Differential visualization of transparent testa mutants in Arabidopsis thaliana

Sheahan, John J.; Rechnitz, G. A.

doi:10.1021/ac00055a022

Cited by 32 publications

(23 citation statements)

References 11 publications

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“…To confirm these findings in Arabidopsis seedlings under the growth conditions used in our laboratory, intact 4-day-old La-er plants were stained with the flavonoid-specific dye DPBA, which displays an emission intensity 1-2 orders of magnitude greater when bound to flavonols or dihydroflavonols than to other flavonoids (39,40). Optical sectioning by CLSM revealed intense staining in the elongation zone and root tip of wild-type seedlings (Fig.…”

Section: Localization Of Flavonoid Endmentioning

confidence: 59%

Nuclear Localization of Flavonoid Enzymes in Arabidopsis

Saslowsky¹,

Warek²,

Winkel

2005

Journal of Biological Chemistry

208

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Flavonoids represent one of the oldest, largest, and most diverse families of plant secondary metabolites. These compounds serve a wide range of functions in plants, from pigmentation and UV protection to the regulation of hormone transport. Flavonoids also have interesting pharmacological activities in animals that are increasingly being characterized in terms of effects on specific proteins or other macromolecules. Although flavonoids are found in many different locations both inside and outside the cell, biosynthesis has long been believed to take place exclusively in the cytoplasm. Recent reports from a number of different plant species have documented the presence of flavonoids in nuclei, raising the possibility of novel mechanisms of action for these compounds. Here we present evidence that not only flavonoids, but also at least two of the biosynthetic enzymes, are located in the nucleus in several cell types in Arabidopsis. This is the first indication that differential targeting of the biosynthetic machinery may be used to regulate the deposition of plant secondary products at diverse sites of action within the cell.Flavonoids represent a major class of plant secondary metabolites that include the flavonols, anthocyanins, proanthocyanidins (condensed tannins), and isoflavonoids. These compounds are well known for their roles in flower pigmentation, UV protection, signaling, male fertility, and defense against microbial pathogens (1-3). Flavonoids are also of significant interest as antioxidant and anticancer agents in the human diet (4 -7), and nutritional engineering is being used to modify the types and amounts of flavonoids produced by vegetable crops such as tomato and soybean (8 -10).Flavonoids are synthesized via a well-characterized biosynthetic pathway that has been localized to the cytoplasm in many different plant species (11-13). The products are then deposited in a variety of cellular locations. In some tissues, such as the epidermis of leaves and flowers and endothelium of the developing seed coat, flavonoids are transported primarily to the vacuole by processes that appear to involve multidrug resistance-associated protein or multidrug and toxic compound extrusion proteins (14 -16). In other tissues, a significant proportion of flavonoids are deposited in the cell wall (17)(18)(19)(20) or secreted (21-23). Vesicles apparently involved in transport of flavonoids to the cell periphery have been described in sorghum plants responding to fungal infection (24) and in maize cells induced to accumulate anthocyanins (25). This localization may occur via alternative secretory pathways not involving the Golgi (26). Flavonoids that regulate basipetal auxin transport in the root may also be moved across the cell in some manner because flavonoid enzymes have been localized to the opposite end of the cell from the auxin transport proteins, with which flavonoids are proposed to interact (27). A large proportion of flavonoids can even remain in the cytoplasm in some tissues; for example, interaction of fla...

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Section: Localization Of Flavonoid Endmentioning

confidence: 59%

Nuclear Localization of Flavonoid Enzymes in Arabidopsis

Saslowsky¹,

Warek²,

Winkel

2005

Journal of Biological Chemistry

208

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“…For the histochemical detection of flavan-3-ols, vanillin staining was handled as described previously . Flavonol localization was assayed with diphenylboric acid 2-aminoethyl ester staining according to Sheahan and Rechnitz (1993).…”

Section: Microscopymentioning

confidence: 99%

TRANSPARENT TESTA10 Encodes a Laccase-Like Enzyme Involved in Oxidative Polymerization of Flavonoids in Arabidopsis Seed Coat

Pourcel

Routaboul

Kerhoas³

et al. 2005

The Plant Cell

407

424

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The Arabidopsis thaliana transparent testa10 (tt10) mutant exhibits a delay in developmentally determined browning of the seed coat, also called the testa. Seed coat browning is caused by the oxidation of flavonoids, particularly proanthocyanidins, which are polymers of flavan-3-ol subunits such as epicatechin and catechin. The tt10 mutant seeds accumulate more epicatechin monomers and more soluble proanthocyanidins than wild-type seeds. Moreover, intact testa cells of tt10 cannot trigger H 2 O 2 -independent browning in the presence of epicatechin and catechin, in contrast with wild-type cells. UVvisible light detection and mass spectrometry revealed that the major oxidation products obtained with epicatechin alone are yellow dimers called dehydrodiepicatechin A. These products differ from proanthocyanidins in the nature and position of their interflavan linkages. Flavonol composition was also affected in tt10 seeds, which exhibited a higher ratio of quercetin rhamnoside monomers versus dimers than wild-type seeds. We identified the TT10 gene by a candidate gene approach. TT10 encodes a protein with strong similarity to laccase-like polyphenol oxidases. It is expressed essentially in developing testa, where it colocalizes with the flavonoid end products proanthocyanidins and flavonols. Together, these data establish that TT10 is involved in the oxidative polymerization of flavonoids and functions as a laccase-type flavonoid oxidase.

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“…The specificity of this dye for flavonoids was previously demonstrated, as tt4 mutants make no flavonoids and have no fluorescence upon DPBA treatment (Sheahan and Rechnitz, 1993;Peer et al, 2001;Buer and Muday, 2004). To determine which aglycones generated fluorescence when complexed with DPBA, we assessed DPBAconjugated fluorescence with N, dihydrokaempferol (DHK), dihydroquercetin (DHQ), K, and Q using spectrofluorometry (Supplemental Fig.…”

Section: Dpba Binds To Flavonoids Generating Intense Fluorescence Inmentioning

confidence: 99%

“…Arabidopsis transparent testa4 (tt4) mutants treated with DPBA have only dim green fluorescence ), as they lack flavonoids (Sheahan and Rechnitz, 1993;Burbulis et al, 1996;Sheahan et al, 1998). DPBA generates compoundspecific fluorescence Wuyts et al, 2006).…”

mentioning

confidence: 99%

Flavonoids Are Differentially Taken Up and Transported Long Distances in Arabidopsis

Buer

Muday²,

Djordjevic³

2007

Plant Physiology

232

169

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Flavonoids are synthesized in response to developmental and environmental signals and perform many functions in plants. Arabidopsis (Arabidopsis thaliana) roots grown in complete darkness do not accumulate flavonoids since the expression of genes encoding enzymes of flavonoid biosynthesis is light dependent. Yet, flavonoids accumulate in root tips of plants with light-grown shoots and light-shielded roots, consistent with shoot-to-root flavonoid movement. Using fluorescence microscopy, a selective flavonoid stain, and localized aglycone application to transparent testa mutants, we showed that flavonoids accumulated in tissues distal to the application site, indicating uptake and movement systems. This was confirmed by timecourse fluorescence experiments and high-performance liquid chromatography. Flavonoid applications to root tips resulted in basipetal movement in epidermal layers, with subsequent fluorescence detected 1 cm from application sites after 1 h. Flavonoid application to midroot or cotyledons showed movement of flavonoids toward the root tip mainly in vascular tissue. Naringenin, dihydrokaempferol, and dihydroquercetin were taken up at the root tip, midroot, or cotyledons and traveled long distances via cell-to-cell movement to distal tissues, followed by conversion to quercetin and kaempferol. In contrast, kaempferol and quercetin were only taken up at the root tip. Using ATP-binding cassette (ABC) transporter and H 1 -ATPase inhibitors suggested that a multidrug resistance-associated protein ABCC transporter facilitated flavonoid movement away from the application site.

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Differential visualization of transparent testa mutants in Arabidopsis thaliana

Cited by 32 publications

References 11 publications

Nuclear Localization of Flavonoid Enzymes in Arabidopsis

Nuclear Localization of Flavonoid Enzymes in Arabidopsis

TRANSPARENT TESTA10 Encodes a Laccase-Like Enzyme Involved in Oxidative Polymerization of Flavonoids in Arabidopsis Seed Coat

Flavonoids Are Differentially Taken Up and Transported Long Distances in Arabidopsis

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