Early steps in proanthocyanidin biosynthesis in the model legume Medicago truncatula

Abstract

Oligomeric proanthocyanidins (PAs) composed primarily of epicatechin units accumulate in the seed coats of the model legume Medicago truncatula, reaching maximal levels at around 20 d after pollination. Genes encoding the single Medicago anthocyanidin synthase (ANS; EC 1.14.11.19) and leucoanthocyanidin reductase (LAR; EC 1.17.1.3) were cloned and the corresponding enzymes functionally identified. Recombinant MtANS converted leucocyanidin to cyanidin, and, more efficiently, dihydroquercetin to the flavonol quercetin. Levels of transcripts encoding dihydroflavonol reductase, ANS, and anthocyanidin reductase (ANR), the enzyme responsible for conversion of anthocyanidin to (-)-epicatechin, paralleled the accumulation of PAs in developing seeds, whereas LAR transcripts appeared to be more transiently expressed. LAR, ANS, and ANR proteins were localized to the cytosol in transfected tobacco (Nicotiana tabacum) leaves. Antisense down-regulation of ANS in M. truncatula resulted in reduced anthocyanin and PA levels, but had no impact on flavonol levels. Transgenic tobacco plants constitutively overexpressing MtLAR showed reduced anthocyanin content, but no catechin or increased levels of PAs were detected either in leaves or in flowers. Our results confirm previously ascribed in vivo functions for ANS and ANR. However, the apparent lack of catechin in M. truncatula PAs, the poor correlation between LAR expression and PA accumulation, and the lack of production of catechin monomers or oligomers in transgenic plants overexpressing MtLAR question the role of MtLAR in PA biosynthesis in Medicago.

Figure 1.

Schematic representation of the biosynthetic pathway for anthocyanins and PAs.

Figure 2.

PA levels in M. truncatula ‘Jemalong A17’. A, Levels of soluble PAs in a range of tissues from mature plants. B, Levels of insoluble PAs in a range of tissues from mature plants. C, Levels of soluble PAs in seeds at various stages of development (dap). D, Levels of insoluble PAs in seeds at various stages of development. Soluble PAs were determined by reaction with DMACA reagent, insoluble PAs by butanol-HCl hydrolysis and estimation of resulting anthocyanidin.

Figure 3.

Composition of PAs in the seed coat of M. truncatula. A, Size heterogeneity of the soluble PA fraction from mature seed coats as determined by normal-phase HPLC and postcolumn derivatization with DMACA reagent. Numbers with arrows indicate the estimated number of units based on parallel analysis of size standards from D. uncinatum. B, Size standards of the anthocyanidin products dephinidin, cyanidin, and pelargonidin resolved by HPLC. C, HPLC analysis of anthocyanidins released by acid-butanol hydrolysis of soluble PAs from developing seed coats. D, Catechin and epicatechin standards analyzed by HPLC; these arise from starter units following phloroglucinolysis of intact PAs. E, Analysis of the products (starter and extension units) released by phloroglucinolysis of intact PAs from mature seed coats; the phloroglucinol adducts arise from extension units. F, Mean DP of PAs in seeds at various stages of development.

Figure 4.

Alignment of deduced amino acid sequences of LAR genes. Sequences are from M. truncatula (ABE90657), grape (CAI56326), D. uncinatum (Q9SEV0), and L. corniculatus (ABC71327). Identical amino acids are indicated by white letters on a black background, conservative amino acids by white on a dark gray background, and similar amino acids by black on a light gray background. The RFLP, ICCN, and THD motifs are boxed.

Figure 5.

Expression and assay of recombinant MtLAR. A, Analysis of MtLAR protein on a 12.5% SDS-PAGE gel. M, Protein molecular weight markers; 1, total protein from E. coli M15 containing pQE30-MtLAR induced by IPTG; 2, purified recombinant MtLAR protein used for enzyme activity assay. B, HPLC analysis of an authentic standard of (+)-catechin. C, HPLC analysis of product from incubation of leucocyanidin with protein extract from vector control. D, Product from incubation of purified recombinant MtLAR with ³H-leucocyanidin monitored by UV absorption. E, Product from incubation of purified recombinant MtLAR with ³H-leucocyanidin monitored by scintillation counting.

Figure 6.

Expression and assay of recombinant MtANS. A, Analysis of MtANS protein on a 12.5% SDS-PAGE gel. M, Protein molecular weight markers; 1, total protein from E. coli M15 containing pQE30-MtANS induced by IPTG; 2, purified recombinant MtANS protein used for enzyme activity assay. B, HPLC analysis of an authentic standard of cyanidin. C, HPLC analysis of product from incubation of leucocyanidin with protein extract from vector control. D, Product from incubation of purified recombinant MtANS protein with ³H-leucocyanidin, monitored by UV absorption. E, Product from incubation of purified recombinant MtANS protein with ³H-leucocyanidin, monitored by scintillation counting.

Figure 7.

Transcript levels of flavonoid/PA pathway genes in different tissues of M. truncatula. A, Affymetrix microarray analysis of RNA from the six tissues shown. B, Quantitative real-time PCR analysis of ANS, LAR, and ANR transcript levels in pods, whole seeds, isolated seed coats, and seeds without seed coats. C to H, Relative transcript levels of the indicated genes (C, LAR; D, ANS; E, ANR; F, DFR1; G, DFR2; H, F3′H) in seed coats at various stages of seed development (dap) as determined by Affymetrix microarray analysis. Letters reflect differences that were statistically significant at P = 0.05 by t test.

Figure 8.

Anthocyanin and PA levels in flowers of transgenic tobacco constitutively expressing MtLAR. A, Anthocyanin levels as determined by extraction and UV absorption. B, Soluble PA levels as determined by extraction and reaction with DMACA reagent. C, Reverse-phase HPLC analysis (with postcolumn derivatization with DMACA) of the soluble PA fractions. Numbers refer to independent transgenic lines. N is empty-vector control line.

Figure 9.

Anthocyanin and PA levels in transgenic M. truncatula ‘R108’ plants expressing an antisense MtANS transgene. A, Visible phenotypes of the adaxial side (top) and abaxial side (bottom) of leaves from plants transformed with an empty-vector control (left) or expressing the MtANS antisense construct (right). Note the loss of visible anthocyanin pigmentation (regions marked with white arrows) in the antisense line. B, Anthocyanin content of leaf tissue from five independent transgenic antisense (designated A) lines or empty-vector control (N). C, Soluble PA levels in seeds of the above lines. D, Insoluble PA levels in seeds of the above lines. E, Levels of flavonols in leaves of the above lines. Soluble PAs were determined by reaction with DMACA reagent, insoluble PAs by butanol-HCl hydrolysis, and estimation of resulting anthocyanidin. Flavonols were determined by HPLC analysis (see Supplemental Fig. S8).