Characterization of tt15, a novel transparent testa mutant of Arabidopsis thaliana (L.) Heynh.

The Arabidopsis thaliana seed coat typically has a brown color due to the accumulation of flavonoid pigments in the testa. Mutants of A. thaliana with defects in pigment biosynthesis often produce seeds that are olive brown or even yellow in appearence, and the responsible genetic loci are referred to as TRANSPARENT TESTA (TT). Large-scale screening for mutants affected in seed development and complementation analysis of a candidate mutant line with all published A. thalianatt mutants identified a new tt locus designated tt15. The tt15 mutation maps to the lower part of chromosome 1. Mutant plants produced pale greenish-brown seeds whose dormancy was slightly reduced. The phenotype was consistent with the maternal origin of the testa. Analysis of pigment accumulation and the study of expression patterns of genes involved in flavonoid biosynthesis in tt15 plants and seeds indicated a seed-specific phenotype. Most notable was a reduction of the cyanidin and quercetin content of tt15 seeds. Received: 2 October 1998 / Accepted: 10 October 1998     

Gene Silencing of BnTT10 Family Genes Causes Retarded Pigmentation and Lignin Reduction in the Seed Coat of Brassica napus

Yellow-seed (i.e., yellow seed coat) is one of the most important agronomic traits of Brassica plants, which is correlated with seed oil and meal qualities. Previous studies on the Brassicaceae, including Arabidopsis and Brassica species, proposed that the seed-color trait is correlative to flavonoid and lignin biosynthesis, at the molecular level. In Arabidopsis thaliana, the oxidative polymerization of flavonoid and biosynthesis of lignin has been demonstrated to be catalyzed by laccase 15, a functional enzyme encoded by the AtTT10 gene. In this study, eight Brassica TT10 genes (three from B. napus, three from B. rapa and two from B. oleracea) were isolated and their roles in flavonoid oxidation/polymerization and lignin biosynthesis were investigated. Based on our phylogenetic analysis, these genes could be divided into two groups with obvious structural and functional differentiation. Expression studies showed that Brassica TT10 genes are active in developing seeds, but with differential expression patterns in yellow- and black-seeded near-isogenic lines. For functional analyses, three black-seeded B. napus cultivars were chosen for transgenic studies. Transgenic B. napus plants expressing antisense TT10 constructs exhibited retarded pigmentation in the seed coat. Chemical composition analysis revealed increased levels of soluble proanthocyanidins, and decreased extractable lignin in the seed coats of these transgenic plants compared with that of the controls. These findings indicate a role for the Brassica TT10 genes in proanthocyanidin polymerization and lignin biosynthesis, as well as seed coat pigmentation in B. napus.     

A Large Insertion in bHLH Transcription Factor BrTT8 Resulting in Yellow Seed Coat in Brassica rapa

Yellow seed is a desirable quality trait of the Brassica oilseed species. Previously, several seed coat color genes have been mapped in the Brassica species, but the molecular mechanism is still unknown. In the present investigation, map-based cloning method was used to identify a seed coat color gene, located on A9 in B. rapa. Blast analysis with the Arabidopsis genome showed that there were 22 Arabidopsis genes in this region including at4g09820 to at4g10620. Functional complementation test exhibited a phenotype reversion in the Arabidopsis thaliana tt8-1 mutant and yellow-seeded plant. These results suggested that the candidate gene was a homolog of TRANSPARENT TESTA8 (TT8) locus. BrTT8 regulated the accumulation of proanthocyanidins (PAs) in the seed coat. Sequence analysis of two alleles revealed a large insertion of a new class of transposable elements, Helitron in yellow sarson. In addition, no mRNA expression of BrTT8 was detected in the yellow-seeded line. It indicated that the natural transposon might have caused the loss in function of BrTT8. BrTT8 encodes a basic/helix-loop-helix (bHLH) protein that shares a high degree of similarity with other bHLH proteins in the Brassica. Further expression analysis also revealed that BrTT8 was involved in controlling the late biosynthetic genes (LBGs) of the flavonoid pathway. Our present findings provided with further studies could assist in understanding the molecular mechanism involved in seed coat color formation in Brassica species, which is an important oil yielding quality trait.     

TRANSPARENT TESTA1 interacts with R2R3-MYB factors and affects early and late steps of flavonoid biosynthesis in the endothelium of Arabidopsis thaliana seeds

Wild type seed coats of Arabidopsis thaliana are brown due to the accumulation of proanthocyanidin pigments (PAs). The pigmentation requires activation of phenylpropanoid biosynthesis genes and mutations in some of these genes cause a yellow appearance of seeds, termed transparent testa (tt) phenotype. The TT1 gene encodes a WIP‐type zinc finger protein and is expressed in the seed coat endothelium where most of the PAs accumulate in wild type plants. In this study we show that TT1 is not only required for correct expression of PA‐specific genes in the seed coat, but also affects CHS, encoding the first enzyme of flavonoid biosynthesis. Many steps of this pathway are controlled by complexes of MYB and BHLH proteins with the WD40 factor TTG1. We demonstrate that TT1 can interact with the R2R3 MYB protein TT2 and that ectopic expression of TT2 can partially restore the lack in PA production in tt1. Reduced seed coat pigmentation was obtained using a TT1 variant lacking nuclear localisation signals. Based on our results we propose that the TT2/TT8/TTG1 regulon may also comprise early genes like CHS and discuss steps to further unravel the regulatory network controlling flavonoid accumulation in endothelium cells during A. thaliana seed development.     

Map-based cloning and characterization of a gene controlling hairiness and seed coat color traits in <Emphasis Type="Italic">Brassica rapa</Emphasis>

A glabrous, yellow-seeded doubled haploid (DH) line and a hairy, black-seeded DH line in Chinese cabbage (B. rapa) were used as parents to develop a DH line population that segregated for both hairiness and seed coat color traits. The data showed that both traits completely co-segregated each other, suggesting that one Mendelian locus controlled both hairiness and seed coat color in this population. A fine genetic map was constructed and a SNP marker that was located inside a Brassica ortholog of TRANSPARENT TESTA GLABRA 1 (TTG1) in Arabidopsis showed complete linkage to both the hairiness and seed coat color gene, suggesting that the Brassica TTG1 ortholog shared the same gene function as its Arabidopsis counterpart. Further sequence analysis of the alleles from hairless, yellow-seeded and hairy, black-seeded DH lines in B. rapa showed that a 94-base deletion was found in the hairless, yellow-seeded DH lines. A nonfunctional truncated protein in the hairless, yellow-seeded DH lines in B. rapa was suggested by the coding sequence of the TTG1 ortholog. Both of the TTG1 homologs from the black and yellow seeded B. rapa lines were used to transform an Arabidopsis ttg1 mutant and the results showed that the TTG1 homolog from the black seeded B. rapa recovered the Arabidopsis ttg1 mutant, while the yellow seeded homolog did not, suggesting that the deletion in the Brassica TTG1 homolog had led to the yellow seeded natural mutant. This was the first identified gene in Brassica species that simultaneously controlled both hairiness and seed coat color traits.     

Involvement of AtLAC15 in lignin synthesis in seeds and in root elongation of Arabidopsis

Laccase, EC 1.10.3.2 or p-diphenol:dioxygen oxidoreductase, has been proposed to be involved in lignin synthesis in plants based on its in vitro enzymatic activity and a close correlation with the lignification process in plants. Despite many years of research, genetic evidence for the role of laccase in lignin synthesis is still missing. By screening mutants available for the annotated laccase gene family in Arabidopsis, we identified two mutants for a single laccase gene, AtLAC15 (At5g48100) with a pale brown or yellow seed coat which resembled the transparent testa (tt) mutant phenotype. A chemical component analysis revealed that the mutant seeds had nearly a 30% decrease in extractable lignin content and a 59% increase in soluble proanthocyanidin or condensed tannin compared with wild-type seeds. In an in vitro enzyme assay, the developing mutant seeds showed a significant reduction in polymerization activity of coniferyl alcohol in the absence of H₂O₂. Among the dimers formed in the in vitro assay using developing wild-type seeds, 23% of the linkages were β-O-4 which resembles the major linkages formed in native lignin. The evidence strongly supports that AtLAC15 is involved in lignin synthesis in plants. To our knowledge, this is the first genetic evidence for the role of laccase in lignin synthesis. Changes in seed coat permeability, seed germination and root elongation were also observed in the mutant.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Allelic Variation of BnaC.TT2.a and Its Association with Seed Coat Color and Fatty Acids in Rapeseed (Brassica napus L.)

Efficient molecular markers for the selection of rapeseed genetic materials with high seed oil content and ideal fatty acid (FA) composition are preferred by rapeseed breeders. Recently, we reported the molecular mechanism of TRANSPARENT TESTA 2 (TT2) in inhibiting seed FA biosynthesis in Arabidopsis. However, evidence showing the association of rapeseed TT2 homologs and seed FA production are still insufficient. In this study, we collected 83 rapeseed (Brassica napus L.) landraces from different geographical backgrounds to conduct association mapping of BnaC.TT2.a in relation to seed coat color and FA biosynthesis. Population background was corrected by 84 pairs of SSR markers that were uniformly distributed among the linkage groups of the Tapidor-Ningyou-7 DH population. A single copy of BnaC.TT2.a for single nucleotide polymorphism (SNP) assay was cloned by a pair of previously reported specific primers. From the analysis of BnaC.TT2.a allelic variations using GLM+Q model, four SNPs on intron 1 of BnaC.TT2.a that were associated with seed FA were discovered. Moreover, an InDel at position 738 on exon 3 of BnaC.TT2.a indicated a change of protein function that was significantly associated with seed coat color, linoleic acid (C18:2), and total FA content. These findings revealed the role of BnaC.TT2.a in regulating the seed color formation and seed FA biosynthesis in rapeseed, thereby suggesting effective molecular markers for rapeseed breeding.     

Medicago glucosyltransferase UGT72L1: potential roles in proanthocyanidin biosynthesis

In the first reaction specific for proanthocyanidin (PA) biosynthesis in Arabidopsis thaliana and Medicago truncatula, anthocyanidin reductase (ANR) converts cyanidin to (?)-epicatechin. The glucosyltransferase UGT72L1 catalyzes formation of epicatechin 3′-O-glucoside (E3′OG), the preferred substrate for MATE transporters implicated in PA biosynthesis in both species. The mechanism of PA polymerization is still unclear, but may involve the laccase-like polyphenol oxidase TRANSPARENT TESTA 10 (TT10). We have employed a combination of cell biological, biochemical and genetic approaches to evaluate this PA pathway model. The promoter regions of UGT72L1 and MtANR share common cis-acting elements and direct overlapping, but partially distinct, expression patterns. UGT72L1 and MtANR are localized in the cytosol, whereas TT10 is localized to the vacuole. Over-expression of UGT72L1 in M. truncatula hairy roots results in increased accumulation of PA-like compounds, and loss of function of UGT72L1 partially reduces epicatechin, E3′OG and extractable PA levels in M. truncatula seeds. Expression of UGT72L1 in A. thaliana leads to a massive increase in E3′OG in immature seed, but reduced levels of extractable PAs. However, when UGT72L1 was expressed in the Arabidopsis tt10 mutant, extractable PA levels increased and seed coat browning was delayed. Our results suggest that glycosylation of epicatechin is important for both PA precursor transport and assembly, but that additional redundant pathways may exist.     

Isolation and Characterization of Mutants Defective in Seed Coat Mucilage Secretory Cell Development in Arabidopsis

In Arabidopsis, fertilization induces the epidermal cells of the outer ovule integument to differentiate into a specialized seed coat cell type producing extracellular pectinaceous mucilage and a volcano-shaped secondary cell wall. Differentiation involves a regulated series of cytological events including growth, cytoplasmic rearrangement, mucilage synthesis, and secondary cell wall production. We have tested the potential of Arabidopsis seed coat epidermal cells as a model system for the genetic analysis of these processes. A screen for mutants defective in seed mucilage identified five novel genes (MUCILAGE-MODIFIED [MUM]1–5). The seed coat development of these mutants, and that of three previously identified ones (TRANSPARENT TESTA GLABRA1, GLABRA2, and APETALA2) were characterized. Our results show that the genes identified define several events in seed coat differentiation. Although APETALA2 is needed for differentiation of both outer layers of the seed coat, TRANSPARENT TESTA GLABRA1, GLABRA2, and MUM4 are required for complete mucilage synthesis and cytoplasmic rearrangement. MUM3 and MUM5 may be involved in the regulation of mucilage composition, whereas MUM1 and MUM2 appear to play novel roles in post-synthesis cell wall modifications necessary for mucilage extrusion.     

Functional characterization of a UDP-glucose:flavonoid 3-O-glucosyltransferase from the seed coat of black soybean (Glycine max (L.) Merr.)

The seed coats of black soybean (Glycine max (L.) Merr.) accumulate red (cyanidin-), blue (delphinidin-), purple (petunidin-), and orange (pelargonidin-based) anthocyanins almost exclusively as 3-O-glucosides; however, the responsible enzyme has not been identified. In this study, the full-length cDNA which encodes the enzyme that catalyzes the final step in anthocyanin biosynthesis, namely UDP-glucose:flavonoid 3-O-glucosyltransferase (UGT78K1), was isolated from the seed coat tissue of black soybean using rapid amplification of cDNA ends (RACE). Of the 28 flavonoid substrates tested, the purified recombinant protein glucosylated only anthocyanidins and flavonols, and demonstrated strict 3-OH regiospecificity. Galactose could also be transferred with relatively low activity to the 3-position of cyanidin or delphinidin in vitro. These findings are consistent with previous reports of mainly 3-O-glucosylated and minor amounts of 3-O-galactosylated anthocyanins in the seed coat of black soybean. The recombinant enzyme exhibited pronounced substrate inhibition by cyanidin at 100 μM acceptor concentration. Transfer of UGT78K1 into the Arabidopsis T-DNA mutant (ugt78d2) deficient in anthocyanidin and flavonol 3-O-glucosyltransferase activity, restored the accumulation of anthocyanins and flavonols, suggesting the in vivo function of the enzyme as a flavonoid 3-O-glucosyltransferase. Genomic and phylogenetic analyses suggest the existence of three additional soybean sequences with high similarity to UGT78K1. RT-PCR confirmed the co-expression of one of these genes (Glyma08g07130) with UGT78K1 in the seed coat of black soybean, suggesting possible functional redundancies in anthocyanin biosynthesis in this tissue.     

QTL Mapping of Seed Coat Color for Yellow Seeded Brassica napus

The development of yellow-seeded varieties of Brassica napus for improving the oilseed quality characteristics of lower fiber content and higher protein and oil content has been a major focus of breeding researches worldwide in recent years. With the black-seeded ‘Youyan 2’ as male and the yellow-seeded GH₀₆ as female parents respectively, F₂ population of 132 individuals were obtained. A linkage map was constructed with 164 markers including 125 AFLP, 37 SSR, 1 RAPD and 1 SCAR markers distributed over 19 linkage groups covering approximately 2 549.8 cM with an average spacing of 15.55 cM. Two loci located on the 5th and 19th group were detected for the trait of seed coat color based on the linkage group using multiple interval mapping method and explained 46% and 30.9% of the phenotypic variation, respectively.