphological difference between the OE and RNAi callus. On the basis of a quantitative real-time PCR (qRT-PCR) assay, the abundance of PabHLH transcript proved to be >5-fold higher in the three OE calli (OE-1, 2 and 3) than in those of the wild type (WT), while the abundance in the three RNAi calli (RNAi-1, 2, 3) was lower than in the WT callus (Fig. 7B). Analysis of the bibenzyl and bisbibenzyl content of the OE, RNAi and WT callus showed that the level of IMC (iso-marchantin C) and neo-marchantin A (NMA) in the OE callus was about 2-fold that in the WT callus, whereas their levels in the RNAi callus were 6.5% and 17%, respectively, of the WT levels. The content of riccardin C (RC) and PE (perrottetin E) was marginally increased by PabHLH overexpression and slightly reduced by PabHLH suppression. There was no genotypic variation for the content of lucularic acid (Fig. 7C, D). An examination of which genes putatively responsible for bisbibenzyl biosynthesis were regulated by PabHLH showed that PaPAL, Pa4CL1 and PaSTCS1 were all up-regulated in the OE callus, as were the Cyt P450-encoding genes P450-3 and P450-4 (Fig. 8). In the RNAi callus, PaPAL, Pa4CL1, PaSTCS1, P450-3 and P450-4 were all down-regulated. The abundance of transcript produced by PaC4H, Pa4CL2, P450-1 and P450-2 was unaffected by the presence of either transgene. Fig. 7 View largeDownload slide PabHLH transcription and bisbibenzyl content in transgenic P. appendiculatum callus. (A) a, Prior to transfection with A. tumefaciens; b, callus co-cultured with A. tumefaciens; c, 2 weeks after transfer to a selective medium; d, clones grown on a selective medium for 3 weeks. (B) The abundance of PabHLH transcript in transgenic callus. (C, D) The bisbibenzyl content of transgenic callus. (C) Representative HPLC profiles of methanolic extracts from the WT, the OE and RNAi transgenic callus. The major bisbibenzyls identified were: P1, lucularic acid; P2, RC; P3, PE; P4, IMC; P5, NMA. (D) Comparison of the bisbibenzyl content in clones of WT vs. OE (left) and WT vs. RNAi (right). Data are shown in the form mean ± SD (n = 3). *,**Means differ significantly from the WT level at P < 0.05 and <0.01, respectively. Fig. 7 View largeDownload slide PabHLH transcription and bisbibenzyl content in transgenic P. appendiculatum callus. (A) a, Prior to transfection with A. tumefaciens; b, callus co-cultured with A. tumefaciens; c, 2 weeks after transfer to a selective medium; d, clones grown on a selective medium for 3 weeks. (B) The abundance of PabHLH transcript in transgenic callus. (C, D) The bisbibenzyl content of transgenic callus. (C) Representative HPLC profiles of methanolic extracts from the WT, the OE and RNAi transgenic callus. The major bisbibenzyls identified were: P1, lucularic acid; P2, RC; P3, PE; P4, IMC; P5, NMA. (D) Comparison of the bisbibenzyl content in clones of WT vs. OE (left) and WT vs. RNAi (right). Data are shown in the form mean ± SD (n = 3). *,**Means differ significantly from the WT level at P < 0.05 and <0.01, respectively. Fig. 8 View largeDownload slide Transcriptional profiling of genes associated with bisbibenzyl biosynthesis in transgenic P. appendiculatum callus, as derived by qRT-PCR. Data are shown in the form mean ± SD (n = 3). *,**Means differ significantly from the WT level at P < 0.05 and <0.01, respectively. Fig. 8 View largeDownload slide Transcriptional profiling of genes associated with bisbibenzyl biosynthesis in transgenic P. appendiculatum callus, as derived by qRT-PCR. Data are shown in the form mean ± SD (n = 3). *,**Means differ significantly from the WT level at P < 0.05 and <0.01, respectively. The accumulation of bisbibenzyls and flavonoids in transgenic P. appendiculatum thallus Pagiochasma appendiculatum axenically cultured thalli were transformed by A. tumefaciens harboring the OE and RNAi transgenes, and also by an empty vector to provide a control (VC) (Fig. 9A). The amplification of the putative transformants’ gDNA confirmed the presence of PabHLH in each selection (Supplementary Fig. S2). In each of the three selected transgenic OE thalli (OE-1', -2' and -3'), the abundance of PabHLH transcript was increased by at least 3.5-fold, while in the three RNAi calli (RNAi-1', -2' and -3') it fell to between 50% and 60% of the level found in the VC thallus (Fig. 9B). The content of riccardin D (RD) in the OE thallus was 140% of that in the VC thallus, while in the RNAi thallus, it was about 80% (Fig. 9C, D). Similar to in the transgenic callus, the genes PaPAL, Pa4CL1, PaSTCS1, P450-3 and P450-4 (especially Pa4CL1 and P450-4) were all up-regulated in the OE material, and were down-regulated in the RNAi thalli; the abundance of PaC4H, Pa4CL2, P450-1 and P450-2 transcript did not vary genotypically (Fig. 10). The OE thalli accumulated a higher flavonoid content than did the VC thallus, which in turn accumulated more than the RNAi thalli (Fig. 9E). Among the genes involved in flavonoid synthesis, PaCHS and PaFNSI, were up-regulated in the OE and down-regulated in the RNAi thalli; the intensity of transcription of PaCHI was invariant (Fig. 10). Taken together, these results demonstrated clearly that PabHLH is a positive TF contributing to the production of bisbibenzyls in P. appendiculatum, and also indicated that the accumulation of bisbibenzyls was along with the biosynthesis of flavonoids which was the regulated object of bHLH subgroup IIIf in higher plants. Fig. 9 View largeDownload slide PabHLH transcript abundance and the content of riccardin D and flavonoids in thallus carrying either an empty vector (VC) or the OE or RNAi transgenes. (A) a, Prior to transfection with A. tumefaciens; b, thallus co-cultured with A. tumefaciens; c, 2 weeks after transfer to a selective medium; d, clones grown on a selective medium for 3 weeks. (B) The abundance of PabHLH transcript in transgenic thallus. (C–E) The riccardin D and flavonoid content of transgenic thallus. (C) Representative HPLC profiles of methanolic extracts from VC, OE and RNAi transgenic callus. RD, authentic riccardin D; IS, internal standard (baicalein). (D, E) The content in transgenic thallus of (D) riccardin D and (E) flavonoids. Data are shown in the form mean ± SD (n = 3). *,**Means differ significantly from the VC level at P < 0.05 and <0.01, respectively. Fig. 9 View largeDownload slide PabHLH transcript abundance and the content of riccardin D and flavonoids in thallus carrying either an empty vector (VC) or the OE or RNAi transgenes. (A) a, Prior to transfection with A. tumefaciens; b, thallus co-cultured with A. tumefaciens; c, 2 weeks after transfer to a selective medium; d, clones grown on a selective medium for 3 weeks. (B) The abundance of PabHLH transcript in transgenic thallus. (C–E) The riccardin D and flavonoid content of transgenic thallus. (C) Representative HPLC profiles of methanolic extracts from VC, OE and RNAi transgenic callus. RD, authentic riccardin D; IS, internal standard (baicalein). (D, E) The content in transgenic thallus of (D) riccardin D and (E) flavonoids. Data are shown in the form mean ± SD (n = 3). *,**Means differ significantly from the VC level at P < 0.05 and <0.01, respectively. Fig. 10 View largeDownload slide Transcriptional profiling of genes associated with riccardin D and ﬂavonoid synthesis in transgenic P. appendiculatum thallus, as derived by qRT-PCR. Data are shown as mean ± SD (n = 3). *,**Means differ significantly from the VC level at P < 0.05 and <0.01, respectively. Fig. 10 View largeDownload slide Transcriptional profiling of genes associated with riccardin D and ﬂavonoid synthesis in transgenic P. appendiculatum thallus, as derived by qRT-PCR. Data are shown as mean ± SD (n = 3). *,**Means differ significantly from the VC level at P < 0.05 and <0.01, respectively. Transgenic thallus suppresses the growth of Candida albicans RD is known to express antibiosis against the fungal pathogen C. albicans (Cheng et al. 2009, Wu et al. 2010). When C. albicans was cultured on a medium containing crude extracts prepared from OE, RNAi and VC thallus, its mycelial growth was most strongly inhibited by the OE extract (Fig. 11). The fungi which developed on this Spider medium produced no hyphae and their outer surface remained smooth (Fig. 11B, C). The effect of a 6 h exposure to RD in RPMI 1640 medium was the suppression of hypha formation (Fig. 11A). We concluded that PabHLH-OE thallus interfered with the hyphal formation of C. albicans through accumulation of RD in P. appendiculatum thallus. Fig. 11 View largeDownload slide In vitro assay of the anti-fungal activity of riccardin D present in the crude extract of transgenic P. appendiculatum thallus. (A, B) Hyphal growth of C. albicans treated with crude extract from the VC, OE and RNAi transgenics. (A) Inoculated on solidified Spider medium; images taken after 5 d of incubation. (B) Inoculated into liquid RPMI 1640 medium; images taken 6 h after inoculation. (C) The diameter of the fungal colonies formed on the solidified medium. Data are shown in the form mean ± SD (n = 3). **Means differ significantly from the OE level at P < 0.01. NA, a negative control medium containing no plant extract. Fig. 11 View largeDownload slide In vitro assay of the anti-fungal activity of riccardin D present in the crude extract of transgenic P. appendiculatum thallus. (A, B) Hyphal growth of C. albicans treated with crude extract from the VC, OE and RNAi transgenics. (A) Inoculated on solidified Spider medium; images taken after 5 d of incubation. (B) Inoculated into liquid RPMI 1640 medium; images taken 6 h after inoculation. (C) The diameter of the fungal colonies formed on the solidified medium. Data are shown in the form mean ± SD (n = 3). **Means differ significantly from the OE level at P < 0.01. NA, a negative control medium containing no plant extract. Discussion The bryophytes are the most primitive terrestrial plants, having been the pioneers to evolve ways to survive outside of the marine/aqueous environment. The liverworts produce a variety of metabolites, among which the bibenzyls and cyclic bisbibenzyls are structurally unique; some of these compounds possess beneficial biological activity (Lou et al. 2002, Xie et al. 2010, Asakawa and Ludwiczuk 2012). Higher plant bHLH TFs are responsible for a number of important biological functions (Pires and Dolan 2010) and, in particular, those assigned to subgroup IIIf regulate flavonoid metabolism (Heim et al. 2003). Similar to flavonoids, cyclic bisbibenzyls also come from the phenylpropanoid and polyketide pathway, as known through isotope tracking experiments (Friederich, et al. 1999a, Friederich, et al. 1999b). The biosynthesis of cyclic bisbibenzyls and flavonoids is driven by a general set of enzyme systems in liverworts. Hence it was thought likely that bisbibenzyl biosynthesis would be regulated in the liverworts by a member of the bHLH TF family. The variation in bisbibenzyl content between P. appendiculatum thallus growing in the greenhouse, axenically cultured thallus and callus was correlated with the abundance of PabHLH transcript. An important property of plant bHLHs is their transcriptional responsiveness to stress (Kennck and Crane 1997, Yamaguchi-Shinozaki and Shinozaki 2006). Here, PabHLH was strongly induced when the plant material was challenged by exposure to either SA or MeJA (Fig. 3). As previously demonstrated, hormone stimulation not only up-regulates PaPAL, but also enhances the content of bisbibenzyls (Yu et al. 2014). The PabHLH sequence was shown to harbor a conserved bHLH domain (Fig. 4B), even though the rest of its sequence was only distantly related to those of the plant bHLHs AtTT8, IpIVS, PhAN1 and VvMYC1. Since protein architecture is generally well conserved within a specific subfamily (Hichri et al. 2011), it seemed reasonable to infer phylogeny here based on the sequence of just the bHLH domain. On this basis, it was possible to categorize PabHLH as a subgroup IIIf member (Fig. 5). Other members of this subgroup, notably AtTT8 (Park et al. 2004) and IpIVS (Park et al. 2004, Park et al. 2007), control both the anthocyanin and proanthocyanidin pathways, while both PhAN1 and VvMYC1 act to promote the accumulation of anthocyanin (Quattrocchio et al. 1993, Hichri et al. 2010). It is reported that the bHLH genes regulating anthocyanin synthesis are divided into two subgroups. Usually, the bHLH domain in one subgroup is separated by an intron, while the other is not. The PabHLH genomic sequence featured three exons, while the sequence encoding its bHLH domain was intron free (Fig. 4A), thereby mirroring the structure of IpIVS, PhAN1 (Spelt et al. 2000) and AtTT8 (Nesi et al. 2000), but not that of either JAF13 and ZmIn1, which both feature a two-exon bHLH domain. The suggestion is therefore that PabHLH is an ortholog of AtTT8, PhAN1 and IpIVS. Both the localization and transactivational activity of PabHLH were consistent with its functioning as a TF (Fig. 6). Thus, a body of evidence has been provided to support the notion that PabHLH is a subgroup IIIf TF involved in the regulation of bisbibenzyl biosynthesis in P. appendiculatum. The most abundant bisbibenzyls formed in P. appendiculatum callus were IMC and NMA, both of which are derived from the condensation of a C–O bond linking the two bibenzyl moieties; the predominant bisbibenzyl present in thallus was RD, which forms from the condensation of a C–C bond. The accumulation of bisbibenzyls was enhanced by the overexpression of PabHLH in the callus; the transgenic cells produced more IMC and NMA than did WT tissue, and RC and PE contents were slightly increased. Liverworts produce a variety of bisbibenzyls, and their biosynthesis might be regulated by different TFs. The RD content of the OE thallus was higher than that in the VC thallus. The knock-down of PabHLH, achieved by introducing an RNAi construct, led to a pronounced fall in the accumulation of bisbibenzyls in both callus and thallus samples (Figs. 7D, 9D). As a consequence of the induced variation in the content of RD, OE thalli significantly inhibited the growth of C. albicans hyphae, while the RNAi thalli were more permissive of fungal growth (Fig. 11). Different bHLHs have different regulating mechanisms, and even the same bHLH has differential regulating mechanisms in various plants. For example, in the MJOr mutant of the dahlia variety ‘Michael J’, the DvIVS TF activates the genes DvCHS1, DvF3H, DvDFR and DvANS to synthesize anthocyanidin, flavone and butein, whereas in the MJY mutant, none of these genes is activated (Ohno et al. 2011). Here, it was possible to show that PabHLH controls the biosynthesis of bisbibenzyls via its regulation of PAL, 4CL1, STCS1, P450-3 and P450-4 (Figs. 8, 10). The gene encoding the STCS1 enzyme, considered as a branching point within the synthetic pathway leading to lucularic acid (Eckermann et al. 2003), was up-regulated in the OE and down-regulated in the RNAi materials, but there was no evidence for variation in lucularic acid content in the callus (the lucularic acid content in the thallus was low). Potentially, lucularic acid was consumed to form downstream target bisbibenzyl by efficient enzymatic reactions. As was the case with the bisbibenzyls, the flavonoid content of the thallus was higher in the OE than in the WT material, and lower in the RNAi material (Fig. 9E), in keeping with previous analyses of flavonoid accumulation in higher plants (Hichri et al. 2010). The abundance of PabHLH transcript also influenced the transcription of CHS and FNSI (Fig. 10), both of which encode key components involved in flavonoid accumulation. Both the upstream and downstream structural genes of bisbibenzyl and flavonoid synthesis appear to be activated under regulation by PabHLH in liverworts. This study has presented evidence suggesting that PabHLH acts as a positive regulator of bisbibenzyl biosynthesis in P. appendiculatum. The implication is that genetic engineering targeting TFs such as PabHLH could represent a viable strategy for sustainably generating bisbibenzyl compounds of interest to the pharmaceutical industry. The present priority is focused on identifying the downstream interactors of PabHLH, since this information will aid in the harnessing of bisbibenzyl biosynthesis through the genetic transformation of liverworts. Materials and Methods Plant materials and phytohormone treatment Plagiochasma appendiculatum thalli were maintained in a growth chamber held at 22°C supplying a 12 h photoperiod. Axenic thallus and callus were cultured, respectively, on half-strength Murashige and Skoog (MS) medium supplemented with 0.5 mg l−1 6-benzyladenine, and full-strength MS medium containing 1 mg l−1 2,4-D. Thallus and callus tissue which developed after a culture period of 2 weeks was used for transformation. The presence of JA and SA in P. appendiculatum was checked by ZOONBIO BIOTECHNOLOGY company (for details, see Supplementary method S1). Two-month-old thalli were exposed to either 100 μM MeJA or 100 μM SA for 0, 6, 12, 24, 36, 48, 60 and 72 h to assess their phytohormonal response. The samples were snap-frozen in liquid nitrogen and stored at –80°C until required. Nucleic acid isolation and gene expression profiling Genomic DNA was extracted from fresh P. appendiculatum thallus and callus following the CTAB (cetyltrimethylammonium bromide) method (Porebski et al. 1997). Total RNA was extracted using an RNAprep pure plant kit (Tiangen), following the manufacturer’s protocol. The quality and concentration of the resulting gDNAs and RNAs were monitored by both agarose gel electrophoresis and spectrophotometry (BioPhotometerplus, Eppendorf). The first cDNA strand was generated from a 1 μg aliquot of RNA using a PrimeScript RT Master Mix kit (TAKARA), and this was subsequently used as the template for PCRs to assess transcript abundance. A gene encoding a P. appendiculatum elongation factor (Yu et al. 2015) was used as the reference sequence. A segment of the PabHLH sequence was amplified using the primer pair PabHLH-RTF/R (Supplementary Table S2) via a qRT-PCR assay. The qRT-PCRs were based on PrimeSTAR®Max DNA Polymerase (TAKARA) using the manufacturer’s protocol. Isolation of PabHLH cDNA and gDNA A 3' fragment of the PabHLH coding region sequence was obtained from a sequenced normalized P. appendiculatum thallus cDNA library (Cheng et al. 2013). The full-length cDNA sequence was derived using the 5'-RACE technique, based on a Smarter™ Race cDNA amplification kit (Clontech) in conjunction with the primers PabHLHGSP and PabHLHNGSP (sequences given in Supplementary Table S1). The full-length PabHLH sequence was reconstructed from those of overlapping partial cDNA fragments. Subsequently, the primer pair PabHLH-qF/R (Supplementary Table S1) was designed to amplify the full-length cDNA and gDNA sequences. Amplicons of the expected length were cloned into pMD19-T (TAKARA) for validation by sequencing. Identification, multiple sequence alignment and phylogenetic analysis of PabHLH A functional annotation of the predicted PabHLH polypeptide was inferred from the outcome of a Blast alignment (www.ncbi.nlm.nih.gov/BLAST/) against publicly available nucleotide and protein data sets. Its theoretical pI and molecular weights were determined using the ExPASy Compute pI/Mw tool (web.expasy.org/compute_pi/). The deduced bHLH domain protein sequence of PabHLH was aligned with that of a collection of other plant bHLHs using DNAMAN v5.2.2 software (LynnonBiosoft). The 66 plant comparator sequences were obtained from A. thaliana, Vitis vinifera, Oryza sativa, Petunia hybrida, Zea mays, Malus domestica, Perilla frutescens, Ipomoea purpurea, Gentiana triﬂora and Pisum sativum. A phylogenetic tree was constructed based on the residues spanning the bHLH core domain. MEGA v4.0 software (Tamura et al. 2007) was used to build a Neighbor–Joining tree. The statistical reliability of nodes of the tree was assessed by bootstrap analyses with 1,000 replicates. Subcellular localization and transactivation assay The sequence of the ORF of PabHLH, lacking its stop codon, was PCR-amplified using the primer pair PabHLH-F/GFPR (Supplementary Table S1), and the resulting amplicon was inserted into the modified pCAMBIA1301-GFP to create a p35S::PabHLH-GFP construct. After validation by sequencing, the construct was transiently expressed in onion epidermal cells using an Agrobacterium tumefaciens-mediated method (Ishizaki et al. 2008); control transformants were created using a p35S::GFP plasmid. The location of the fusion protein in transformed cells was observed via confocal laser scanning microscopy (LSM700, Zeiss). The full-length PabHLH ORF and truncated ORF fragments corresponding to residues 1–160, 1–273, 161–521 and 274–521 were PCR-amplified with primers which included terminal EcoRI and BamHI restriction sites (Supplementary Table S1), and the resulting amplicons were then introduced into pGBKT7 (Clontech). Recombinant plasmids were transformed separately into yeast strain AH109 (Clontech), as described elsewhere (He et al. 2011). Transformants were grown on SD medium lacking tryptophan (SD-Trp) for positive clone selection and then on SD medium lacking tryptophan, histidine and adenine (SD-Trp-His-Ade) for the transactivation assay. Construction of a PabHLH overexpression vector and an RNAi suppression transgene To construct a PabHLH overexpression vector, the coding sequence of PabHLH was amplified using the primer pair PabHLHF/R (Supplementary Table S1). The RNAi construct designed to down-regulate PabHLH was assembled in the pBluescriptSK(–) vector (Stratagene). The sense and antisense arms of the inverted repeat element were based on the sequence of a PabHLH cDNA 5' fragment. The sense arms, including a SmaI and a XbaI restriction site, were PCR-amplified using the primer pair PabHLH-RNAiSF/R (Supplementary Table S1). The primer pair PabHLH-RNAiASF/R (Supplementary Table S1) was used to amplify the antisense arms, thereby introducing a NotI and a SacI restriction site to facilitate hairpin formation. Each fragment was then cloned sequentially into pBluescriptSK(–). The final pBluescriptSK(–) plasmids, housing either the RNAi sense and antisense arms or the overexpression sequence, were digested with the appropriate restriction enzymes and cloned into the modified pCAMBIA1301 to ensure that the transgene was under the control of the Cauliflower mosaic virus (CaMV) 35S promoter. The constructs were finally transferred into A. tumefaciens strain EHA105 using the freeze–thaw method (Danzer et al. 2015). Transformation of PabHLH into P. appendiculatum callus and thallus A 1 ml aliquot of an overnight A. tumefaciens culture was inoculated into 100 ml of yeast extract peptone medium supplemented with 50 mg l−1 kanamycin and 50 mg l−1 rifamycin, and cultured until the OD600 had reached 1.5–2.0. The cells were then harvested by centrifugation and resuspended in 50 ml of liquid MS medium supplemented with 100 μM acetosyringone (Sigma-Aldrich). Plagiochasma appendiculatum axenically cultured callus or thallus grown for 2 weeks was immersed in a suspension of the bacterial cells, held for 1 h with gentle shaking and then plated onto solidified MS medium containing 100 μM acetosyringone and left in the dark for 2–3 d. The samples were subsequently rinsed four times in sterile water, then transferred to a medium containing 200 mg l−1 cefotaxime and 25 mg l−1 hygromycin to select for transgenic cells. After a further 2–3 weeks, surviving callus/thallus were transferred to a medium containing 50 mg l−1 hygromycin, on which they were subcultured at 2 week intervals. Genomic DNA and RNA were extracted from transgenic and WT material: the former was used as a template in a PCR designed to identify the presence of hptII (primer pair Hpt1/Hpt6a; see Supplementary Table S1), and the latter was reverse-transcribed to cDNA as described above and used as an RT-PCR template to quantify the abundance of the transcripts. Bisbibenzyl extraction and HPLC analysis The bisbibenzyl content of P. appendiculatum callus/thallus was derived from a 100 mg FW sample of both transgenic and WT callus and from a 10 mg DW sample of powdered thallus; each was extracted overnight with shaking in 100 μl of methanol. After centrifugation (15,000×g, 10 min), the supernatant was injected into an Agilent (Agilent Technologies) 1260 HPLC device equipped with a 250 mm×4.6 mm, 5 μm C18 column, and the products were detected at 280 nm. The eluate was a mixture of 0.1% (v/v) glacial acetic acid in water (A) and acetonitrile (B). The gradient was altered in a linear fashion from 60% A/40% B to 33% A/67% B over 45 min, then held at 10% A/90% B for 5 min, followed by 60% A/40% B for 5 min. The solvent flow rate was 1 ml min−1. The standards employed were lucularic acid, RC, RD, PE, IMC and NMA, all of which had been purified in-house. A standard curve of each reference compound was made to quantify the concentrations of bisbibenzyls in the form μg g–1 FW or mg g–1 DW. Determination of total flavonoid content The extraction and quantification of total flavonoids from transgenic thallus were performed using a colorimetric method (Wang et al. 2016). A 10 mg aliquot of powdered plant material was suspended in 400 μl of 1% (v/v) HCl-methanol and ultrasonicated for 1 h. The extract was thoroughly mixed with 400 μl of chloroform and 200 μl of deionized water to remove Chl. After centrifugation (15,000×g, 10 min), the upper aqueous phase was subjected to UV spectral analysis (Muir et al. 2001). Total flavonoid contents were reported in the form A340 value per g DW. Transcriptional profiling of genes involved in bisbibenzyl and flavonoid synthesis The transcription of PaPAL, PaC4H, Pa4CL1, Pa4CL2, PaCHS, PaCHI, PaFNSI and PaSTCS1, all of which encode proteins related to bisbibenzyl and/or flavonoid synthesis, was monitored using qRT-PCR. In addition, four genes encoding Cyt P450s, which were abundantly expressed in thallus as opposed to in callus (data not shown), were profiled. The primer sequences used for the qRT-PCRs are given in Supplementary Table S2. In vitro assays for anti-fungal activity An in vitro assay for anti-fungal activity was performed using either a solidified Spider medium or a liquid RPMI 1640 medium. Aliquots (20 mg DW) of transgenic thallus were ground into fine powder and extracted by immersion in 100 μl of methanol followed by sonication for 1 h. After centrifugation (15,000×g, 10 min), the supernatants were vacuum-dried in a SpeedVac device (Thermo Savant) and the residues were redissolved in 100 μl of dimethylsulfoxide (DMSO). The DMSO solution was degermed by passing through a 0.2 μm filter and added to the growth medium. For the morphological transition test using solidified Spider medium, a 100 μl aliquot of C. albicans strain SC5314 (2×102 cells ml−1 in phosphate-buffered saline) was dispersed over the plate, which was then cultured at 25°C for 5 d. For the assay of C. albicans growth in a liquid medium, the same strain was cultured (2×102 cells ml−1) in RPMI 1640 medium at 37°C for 6 h. Hyphal growth was monitored by light microscopy. Accession numbers Grapevine (V. vinifera) bHLH sequences were retrieved from the Plant TFDB (http://planttfdb.cbi.pku.edu.cn/), and the following sequences from the NCBI database: BPEp (Q0JXE7), BEE1 (Q8GZ13), AtRHD6 (Q9C707), AtRSL1 (Q9FJ00), RSL2 (OAO99783), RSL3 (Q7XHI9), RSL4 (Q8LEG1), RSL5 (Q3E7L7), SPT (Q9FUA4), ALC (Q9FHA2), PIL1 (Q8L5W8), PIF1 (Q8GZM7), PIF3 (O80536), PIF4 (Q8W2F3), FAMA (Q56YJ8), SPEECHLESS (Q700C7), MUTE (Q9M8K6), OsMUTE (XP015638702), ORG2 (Q9M1K1), ORG3 (Q9M1K0), OsIRO2 (FAA00382), HvIRO2 (BAF30424), RGE1 (Q9FXA3), ICE1 (Q9LSE2), SCRM2 (AQ9LPW3), AMS (Q9ZVX2), DYT1 (O81900), FIT (Q0V7X4), AtMYC2 (Q39204), ATR2 (Q9FIP9), PsGBF (ABD59338), AtAIB (Q9ZPY8), VvMYCA1 (EF193002), MdbHLH33 (ABB84474), AtMYC1 (Q8W2F1), EGL3 (Q9CAD0), GL3 (Q9FN69), PhJAF13 (AAC39455), PfMYC-RP (BAA75513), AmDEL (AAA32663), ZmLc (AAA33504), ZmB (CAA40544), OsRc (BAF42667), ZmIN1 (AAB03841), IpIVS (BAD18982), GtbHLH1 (BAH03387), PhAN1 (AAG25928), VvMYC1 (EU447172) and AtTT8 (Q9FT81). 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Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations 4CL 4-coumarate:coenzyme A ligase bHLH basic helix–loop–helix C4H cinnamic acid 4-hydroxylase gDNA , genomic DNA GFP , gren fluorescent protein IMC , iso-marchantin C MeJA methyl jasmonate MS Murashige and Skoog NMA neo-marchantin A OE overexpressed ORF open reading frame PAL phenylalanine ammonia lyase PE perrottetin E qRT-PCR quantitative real-time PCR RACE rapid amplification of cDNA ends RC riccardin C RD riccardin D RNAi RNAi interference SA salicylic acid STCS stilbene carboxylate synthase TF transcription factor VC vector control WT wild type Footnote Footnote The nucleotide sequence reported in this paper has been submitted to the NCBI database with accession number MF983804 © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. 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Plant and Cell Physiology – Oxford University Press
Published: Mar 8, 2018
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