A transcript profiling approach reveals an epicatechin-specific glucosyltransferase expressed in the seed coat of Medicago truncatula

Abstract

Expression of the Arabidopsis TRANSPARENT TESTA 2 (TT2) MYB family transcription factor leads to massive accumulation of proanthocyanidins (PAs) in hairy roots of Medicago truncatula. Microarray analysis showed that TT2 induces genes for flavonoid/PA biosynthesis, transcription factors, and a large number of genes of unknown function. A second microarray dataset identified genes that were preferentially expressed in the M. truncatula seed coat. Comparison of the two datasets defines target genes for steps that are yet unidentified in PA biosynthesis and accumulation. Of these genes, a glycosyltransferase, UGT72L1, was active specifically toward the PA precursor (؊)؊epicatechin, and its expression pattern in developing seeds correlated with the presence of epicatechin glucoside and accumulation of PAs. UGT72L1 may be involved in the production of epicatechin 3 -O-glucoside in the seed coat as a key step in PA biosynthesis or its regulation.glucosyltransferase ͉ model legume ͉ proanthocyanidin

MYB5 and MYB14 Play Pivotal Roles in Seed Coat Polymer Biosynthesis in Medicago truncatula

“…MtPAR, another TT2-related MYB transcription factor in M. truncatula, also promotes PA accumulation in M. truncatula hairy roots and alfalfa plants (Verdier et al, 2012). PA accumulation is also induced by strong expression of Arabidopsis TT2 in M. truncatula hairy roots (Pang et al, 2008). These results indicate that there is some degree of promiscuity with regard to the MYB components of the MBW complex, at least in heterologous systems.…”

“…ANR was induced more than 250-fold in MYB5-overexpressing hairy roots and 985-fold in MYB14-overexpressing hairy roots. These extremely high values mirror the effects of high overexpression of AtTT2 in M. truncatula hairy roots (Pang et al, 2008) and are, in part, explicable in terms of extremely low ANR expression in control tissues. As in AtTT2-expressing M. truncatula hairy roots, UGT72L1, a glycosyltransferase specific for epicatechin (Pang et al, 2008), was also highly induced in MYB5-and MYB14-expressing hairy roots.…”

“…These extremely high values mirror the effects of high overexpression of AtTT2 in M. truncatula hairy roots (Pang et al, 2008) and are, in part, explicable in terms of extremely low ANR expression in control tissues. As in AtTT2-expressing M. truncatula hairy roots, UGT72L1, a glycosyltransferase specific for epicatechin (Pang et al, 2008), was also highly induced in MYB5-and MYB14-expressing hairy roots. Besides PA pathway-specific genes, genes encoding enzymes functioning upstream of ANR or LAR, such as CHS, F39H, and DFR, which could potentially be involved in both anthocyanin and PA biosynthesis, were also induced by MYB5 and MYB14.…”

“…For seeds, plant materials were first ground to a powder in liquid nitrogen and then freeze dried for 16 h. Approximately 50 mg of freeze-dried materials was then extracted. Extraction was three times with 1 mL of 70% (v/v) acetone and 0.5% (v/v) acetic acid, as described by Pang et al (2008). Assay of soluble PAs (with DMACA reagent) and insoluble PAs (with butanol-HCl) was performed as described previously (Pang et al, 2008).…”

“…epicatechin) by anthocyanidin reductase (ANR; Xie et al, 2003Xie et al, , 2004. Epicatechin can also be glycosylated and then transported to the vacuole by a multidrug and toxic compound extrusion transporter (Pang et al, 2008;Zhao and Dixon, 2009), where it is believed to be polymerized by a laccase to form PA oligomers (Pourcel et al, 2005).…”