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• W2069611962 abstract "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 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 (1Winkel-Shirley B. Plant Physiol. 2001; 126: 485-493Crossref PubMed Scopus (2567) Google Scholar, 2Dixon R.A. Steele C.L. Trends Plant Sci. 1999; 4: 394-400Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar, 3Harborne J.B. Williams C.A. Phytochemistry. 2000; 55: 481-504Crossref PubMed Scopus (3229) Google Scholar). Flavonoids are also of significant interest as antioxidant and anticancer agents in the human diet (4Havsteen B.H. Pharmacol. Ther. 2002; 96: 67-202Crossref PubMed Scopus (2094) Google Scholar, 5Rice-Evans C. Curr. Med. Chem. 2001; 8: 797-807Crossref PubMed Scopus (757) Google Scholar, 6Vom Endt D. Kijne J.W. Memelink J. Phytochemistry. 2002; 61: 107-114Crossref PubMed Scopus (259) Google Scholar, 7Stevens J.F. Page J.E. Phytochemistry. 2004; 65: 1317-1330Crossref PubMed Scopus (544) Google Scholar), and nutritional engineering is being used to modify the types and amounts of flavonoids produced by vegetable crops such as tomato and soybean (8Yu O. Shi J. Hession A.O. Maxwell C.A. McGonigle B. Odell J.T. Phytochemistry. 2003; 63: 753-763Crossref PubMed Scopus (208) Google Scholar, 9Verhoeyen M.E. Bovy A. Collins G. Muir S. Robinson S. de Vos C.H. Colliver S. J. Exp. Bot. 2002; 53: 2099-2106Crossref PubMed Scopus (182) Google Scholar, 10Bovy A. de Vos R. Kemper M. Schijlen E. Almenar Pertejo M. Muir S. Collins G. Robinson S. Verhoeyen M. Hughes S. Santos-Buelga C. van Tunen A. Plant Cell. 2002; 14: 2509-2526Crossref PubMed Scopus (403) Google Scholar). Flavonoids are synthesized via a well-characterized biosynthetic pathway that has been localized to the cytoplasm in many different plant species (11Winkel B. Annu. Rev. Plant Biol. 2004; 55: 85-107Crossref PubMed Scopus (491) Google Scholar, 12Winkel-Shirley B. Physiol. Plant. 1999; 107: 142-149Crossref Scopus (240) Google Scholar, 13Hrazdina G. Jensen R.A. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1992; 43: 241-267Crossref Scopus (189) Google Scholar). 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 (14Goodman C.D. Casati P. Walbot V. Plant Cell. 2004; 16: 1812-1826Crossref PubMed Scopus (330) Google Scholar, 15Debeaujon I. Peeters A.J.M. Léon-Kloosterziel K.M. Koornneef M. Plant Cell. 2001; 13: 853-871Crossref PubMed Scopus (427) Google Scholar, 16Mathews H. Clendennen S.K. Caldwell C.G. Liu X.L. Connors K. Matheis N. Schuster D.K. Menasco D.J. Wagoner W. Lightner J. Wagner D.R. Plant Cell. 2003; 15: 1689-1703Crossref PubMed Scopus (428) Google Scholar). In other tissues, a significant proportion of flavonoids are deposited in the cell wall (17Markham K.R. Gould K.S. Ryan K.G. Phytochemistry. 2001; 58: 403-413Crossref PubMed Scopus (51) Google Scholar, 18Hutzler P. Rischbach R. Heller W. Jungblut T.P. Reuber S. Schmitz R. Veit M. Weissenbck G. Schmitzler J-P. J. Exp. Bot. 1998; 49: 953-965Crossref Scopus (311) Google Scholar, 19Schnitzler J.P. Jungblut T.P. Heller W. Kofferlein M. Hutzler P. Heinzmann U. Schmelzer E. Ernst D. Langebartels C. Sandermann H. New Phytol. 1996; 132: 247-258Crossref Scopus (166) Google Scholar, 20Marchand L. Charest P.M. Ibrahim R.K. J. Plant Physiol. 1987; 131: 339-348Crossref Scopus (9) Google Scholar) or secreted (21Wollenweber E. Dietz V.H. Phytochemistry. 1981; 20: 869-932Crossref Scopus (322) Google Scholar, 22Cuadra P. Harborne J.B. Z. Naturforsch. C. 1996; 51: 671-680Crossref Scopus (32) Google Scholar, 23Kuras M. Stefanowska-Wronka M. Lynch J.M. Zobel A.M. Ann. Bot. 1999; 84: 135-143Crossref Scopus (25) Google Scholar). Vesicles apparently involved in transport of flavonoids to the cell periphery have been described in sorghum plants responding to fungal infection (24Snyder B.A. Nicholson R.L. Science. 1990; 248: 1637-1639Crossref PubMed Scopus (277) Google Scholar) and in maize cells induced to accumulate anthocyanins (25Grotewold E. Chamberlin M. Snook M. Siame B. Butler L. Swenson J. Maddock S. St. Clair G. Bowen B. Plant Cell. 1998; 10: 721-740Crossref PubMed Scopus (285) Google Scholar). This localization may occur via alternative secretory pathways not involving the Golgi (26Lin Y. Irani N.G. Grotewold E. BMC Plant Biol. 2003; 3: 10.11-10.12Crossref Scopus (37) Google Scholar). 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 (27Saslowsky D. Winkel-Shirley B. Plant J. 2001; 27: 37-48Crossref PubMed Google Scholar). A large proportion of flavonoids can even remain in the cytoplasm in some tissues; for example, interaction of flavonoids with cytoplasmic proteins has been proposed to contribute to yellow flower color in lisianthus (17Markham K.R. Gould K.S. Ryan K.G. Phytochemistry. 2001; 58: 403-413Crossref PubMed Scopus (51) Google Scholar, 18Hutzler P. Rischbach R. Heller W. Jungblut T.P. Reuber S. Schmitz R. Veit M. Weissenbck G. Schmitzler J-P. J. Exp. Bot. 1998; 49: 953-965Crossref Scopus (311) Google Scholar). Several recent reports describe the accumulation of flavonoids in the nucleus in such diverse species as Arabidopsis thaliana, Brassica napus, Flaveria chloraefolia, Picea abies, Tsuga canadensis, and Taxus baccata (18Hutzler P. Rischbach R. Heller W. Jungblut T.P. Reuber S. Schmitz R. Veit M. Weissenbck G. Schmitzler J-P. J. Exp. Bot. 1998; 49: 953-965Crossref Scopus (311) Google Scholar, 23Kuras M. Stefanowska-Wronka M. Lynch J.M. Zobel A.M. Ann. Bot. 1999; 84: 135-143Crossref Scopus (25) Google Scholar, 28Buer C.S. Muday G.K. Plant Cell. 2004; 16: 1191-1205Crossref PubMed Scopus (323) Google Scholar, 29Grandmaison J. Ibrahim R.K. J. Plant Physiol. 1996; 147: 653-660Crossref Scopus (35) Google Scholar, 30Feucht W. Treutter D. Polster J. Plant Cell Rep. 2004; 22: 430-436Crossref PubMed Scopus (68) Google Scholar, 31Peer W.A. Brown D.E. Tague B.W. Muday G.K. Taiz L. Murphy A.S. Plant Physiol. 2001; 126: 536-548Crossref PubMed Scopus (273) Google Scholar). This is somewhat surprising because flavonoids are well known to bind to proteins and nucleic acids and could therefore interfere with essential biological processes (32Nijveldt R.J. van Nood E. van Hoorn D.E. Boelens P.G. van Norren K. van Leeuwen P.A. Am. J. Clin. Nutr. 2001; 74: 418-425Crossref PubMed Scopus (2417) Google Scholar, 33Stafford H.A. Rec. Adv. Phytochem. 1974; 8: 53-79Crossref Scopus (72) Google Scholar). Although the physiological roles of flavonoids in plant cell nuclei are far from clear, it has been suggested that these compounds may serve to protect DNA from UV and oxidative damage (30Feucht W. Treutter D. Polster J. Plant Cell Rep. 2004; 22: 430-436Crossref PubMed Scopus (68) Google Scholar) or directly or indirectly control the transcription of genes required for growth and development including those encoding auxin transport proteins (23Kuras M. Stefanowska-Wronka M. Lynch J.M. Zobel A.M. Ann. Bot. 1999; 84: 135-143Crossref Scopus (25) Google Scholar, 28Buer C.S. Muday G.K. Plant Cell. 2004; 16: 1191-1205Crossref PubMed Scopus (323) Google Scholar, 29Grandmaison J. Ibrahim R.K. J. Plant Physiol. 1996; 147: 653-660Crossref Scopus (35) Google Scholar). The flavonoid biosynthetic machinery has, until now, been found exclusively in the cytoplasm, where it appears to be organized as a multienzyme complex both at the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; CHI, chalcone isomerase (EC 5.5.1.6); CHS, chalcone synthase (EC 2.3.1.74); CLSM, confocal laser scanning microcopy; DPBA, diphenylboric acid 2-aminoethyl ester; GFP, green fluorescent protein; MES, 2(N-morpholino)ethanesulfonic acid; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; NLS, nuclear localization signal. and in as-yet-uncharacterized electron-dense particles (11Winkel B. Annu. Rev. Plant Biol. 2004; 55: 85-107Crossref PubMed Scopus (491) Google Scholar, 12Winkel-Shirley B. Physiol. Plant. 1999; 107: 142-149Crossref Scopus (240) Google Scholar, 13Hrazdina G. Jensen R.A. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1992; 43: 241-267Crossref Scopus (189) Google Scholar). Although specific transport mechanisms control the distribution of flavonoids from the cytoplasm to sites of action in the vacuole and at the cell periphery, we now have evidence that flavonoids located in the nucleus may be synthesized in situ. These findings provide the first evidence that differential localization of a biosynthetic pathway may be used to direct the deposition of metabolites at diverse sites of action within the cell. Plant Material—The Landsberg erecta (La-er) ecotype was used as the wild type in all experiments. Seedlings were grown on the surface of agar medium containing Murashige and Skoog salts and sucrose under continuous white light (120 microeinsteins m–2 s–1) for 4 days at 21 °C as described previously (27Saslowsky D. Winkel-Shirley B. Plant J. 2001; 27: 37-48Crossref PubMed Google Scholar). Construction of Transgenic Arabidopsis Plants Expressing Green Fluorescent Protein (GFP) Fusion Proteins—The AtCHS and AtCHI coding regions (34Pelletier M.K. Shirley B.W. Plant Physiol. 1996; 111: 339-345Crossref PubMed Scopus (165) Google Scholar) were amplified using Pfu polymerase (Stratagene) and primers that added NcoI sites for fusion to the 5′ end of mGFP5 and either BglII (AtCHS) or BamHI (AtCHI) sites for fusion to the 3′ end. The fragments were then inserted into the NcoI or BlgII site in pAVA393 (35von Arnim A.G. Deng X.W. Stacey M.G. Gene (Amst.). 1998; 221: 35-43Crossref PubMed Scopus (203) Google Scholar). For mGFP5-CHI, the resulting expression cassette, consisting of the double-enhanced CaMV 35S promoter, Tobacco Etch Virus translational leader, mGFP5-CHI coding region, and nopaline synthase terminator, was then excised using XmaI and SalI and subcloned into corresponding sites in pBIB-KAN (36Becker D. Nucleic Acids Res. 1990; 18: 203Crossref PubMed Scopus (222) Google Scholar). The corresponding region was also subcloned from the empty pAVA393 vector into pBIB-KAN to serve as a control for localization experiments. For the other three fusions, constructs in pAVA393 were digested with KpnI, filled in, digested with SacI, and then cloned into the SmaI and SacI sites in pCAMBIA3300 (Cambia, Canberra, Australia). The five constructs were used to transform Agrobacterium tumefaciens strain GV3101 using a freeze-thaw method (37Chen H. Nelson R.S. Sherwood J.L. BioTechniques. 1994; 16 (670): 664-668PubMed Google Scholar) and then introduced into Arabidopsis La-er plants using the floral dip method (38Clough S.J. Bent A.F. Plant J. 1998; 16: 735-743Crossref PubMed Google Scholar). For the constructs in pBIB-KAN, selection of transformants was on medium containing 0.5× Murashige-Skoog salts and 50 μg/ml kanamycin. For constructs in pCAMBIA3300, selection was carried out by spraying soil-grown plants at 10, 11, 12, and 16 days with a 1:10,000 dilution of glufosinate ammonium concentrate (Basta; AgrEvo Co.), 0.005% Silwet L-77 (Lehle) per the manufacturer's instructions. Immunolocalization, Flavonoid Staining, and Imaging—Methods were as described previously (27Saslowsky D. Winkel-Shirley B. Plant J. 2001; 27: 37-48Crossref PubMed Google Scholar) for immunolocalization using affinity-purified rabbit anti-CHS and chicken anti-CHI antibody preparations, flavonoid staining with diphenylboric acid 2-aminoethyl ester (DPBA), and imaging by electron microscopy or confocal laser scanning microscopy (CLSM). mGFP5 fluorescence was visualized using a LSM510 confocal microscope (Carl Zeiss) using a 1.2 numerical aperture 40× C-Apo water-immersion objective lens, 488 nm argon laser line, and 505–550 nm band pass filter. Nuclear Protein Preparations—Seedlings were immersed in liquid nitrogen in a pre-chilled ceramic mortar and ground with a pestle to a fine powder. The powder (~1.5 g) was transferred to a Dounce homogenizer (Kontes Glass) containing 10 ml of cold nuclei isolation buffer (10 mm MES-KOH, pH 6.3, 330 mmd-sorbitol, 0.1 mm spermine, 0.5 mm spermidine, 2.5 mm EDTA, pH 6.3, 2.5 mm dithiothreitol, 10 mm KCl, 10 mm NaCl, 1 mm MgCl2, and 1× Protease Complete (-EDTA) protease inhibitor mixture (Roche Applied Science)). The suspension was allowed to thaw on ice, and then 10 passes with a loose (0.16 mm) clearance and 10 passes with a tight (0.07 mm) clearance pestle were performed. The resulting homogenate was centrifuged at 380 × g for 6 min at 4 °C to sediment large cellular debris and then filtered through 75 μm NITEX nylon mesh (Precision Woven Screening Media, Briarcliff Manor, NY) to remove smaller cell fragments. The homogenate was centrifuged for 6 min at 4 °C once at 600 × g and then two more times at 700 × g, with the pellet being discarded between each step. This served to remove any large cell wall fragments as well as non-lysed cells as determined by light microscopy. Nuclei were pelleted at 2750 × g for 10 min at 4 °C. The remaining supernatant was centrifuged at 16,000 × g for 10 min at 4 °C to produce the “lysed cell supernatant.” The nuclei-enriched pellet was resuspended in 3 ml of nuclei isolation buffer. To selectively lyse any remaining chloroplasts or mitochondria, 10% Triton X-100 was slowly added to a final concentration of 0.2%. After 5 min of incubation on ice, the suspension was centrifuged at 2750 × g for 10 min at 4 °C. The resulting pellet was washed by resuspension in 3 ml of nuclei isolation buffer and centrifuged at 2750 × g for 10 min at 4 °C. This produced a “nuclei-enriched pellet” with a volume of ~30 μl. Qualitative purity was gauged by resuspending this pellet in 200 μl of nuclei isolation buffer, counterstaining with 1 μg ml–1 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Molecular Probes) for 10 min on ice, and visualizing by differential interference contrast and fluorescence light microscopy (Zeiss Axioskop 2 equipped with a standard DAPI filter set). Immunoblot Analysis—Samples were prepared for electrophoresis by suspending the nuclei-enriched pellet in 200 μl of 1× sample buffer (62.6 mm Tris, pH 6.8, 3.3% SDS, 10% glycerol, 0.7 m β-mercaptoethanol, 0.5 mg ml–1 bromphenol blue, and 0.2 mg ml–1 pyronin Y) or combining 200 μl of the lysed cell supernatant with 100 μlof3× sample buffer. The samples were boiled for 15 min, centrifuged at 16,000 × g for 10 min, and then analyzed by immunoblotting as described previously (27Saslowsky D. Winkel-Shirley B. Plant J. 2001; 27: 37-48Crossref PubMed Google Scholar) using affinity-purified rabbit anti-CHS at a 1:800 dilution, affinity-purified chicken anti-CHI at a 1:400 dilution, mouse anti-histone monoclonal antibody (Chemicon International, Temecula, CA) at a 1:700 dilution, or mouse anti-α-tubulin monoclonal DM1A and 611B1 (Sigma) at a 1:500 and 1:1000 dilution, respectively. Typical protein concentrations were 10 mg ml–1 for the nuclei-enriched samples and 3 mg ml–1 for the lysed cell supernatant as determined using a Bradford assay (Bio-Rad). Localization of Flavonoid End Products—A number of fluorescence microscopy studies have provided evidence for the accumulation of flavonoids in the nuclei of diverse plant species, including Arabidopsis (18Hutzler P. Rischbach R. Heller W. Jungblut T.P. Reuber S. Schmitz R. Veit M. Weissenbck G. Schmitzler J-P. J. Exp. Bot. 1998; 49: 953-965Crossref Scopus (311) Google Scholar, 23Kuras M. Stefanowska-Wronka M. Lynch J.M. Zobel A.M. Ann. Bot. 1999; 84: 135-143Crossref Scopus (25) Google Scholar, 28Buer C.S. Muday G.K. Plant Cell. 2004; 16: 1191-1205Crossref PubMed Scopus (323) Google Scholar, 29Grandmaison J. Ibrahim R.K. J. Plant Physiol. 1996; 147: 653-660Crossref Scopus (35) Google Scholar, 30Feucht W. Treutter D. Polster J. Plant Cell Rep. 2004; 22: 430-436Crossref PubMed Scopus (68) Google Scholar, 31Peer W.A. Brown D.E. Tague B.W. Muday G.K. Taiz L. Murphy A.S. Plant Physiol. 2001; 126: 536-548Crossref PubMed Scopus (273) Google Scholar). 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 (39Murphy A. Peer W.A. Taiz L. Planta. 2000; 211: 315-324Crossref PubMed Scopus (286) Google Scholar, 40Sheahan J.J. Rechnitz G.A. Anal. Chem. 1993; 65: 961-963Crossref Scopus (31) Google Scholar). Optical sectioning by CLSM revealed intense staining in the elongation zone and root tip of wild-type seedlings (Fig. 1a), as previously reported (27Saslowsky D. Winkel-Shirley B. Plant J. 2001; 27: 37-48Crossref PubMed Google Scholar, 39Murphy A. Peer W.A. Taiz L. Planta. 2000; 211: 315-324Crossref PubMed Scopus (286) Google Scholar), and detected only autofluorescence in tt5(86) seedlings, which carry a mutation in the second enzyme of the flavonoid pathway (Fig. 1b) (41Shirley B.W. Hanley S. Goodman H.M. Plant Cell. 1992; 4: 333-347PubMed Google Scholar). Optical sections through DPBA-stained wild-type epidermal (Fig. 1c) and cortex cells (Fig. 1d) showed fluorescence emanating from nuclei throughout the elongation zone and root tip. Fig. 1e illustrates, at higher magnification, DPBA fluorescence in nuclei as well as cell walls and/or plasmalemma and intracellular membranes, similar to previous reports (28Buer C.S. Muday G.K. Plant Cell. 2004; 16: 1191-1205Crossref PubMed Scopus (323) Google Scholar, 31Peer W.A. Brown D.E. Tague B.W. Muday G.K. Taiz L. Murphy A.S. Plant Physiol. 2001; 126: 536-548Crossref PubMed Scopus (273) Google Scholar). No obvious nuclear-specific fluorescence was observed in the hypocotyl/root transition zone, although this is likely to be masked by the high levels of endogenous fluorescence in this region (data not shown). It is not possible to distinguish fluorescence from kaempferol versus quercetin (520 and 543 nm emission, respectively) using CLSM; however, it was previously shown that there is a higher kaempferol/quercetin ratio in the elongation zone and root tip as compared with the hypocotyl/root transition zone (27Saslowsky D. Winkel-Shirley B. Plant J. 2001; 27: 37-48Crossref PubMed Google Scholar, 31Peer W.A. Brown D.E. Tague B.W. Muday G.K. Taiz L. Murphy A.S. Plant Physiol. 2001; 126: 536-548Crossref PubMed Scopus (273) Google Scholar). DPBA staining in the absence of 0.005% Triton X-100 yielded similar but less intense results, and no fluorescence was detected in this region in tt5(86) seedlings when imaged with the parameters used to acquire Fig. 1e (data not shown). It should also be noted that the optical thickness of the images shown in Fig. 1e is 1.5 μm, well below the ~10-μm diameter of nuclei in these cells; thus, the fluorescence is clearly intranuclear, not perinuclear. Immunolocalization of CHS and CHI in Root Cells—To determine whether a link could be established between the nuclear localization of flavonoid end products and the distribution of the flavonoid biosynthetic machinery, co-immunolocalization of CHS and CHI was performed. These enzymes catalyze the first and second committed reactions in the flavonoid pathway, respectively (for a schematic of the pathway, see Ref. 1Winkel-Shirley B. Plant Physiol. 2001; 126: 485-493Crossref PubMed Scopus (2567) Google Scholar). Immunofluorescence CLSM with affinity-purified rabbit anti-CHS and chicken anti-CHI preparations (Fig. 2a) showed co-localization of these enzymes within nuclei of epidermal and cortex cells of the wild-type root tip, as well as in the cytoplasm as previously reported (27Saslowsky D. Winkel-Shirley B. Plant J. 2001; 27: 37-48Crossref PubMed Google Scholar). As with DPBA staining (Fig. 1), the fluorescence emanated from within the nucleus and not from perinuclear ER. These signals were highly specific for CHS and CHI because no fluorescence was detected in CHS (tt4(UV118a)) or CHI (tt5(86)) mutants immunolabeled with anti-CHS or anti-CHI antibodies, respectively (27Saslowsky D. Winkel-Shirley B. Plant J. 2001; 27: 37-48Crossref PubMed Google Scholar). The signals were also not due to bleed-through from the DAPI channel because no fluorescence was detected in the fluorescein isothiocyanate or rhodamine channels for the nuclei of tt4(UV118a) control samples (Fig. 2b). Wild-type seedlings co-labeled with antibodies against CHI and the ER-resident protein BiP (an HSP70) showed that, unlike CHI, BiP is excluded from elongation zone nuclei and exhibits a subcellular distribution consistent with ER localization (Fig. 2c). Together, these results point to specific localization not only of flavonoid end products but also of at least part of the biosynthetic machinery to nuclei of root tip epidermal and cortex cells. To examine this phenomenon at higher resolution, immunotransmission electron microscopy was performed. Employing an indirect immunogold detection methodology, CHS and CHI were again found to co-localize within nuclei of root tip cells (Fig. 3). Although some of the cellular ultrastructure was sacrificed to favor epitope preservation, it is still possible to detect concentric sheets of perinuclear ER as well as the grainy, electron-dense nucleus. Consistent with the immunofluorescence experiments, labeling for both CHS and CHI was observed throughout the cytoplasm and also within the nucleus and at perinuclear ER. Once again, tt4(UV118a) and tt5(86) mutants failed to exhibit any labeling with affinity-purified anti-CHS or anti-CHI antibodies, respectively, and no labeling was observed in other organelles such as mitochondria (27Saslowsky D. Winkel-Shirley B. Plant J. 2001; 27: 37-48Crossref PubMed Google Scholar). In Vivo Localization of CHS and CHI—To examine the subcellular distribution of CHS and CHI in living seedlings, transgenic plants expressing these enzymes fused to the N or C terminus of mGFP5 were produced. Plants harboring a control construct expressing mGFP5 alone displayed fluorescence primarily in the cytoplasm of epidermal and cortex cells in the elongation zone and root tip (Fig. 4a). In contrast, fusion of CHI to the C terminus of mGFP5 (mGFP5-CHI) resulted in accumulation of high levels of GFP fluorescence in the nucleus in several independent lines (Fig. 4b). This was also observed in the epidermal cells of the cotyledon, in which localization to the both the ER and nuclei were clearly visible (Fig. 4c). Similar results were observed with a construct in which CHS was fused to the N terminus of mGFP5 (CHS-mGFP5) (Fig. 4d) and for lines expressing CHI-mGFP5 or mGFP5-CHS, although fluorescence was less intense (data not shown). The combined size of CHI and mGFP5 is 54 kDa, larger than what is believed to pass the nuclear pore complex by passive diffusion (42Keminer O. Peters R. Biophys. J. 1999; 77: 217-228Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar), whereas for CHS and mGFP5, it is even larger (70 kDa). Therefore, this experiment provides further evidence that CHS and CHI are actively sequestered in nuclei of the root tip and elongation zone cells. Detection of CHS and CHI in Isolated Nuclei—As a biochemical complement to the fluorescence microscopy data, we enriched for nuclei from macerated seedlings and assessed the presence of CHS and CHI by immunoblot analysis. Although high levels of CHS and CHI protein were found in the crude cellular lysate, a substantial proportion of each enzyme co-purified with the enriched nuclear fraction (Fig. 5, top two panels). The nuclear marker protein histone H1 was detected exclusively in the nuclei-enriched fraction, whereas the cytosolic marker α-tubulin showed only a very small proportion co-fractionating with nuclei (Fig. 5, bottom two panels), much less than the amount seen for CHS and CHI. α-Tubulin served as a suitable “soluble” cytosolic protein marker for these experiments because most of the steps in the nuclear enrichment protocol were on ice or at 4 °C, conditions that depolymerize microtubules into tubulin monomers (71Moskalewski S. Thyberg J. Friberg U. Cell Tissue Res. 1980; 210: 403-415Crossref PubMed Scopus (25) Google Scholar). Additionally, the centriole is comprised mainly of γ-tubulin, which is not immunoreactive with the monoclonal α-tubulin antibody used in this study. It is unlikely that the CHS and CHI in the nuclei-enriched fraction are due to contamination from other organelles or membrane fragments due to the detergent treatment step employed in these experiments, which serves to selectively lyse chloroplasts and mitochondria. It should be noted that this analysis is not quantitative because it is not possible to recover all nuclei (or all protein, for that matter) from intact organisms. Our finding that at least two of the enzymes of flavonoid metabolism are located not only in the cytoplasm but also in the nuclei of some cells, while surprising, is not without precedent. There are, in fact, a growing number of metabolic enzymes for which dual cytoplasmic/nuclear localization has been observed, both in plants and in other organisms. A number of these enzymes are proposed to have distinct functions in these two locations. For example, mammalian Wiskott-Aldrich syndrome protein controls actin polymerization at the cell periphery but acts as a transcriptional regulator in the nucleus (43Suetsugu S. Takenawa T. J. Biol. Chem. 2003; 278: 42515-42523Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). In yeast, glucokinase has been shown move to the nucleus and alter gene expression in response to different environmental conditions (44Herrero P. Martinez-Campa C. Moreno F. FEBS Lett. 1998; 434: 71-76Crossref PubMed Scopus (82) Google Scholar). Mammalian phospholipid scramblase 1 has a postulated role in the redistribution of phospholipids at the plasma membrane and an undetermined function in the nucleus involving interaction with DNA (45Ben-Efraim I. Zhou Q. Wiedmer T. Gerace L. Sims P.J. Biochemistry. 2004; 43: 3518-3526Crossref PubMed Scopus (57) Google Scholar). Similarly, the glycolytic enzymes phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase have been proposed to function as the primer recognition protein for DNA polymerase-α and as a uracil glycosylase, respectively, in plant and animal nuclei (46Bryant J.A. Brice D.C. Fitchett P.N. Anderson L.E. J. Exp. Bot. 2000; 51: 1945-1947Crossref PubMed Google Scholar, 47Wang X. Sirover M.A. Anderson L.E. Arch. Biochem. Biophys. 1999; 367: 348-353Crossref PubMed Scopus (12) Google Scholar, 48Popanda O. Fox G. Thielmann H.W. Biochim. Biophys. Acta. 1998; 1397: 102-117Crossref PubMed Scopus (110) Google Scholar, 49Mazzola J.L. Sirover M.A. Biochim. Biophys. Acta. 2003; 1622: 50-56Crossref PubMed Scopus (64) Google Scholar). Interestingly, both the cytoplasmic and plastid forms of pea phosphoglycerate kinase are found in the nucleus, indicating that dual distribution can also involve other organelles (50Anderson L.E. Bryant J.A. Carol A.A. Protoplasma. 2004; 223: 103-110P" @default.
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