Tc-MYBPA an Arabidopsis TT2-like transcription factor and functions in the regulation of proanthocyanidin synthesis in Theobroma cacao

Abstract

Background: The flavan-3-ols catechin and epicatechin, and their polymerized oligomers, the proanthocyanidins (PAs, also called condensed tannins), accumulate to levels of up to 15 % of the total weight of dry seeds of Theobroma cacao L. These compounds have been associated with several health benefits in humans. They also play important roles in pest and disease defense throughout the plant. In Arabidopsis, the R2R3 type MYB transcription factor TT2 regulates the major genes leading to the synthesis of PA.

Results: To explore the transcriptional regulation of the PA synthesis pathway in cacao, we isolated and characterized an R2R3 type MYB transcription factor MYBPA from cacao. We examined the spatial and temporal gene expression patterns of the Tc-MYBPA gene and found it to be developmentally expressed in a manner consistent with its involvement in PAs and anthocyanin synthesis. Functional complementation of an Arabidopsis tt2 mutant with Tc-MYBPA suggested that it can functionally substitute the Arabidopsis TT2 gene. Interestingly, in addition to PA accumulation in seeds of the Tc-MYBPA expressing plants, we also observed an obvious increase of anthocyanidin accumulation in hypocotyls. We observed that overexpression of the Tc-MYBPA gene resulted in increased expression of several key genes encoding the major structural enzymes of the PA and anthocyanidin pathway, including DFR (dihydroflavanol reductase), LDOX (leucoanthocyanidin dioxygenase) and BAN (ANR, anthocyanidin reductase).

Conclusion: We conclude that the Tc-MYBPA gene that encodes an R2R3 type MYB transcription factor is an Arabidopsis TT2 like transcription factor, and may be involved in the regulation of both anthocyanin and PA synthesis in cacao. This research may provide molecular tools for breeding of cacao varieties with improved disease resistance and enhanced flavonoid profiles for nutritional and pharmaceutical applications.

Fig. 1

Working model of Anthocyanin and Proanthocyanidin synthesis pathway adapted from [23]. Enzymes are represented in uppercase bold letters; the products in the pathway are given in black boxes. The enzymes involved in the pathway are shown as follows: CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone-3β-hydroxylase; DFR, dihydroflavonol-4-reductase; LDOX, leucoanthocyanidin dioxygenase; FLS, flavonol synthase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; and UFGT, UDP-Glc:flavonoid-3-O-glucosyltransferase

Fig. 2

Comparison of the amino acid sequences of Tc-MYBPA and various plant MYB transcription factors. a Alignment of the deduced amino acid sequences of the R2R3-MYB proteins functioning in anthocyanin and PA synthesis, including Tc-MYBPA (cacao), ZmC1 (maize), VvMybPA1, VvMybPA2, VvMybA1, VvMybA2 (grape), PtMyb134 (poplar), LjTT2a, LjTT2b, LjTT2c (Lotus), and Arabidopsis regulators AtTT2, AtPAP1 and AtPAP2. The R2 and R3 repeats of the MYB domain are indicated above the alignment. Identical amino acids are indicated in black, similar amino acids in gray. Arrowheads indicate amino acids that are conserved in all PA-regulating MYBs but absent in anthocyanin-regulating MYBs. Sequences were aligned using the ClustalW program and were displayed using the GeneDoc program. b Phylogenetic tree showing selected plant MYB transcription factors from GenBank. Human c-myb is included as an outgroup. Functions of the MYB proteins are given on the right side in bold. The alignment was performed using the ClustalW program and the tree was constructed using the neighbor-joining algorithm of the MEGA package (Version 3.1). The scale bar represents 0.1 substitutions per site and the numbers next to each node are bootstrap values from 1000 replicates. The GenBank accession numbers of the MYB proteins are as follows: AtGL1 (P27900), ZmP (P27898), ZmC1 (AAA33482), VvMybA1 (BAD18977), VvMybA2 (BAD18978), AtPAP1 (AAG42001), PhAN2 (AAF66727), LeANT1 (AAQ55181), OsMyb4 (T02988), AmMixta (CAA55725), AtMyb111 (AAK97396), AtMyb12 (NM_130314), PmMybF1 (AAA82943), PhPH4 (AAY51377), AtPAP2 (AAG42002), AtMybWER (CAC01874), VvMyb5a (AAS68190), VvMYB5b (Q58QD0), VvMYBPA1 (AM259485), VvMybPA2 (ACK56131), c-myb (AAB49039), PtMyb134 (FJ573151), PhMyb1 (Z13996), LjTT2a (AB300033), LjTT2b (AB300034), LjTT2c (AB300035), AtTT2 (Q2FJA2), MtMybPAR (ADU78729), TaMyb14 (AFJ53053) Also included in the tree are one putative cacao PA specific MYB (Tc-MYBPA), and three MYB-like proteins found in the cacao EST collections (CL158Contig1, CL8212Contig1 and CL2621Contig1)

Fig. 3

Expression of Tc-MYBPA, TcANR, TcANS and TcLAR genes and accumulation of PAs in Theobroma cacao (Scavina 6; S6) leaves and flowers at various developmental stages. a Transcript levels of Tc-MYBPA, TcANR, TcANS and TcLAR. Expression was determined by semi-quantitative RT-PCR and was calculated relative to the expression of TcActin in each sample. b Levels of soluble PAs expressed as mg PA per g of fresh weight. c Levels of insoluble PAs expressed as mg PA per g of fresh weight. All data are presented as means ± SE, for gene expression data, n ≥ 3, for PA level data, n ≥ 5. FW, Fresh weight

Fig. 4

Expression of Tc-MYBPA, TcANR, TcANS and TcLAR genes and accumulation of PAs in whole pods of Theobroma cacao (Amelonado) during early stages of pod development (from 2 to 6 weeks after pollination). a Transcript levels of TcANR, TcANS and TcLAR. Expression was determined by semi-quantitative RT-PCR and was calculated relative to the expression of TcActin in each sample. b Levels of total soluble PAs expressed as mg PAs per g of fresh weight. c Levels of total insoluble PAs expressed as μg PAs per g of fresh weight. All data are presented as means ± SE. For gene expression data, n ≥ 3, for PA accumulation data, n ≥ 5. FW, Fresh weight

Fig. 5

Expression of Tc-MYBPA, TcANR, TcANS and TcLAR genes and accumulation of PAs in pod exocarp of Theobroma cacao (Amelonado) during pod development (from 8 to 20 weeks after pollination). a Transcript levels of Tc-MYBPA, TcANR, TcANS and TcLAR. Expression was determined by semi-quantitative RT-PCR and was calculated relative to the expression of TcActin in each sample. b Levels of total soluble PAs expressed as mg PAs per g of fresh weight. c Levels of total insoluble PAs expressed as μg PAs per g of fresh weight. All data are presented as means ± SE, for gene expression data, n ≥ 3, for PA level data, n ≥ 5. FW, Fresh weight

Fig. 6

Expression of Tc-MYBPA, TcANR, TcANS and TcLAR genes and accumulation of PAs in ovules of Theobroma cacao (Amelonado) during pod development (from 8 to 20 weeks after pollination). a Transcript levels of Tc-MYBPA, TcANR, TcANS and TcLAR. Expression was determined by semi-quantitative RT-PCR and was calculated relative to the expression of TcActin in each sample. b Levels of total soluble PAs expressed as mg PAs per g of fresh weight. c Levels of total insoluble PAs expressed as ug PAs per g of fresh weight. All data are presented as means ± SE, for gene expression data, n ≥ 3, for PA level data, n ≥ 5. FW, Fresh weight

Fig. 7

Complementation of the PA-deficient tt2 mutant phenotype by constitutively expressing Tc-MYBPA. a 7-day old seedlings of and DMACA stained seeds from Col-0, the tt2 mutant (SALK_005260) and three independent T2 transgenic lines of tt- 35S:Tc-MYBPA. The bar represents 1 mm. b RT-PCR analysis of Tc-MYBPA and AtUbiquitin transcripts in total RNA from the young seedlings shown in (a). PCR products from the Tc-MYBPA-pGEM plasmid were loaded on the last lane as a positive control for the Tc-MYBPA primer set and as a negative control for the AtUbiquitin primer set. C, PA levels in mature seeds of plants shown in (a). PA levels were determined by extraction and DMACA reaction using procyanidin B2 as a standard. All the data are presented as means ± SE, n = 3. **P < 0.01 versus tt2; ***P < 0.001 versus tt2. FW, fresh weight

Fig. 8

Semi-quantitative RT-PCR analysis of expression of flavonoid structural genes in young seedlings of the same Arabidopsis lines analyzed in Fig. 6. DFR, dihydroflavonol reductase; LDOX, leucoanthocyanidin dioxygenase; BAN, banyuls (anthocyanidin reductase); UFGT, UDP-Glc flavonoid glucosyltransferase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavonoid 3’-hydroxylase; FLS, flavonol synthase, UBi, Ubiquitin