Identification and biochemical characterization of mutants in the proanthocyanidin pathway in Arabidopsis

Abstract

Proanthocyanidin (PA), or condensed tannin, is a polymeric flavanol that accumulates in a number of tissues in a wide variety of plants. In Arabidopsis, we found that PA precursors (detected histochemically using OsO(4)) accumulate in the endothelial cell layer of the seed coat from the two-terminal cell stage of embryo development onwards. To understand how PA is made, we screened mature seed pools of T-DNA-tagged Arabidopsis lines to identify mutants defective in the synthesis of PA and found six tds (tannin-deficient seed) complementation groups defective in PA synthesis. Mutations in these loci disrupt the amount (tds1, tds2, tds3, tds5, and tds6) or location and amount of PA (tds4) in the endothelial cell layer. The PA intermediate epicatechin has been identified in wild type and mutants tds1, tds2, tds3, and tds5 (which do not produce PA) and tds6 (6% of wild-type PA), whereas tds4 (2% of wild-type PA) produces an unidentified dimethylaminocinnamaldehyde-reacting compound, indicating that the mutations may be acting on genes beyond leucoanthocyanidin reductase, the first enzymatic reduction step dedicated to PA synthesis. Two other mutants were identified, an allele of tt7, which has a spotted pattern of PA deposition and produces only 8% of the wild-type level of type PA as propelargonidin, and an allele of tt8 producing no PA. Spotted patterns of PA deposition observed in seed of mutants tds4 and tt7-3 result from altered PA composition and distribution in the cell. Our mutant screen, which was not exhaustive, suggests that the cooperation of many genes is required for successful PA accumulation.

Figure 1

The anthocyanin and PA biosynthetic pathways. Enzymatic steps altered in the transparent testa mutants referred to in the text are shown. There is no evidence that Arabidopsis produces tri-hydroxylated intermediates or end products in PA or anthocyanin biosynthesis, so this branch of the pathway is not shown. All steps analyzed so far, with the possible exception of flavonol synthase (FS), are encoded by a single gene in Arabidopsis. CHI, Chalcone isomerase; F3H, flavanone 3-hyroxylase; F3′H, flavanone 3′-hydroxylase; DFR, dihydroflavonol reductase; LDOX, leucoanthocyanidin dioxygenase.

Figure 2

Wild-type and mutant mature seed stained with DMACA. A through I, Pools of mature seed including Ws-2, tds1, tds2, tds3-1, tds4, tds5, tds6, tt8-4, and tt7-3 showing differences in staining with DMACA. J and K, Enlarged ∼images comparing Ws-2, tds4, and tt7-3 (J) and Ws-2, ban, tt4, and tds2 (K). Bars = 0.05 mm (A through I) and 0.025 mm (J and K).

Figure 3

Wild-type and mutant developing seed stained with DMACA. A through I, Pools of developing seed dissected from siliques, including Ws-2, tds1, tds2, tds3-1, tds4, tds5, tds6, tt8-4, and tt7-3, showing DMACA-reacting PA intermediates present in all except tt8-4. Bar = 0.05 mm.

Figure 4

TLC of anthocyanin extracts from mature seed and leaves. A, TLC of acid-hydrolyzed seed anthocyanin extracts showing flavonols, kaempferol (K, yellow), and quercetin (Q, orange) when sprayed with NP reagent. All mutants have both K and Q, except tt4, which has neither, and tt7-3, which has K only. Anthocyanidins are not evident on seed TLC. B, TLC of acid-hydrolyzed leaf anthocyanin extracts, not sprayed with NP reagent, showing K present in leaves, but not Q, and pink cyanidin (Cy) in all mutants except tt4 and tt7-3, which has pelargonidin (Pg).

Figure 5

Quantitation of anthocyanin and PA. A, Quantitation of leaf anthocyanin as a percentage of wild-type values, measured in duplicate. B, Mature seed anthocyanin shown as a percentage of wild type, measured in duplicate. C, PA measured in mature seed for Ws-2 tds4, Col-7, tt7-3, and tds6, measured in duplicate. Results shown as a percentage relative to Ws-2 wild type. Error bars = sd.

Figure 6

TLC analyses of ethyl acetate fractions of PA extracts from developing siliques. The ethyl acetate fraction contains PA intermediates that react with DMACA. Mutants tt8-4, tt7-3, tds5, and tds6 are compared with wild type, tt10, and tt3 as positive and negative controls. Authentic standards of catechin (cat), epicatechin (e-cat), catechin glucoside (cat-glu), and O. viciifolia PA are shown. tt8-5 and tt3 lack the DMACA-reacting intermediate, and tt7-3 has an alternative intermediate, possibly afzelechin or epiafzelechin, due to the lack of F3′H activity. Polymers of PA are not observed in the soluble fraction of PA extracts from developing mutant or wild-type siliques.

Figure 7

HPLC analyses of ethyl acetate fractions of PA extracts from developing siliques. The peak appearing in the Ws-2, tds5, and tds6 traces at 4 min has the same retention time as the epicatechin standard and is DMACA positive, corresponding to the intermediate observed on TLC plates. This peak was purified from Ws-2 and analyzed by HPLC mass spectroscopy. This intermediate is not observed in tt3 or ban extracts. The differences in structure and retention times of the stereoisomers catechin and epicatechin are shown. The traces of extracts from tds1, tds2, and tds3-1 siliques are similar to tds5 and tds6 and are not shown.

Figure 8

Microscopic sections of OsO₄-treated developing seeds showing altered PA accumulation in tt7-3 and tds4. A, B, G, and H, PA accumulation in the endothelial layer of Ws-2 developing seeds, shown as a gray deposit within the vacuole. PA intermediates were evident at the two-terminal cell stage of development (G) and continued to accumulate in the heart stage (A and B). C and D, tt7-3 at the heart stage of development showing small round inclusions of PA in vacuoles. E and F, tds4 at the torpedo stage of development, showing very small PA inclusions or provacuoles, distinct from the main vacuole of the cell (E). en, Endothelium; v, vacuole; n, nucleus. Bar = 30 (A, C, and E), 40 (B, D, and F), and 140 (G, H) μm.