The TRANSPARENT TESTA12 Gene of Arabidopsis Encodes a Multidrug Secondary Transporter-like Protein Required for Flavonoid Sequestration in Vacuoles of the Seed Coat Endothelium

Abstract

Phenolic compounds that are present in the testa interfere with the physiology of seed dormancy and germination. We isolated a recessive Arabidopsis mutant with pale brown seeds, transparent testa12 ( tt12 ), from a reduced seed dormancy screen. Microscopic analysis of tt12 developing and mature testas revealed a strong reduction of proanthocyanidin deposition in vacuoles of endothelial cells. Double mutants with tt12 and other testa pigmentation mutants were constructed, and their phenotypes confirmed that tt12 was affected at the level of the flavonoid biosynthetic pathway. The TT12 gene was cloned and found to encode a protein with similarity to prokaryotic and eukaryotic secondary transporters with 12 transmembrane segments, belonging to the MATE (multidrug and toxic compound extrusion) family. TT12 is expressed specifically in ovules and developing seeds. In situ hybridization localized its transcript in the endothelium layer, as expected from the effect of the tt12 mutation on testa flavonoid pigmentation. The phenotype of the mutant and the nature of the gene suggest that TT12 may control the vacuolar sequestration of flavonoids in the seed coat endothelium. INTRODUCTIONFlavonoids represent a highly diverse group of plant aromatic secondary metabolites. The major forms, which are anthocyanins (red to purple pigments), flavonols (colorless to pale yellow pigments), and proanthocyanidins (PAs), also known as condensed tannins (colorless pigments that brown with oxidation), are present in proportions and amounts that vary according to the plant species, the organ, the stage of development, and environmental growth conditions. Arabidopsis contains flavonoid pigments in vegetative tissues and in seeds (Shirley et al., 1995). In the mature testa, flavonoids were detected in both the endothelium and the three crushed parenchymal layers just above the endothelium . PAs have been shown to accumulate exclusively in the endothelium layer (Devic et al., 1999). Wild-type seed color depends on the stage of development: as long as the testa is transparent, until ‫ف‬ 10 days after pollination, the seed has the color of the underlying endosperm and embryo. At maturity, the testa becomes opaque after the oxidation of flavonoid pigments, which gives the seed its characteristic brown color. Therefore, the color of mutants with seed flavonoid defects ranges from pale yellow (complete lack of visible pigments in the testa) to pale brown, depending on the accumulated intermediate flavonoids .In Arabidopsis, mutants at 21 loci have been isolated on the basis of altered testa pigmentation (Figure 1). The genes disrupted in the transparent testa ( tt ) mutants tt3 , tt4 , tt5 , tt6 , and tt7 (Shirley et al., 1995) and the banyuls ( ban ) mutant (Albert et al., 1997) code for the enzymes dihydroflavonol-4-reductase (Shirley et al., 1992), chalcone synthase (Feinbaum and Ausubel, 1988), chalcone isomerase (Shirley et al., 1992), flavonol 3-hydroxylase (Pelletier and Shirley, 1996; Wisman et al., 1998), flavonol 3 Ј -hydroxylase (Ko...

A Multidrug Resistance–Associated Protein Involved in Anthocyanin Transport in Zea mays

“…The simplest explanation is that these transport mechanisms are compound specific. In bronze2 and An9 mutants and in aZmMrp3 plants, anthocyanins are mislocalized (Marrs et al, 1995;Alfenito et al, 1998; Figure 5C), whereas in Arabidopsis tt12 mutants, the substrates in question are proanthocyanidins (Debeaujon et al, 2001). The fact that tt12 mutants do not affect anthocyanin pigmentation argues that the TT12 transporter is not involved in anthocyanin transport in Arabidopsis.…”

“…The tt12 mutant described by Debeaujon et al (2001) yields seeds that show reduced accumulation of proanthocyanidin pigments in endothelial cells. This phenotype results from the loss of a multidrug and toxic compound extrusion (MATE) protein.…”

The unusual tegumental tissues of the Lunaria annua (Brassicaceae) seed: A developmental study using light and electron microscopy

“…As proposed for root endoderm of Gossypium and Musa (Mueller and Greenwood, 1978), the accumulation of tannins in the pre-sclerenchymatic layer of Lunaria could protect against pathogens (Kraus et al, 2003). Condensed tannins within vacuoles could also play a role in seed dormancy and germination as is the case with Arabidopsis (Debeaujon et al, 2001).When the presclerenchymatic tissue is in the later stages of its development the cytoplasm becomes more osmiophilic, showing signs of degeneration. Such modifications appear related to the lignification of some of the cytoplasmic components, eventually leading to the formation of solid phloroglucinol-positive blocks at maturity (as shown in ESEM images).…”

The Effect of α-Amanitin on the Arabidopsis Seed Proteome Highlights the Distinct Roles of Stored and Neosynthesized mRNAs during Germination

“…The isolation of the transparent testa (tt) mutants in the Ler and Wassilewskija (Ws) backgrounds was described by Koornneef and van der Veen (1980), Koornneef (1990), and Debeaujon et al (2001), and their physiological properties were analyzed by and . Seeds of mutants tt2-1, tt5-1, ttg1-1 (Ler background), and tt12-1 (Ws background) have been used here.…”