Rutin Concentration and Characterization of Rutinosidase in Perennial Buckwheat (Fagopyrum cymosum) and Its Application in Foods.

To evaluate the potential of perennial buckwheat (Fagopyrum cymosum; FC) as a food source, rutin concentration was investigated. FC contains more than 1% (w/w) rutin and 0.03% quercetin in the leaves, flowers, and seeds. In particular, rutin and quercetin concentrations were high in plant seeds. Therefore, FC is useful as a rutin- and quercetin-rich material. In contrast, the FC seed contained a large amount of rutinosidase. Purified rutinosidase in a homogenous mixture consisted of only one isozyme with M.W. of 58.4 KD and low Km for rutin (0.367 mM). The rutin concentration in the FC dough decreased to almost zero, 10 min after the addition of water. Parallel to the decrease in rutin, quercetin was increased, and strong bitterness was generated, whereas steam-heated flour in which rutinosidase was inactivated did not have rutin hydrolysis and bitterness. These results indicate that rutinosidase is a major cause of rutin hydrolysis and bitterness. The in vitro rutinosidase is inactivated at pH 8.0 and 65 °C. Therefore, the control of dough pH and temperature should be useful in preventing rutinosidase activity.

Breeding of Buckwheat for Usage of Sprout and Pre-Harvest Sprouting Resistance.

Buckwheat is recognized as an important traditional crop and supports local economies in several regions around the world. Buckwheat is used, for example, as a cereal grain, noodle and bread. In addition, buckwheat is also used as a sprout or a young seedling. For these foods, sprouting is an important characteristic that affects food quality. For foods made from buckwheat flour, pre-harvest sprouting may decrease yield, which also leads to the deterioration of noodle quality. Breeding buckwheat that is resistant to pre-harvest sprouting is therefore required. Germination and subsequent growth are also important characteristics of the quality of sprouts. Although buckwheat sprouts are the focus because they contain many functional compounds, such as rutin, several problems have been noted, such as thin hypocotyls and husks remaining on sprouts. To date, several new varieties have been developed to resolve these quality issues. In this review, we summarize and introduce research on the breeding of buckwheat related to quality, sprouting and subsequent sprout growth.

Breeding of Buckwheat to Reduce Bitterness and Rutin Hydrolysis.

Buckwheat (Fagopyrum esculentum) is recognized as an important traditional crop in some regions, and its taste is an important characteristic. Of the three cultivated buckwheat species, Tartary buckwheat (Fagopyrum tataricum) and perennial buckwheat (Fagopyrum cymosum) have strong bitterness in their seeds, which has prevented the wider use of the seeds of these varieties. In Tartary buckwheat, some studies have focused on the cause of strong bitterness generation. Tartary buckwheat seeds contain large amounts of the functional compounds rutin and rutinosidase, and rutin hydrolysis by rutinosidase has been found to be the trigger of rutin hydrolysis. Therefore, a variety with only a trace of rutinosidase and with reduced bitterness is required. The rutinosidase in Tartary buckwheat seeds consists of two major isozymes with very similar enzymatic characteristics, which can hydrolyze flour rutin within several minutes after the addition of water. Recently, the trace-rutinosidase variety Manten-Kirari in Tartary buckwheat was developed. The trace-rutinosidase characteristics were dominated by a single recessive gene. In 'Manten-Kirari' dough and foods, such as breads, confectionaries, and noodles, the rutin residual ratio was higher and bitterness was reduced compared to that of the normal-rutinosidase variety. In this review, we summarize the detailed research on the breeding of buckwheat related to reducing bitterness and rutin hydrolysis.

Development of a DNA marker for variety discrimination specific to 'Manten-Kirari' based on an NGS-RNA sequence in Tartary buckwheat (Fagopyrum tataricum).

To discriminate the trace-rutinosidase variety of Tartary buckwheat 'Manten-Kirari', we developed DNA markers based on RNA polymorphism. Specifically, we mapped 17.76 GB RNA sequences, obtained using HiSeq2000, to create 11,358 large contigs constructed de novo from 'Manten-Kirari' RNA derived from GS-FLX+ titanium. From these, we developed eight DNA markers corresponding to single- to four-nucleotide polymorphisms between 'Manten-Kirari' and 'Hokkai T8', which is representative of normal rutinosidase content varieties in Japan. Using these markers, 'Manten-Kirari' was discriminated from 'Hokkai T8' by eight markers, from major Tartary buckwheat varieties by three markers, and from common buckwheats by two markers. We also performed direct PCR from flour and dried noodle made with 'Manten-Kirari' and 'Hokkai T8'. Based on the results, the DNA markers developed are promising for discriminating 'Manten-Kirari'. This is the first study to develop a DNA marker to discriminate varieties in the Polygonaceae family including buckwheat species.

A new buckwheat dihydroflavonol 4-reductase (DFR), with a unique substrate binding structure, has altered substrate specificity.

BACKGROUND: Dihydroflavonol 4-reductase (DFR) is the key enzyme committed to anthocyanin and proanthocyanidin biosynthesis in the flavonoid biosynthetic pathway. DFR proteins can catalyse mainly the three substrates (dihydrokaempferol, dihydroquercetin, and dihydromyricetin), and show different substrate preferences. Although relationships between the substrate preference and amino acids in the region responsible for substrate specificity have been investigated in several plant species, the molecular basis of the substrate preference of DFR is not yet fully understood. RESULTS: By using degenerate primers in a PCR, we isolated two cDNA clones that encoded DFR in buckwheat (Fagopyrum esculentum). Based on sequence similarity, one cDNA clone (FeDFR1a) was identical to the FeDFR in DNA databases (DDBJ/Gen Bank/EMBL). The other cDNA clone, FeDFR2, had a similar sequence to FeDFR1a, but a different exon-intron structure. Linkage analysis in an F2 segregating population showed that the two loci were linked. Unlike common DFR proteins in other plant species, FeDFR2 contained a valine instead of the typical asparagine at the third position and an extra glycine between sites 6 and 7 in the region that determines substrate specificity, and showed less activity against dihydrokaempferol than did FeDFR1a with an asparagine at the third position. Our 3D model suggested that the third residue and its neighbouring residues contribute to substrate specificity. FeDFR1a was expressed in all organs that we investigated, whereas FeDFR2 was preferentially expressed in roots and seeds. CONCLUSIONS: We isolated two buckwheat cDNA clones of DFR genes. FeDFR2 has unique structural and functional features that differ from those of previously reported DFRs in other plants. The 3D model suggested that not only the amino acid at the third position but also its neighbouring residues that are involved in the formation of the substrate-binding pocket play important roles in determining substrate preferences. The unique characteristics of FeDFR2 would provide a useful tool for future studies on the substrate specificity and organ-specific expression of DFRs.

Isolation and characterization of genes encoding leucoanthocyanidin reductase (FeLAR) and anthocyanidin reductase (FeANR) in buckwheat (Fagopyrum esculentum).

Proanthocyanidins (PAs) are a major group of flavonoids synthesized via the phenylpropanoid biosynthesis pathway, however the pathway has not been fully characterized in buckwheat. Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) are involved in the last steps of PA biosynthesis. To isolate the genes for these enzymes from buckwheat we performed PCR using degenerate primers and obtained cDNAs of ANR and LAR, which we designated FeANR and FeLAR1. A search for homologs in a buckwheat genome database with both sequences returned two more LAR sequences, designated FeLAR2 and FeLAR3. Linkage analysis with an F2 segregating population indicated that the three LAR loci were not genetically linked. We detected high levels of PAs in roots and cotyledons of buckwheat seedlings and in buds and flowers of mature plants. FeANR and FeLAR1-3 were expressed in most organs but had different expression patterns. Our findings would be useful for breeding and further analysis of PA synthesis and its regulation in buckwheat.

Acetyl-L-carnitine suppresses thyroid hormone-induced and spontaneous anuran tadpole tail shortening.

Mitochondrial membrane permeability transition (MPT) plays a crucial role in apoptotic tail shortening during anuran metamorphosis. L-carnitine is known to shuttle free fatty acids (FFAs) from the cytosol into mitochondria matrix for ß-oxidation and energy production, and in a previous study we found that treatment with L-carnitine suppresses 3, 3', 5-triiodothyronine (T3 ) and FFA-induced MPT by reducing the level of FFAs. In the present study we focus on acetyl-L-carnitine, which is also involved in fatty acid oxidation, to determine its effect on T3 -induced tail regression in Rana rugosa tadpoles and spontaneous tail regression in Xenopus laevis tadpoles. The ladder-like DNA profile and increases in caspase-3 and caspase-9 indicative of apoptosis in the tails of T3 -treated tadpoles were found to be suppressed by the addition of acetyl-L-carnitine. Likewise, acetyl-L-carnitine was found to inhibit thyroid hormone regulated spontaneous metamorphosis in X. laevis tadpoles, accompanied by decreases in caspase and phospholipase A2 activity, as well as non-ladder-like DNA profiles. These findings support our previous conclusion that elevated levels of FFAs initiate MPT and activate the signaling pathway controlling apoptotic cell death in tadpole tails during anuran metamorphosis.

Multi-modal effects of BMP signaling on Nodal expression in the lateral plate mesoderm during left-right axis formation in the chick embryo.

During development of left-right asymmetry in the vertebrate embryo, Nodal plays a central role for determination of left-handedness. Bone morphogenetic protein (BMP) signaling has an important role for regulation of Nodal expression, although there is controversy over whether BMP signaling has a positive or negative effect on Nodal expression in the chick embryo. As BMP is a morphogen, we speculated that different concentrations might induce different responses in the cells of the lateral plate mesoderm (LPM). To test this hypothesis, we analyzed the effects of various concentrations of BMP4 and NOGGIN on Nodal expression in the LPM. We found that the effect on Nodal expression varied in a complex fashion with the concentration of BMP. In agreement with previous reports, we found that a high level of BMP signaling induced Nodal expression in the LPM, whereas a low level inhibited expression. However, a high intermediate level of BMP signaling was found to suppress Nodal expression in the left LPM, whereas a low intermediate level induced Nodal expression in the right LPM. Thus, the high and the low intermediate levels of BMP signaling up-regulated Nodal expression, but the high intermediate and low levels of BMP signaling down-regulated Nodal expression. Next, we sought to identify the mechanisms of this complex regulation of Nodal expression by BMP signaling. At the low intermediate level of BMP signaling, regulation depended on a NODAL positive-feedback loop suggesting the possibility of crosstalk between BMP and NODAL signaling. Overexpression of a constitutively active BMP receptor, a constitutively active ACTIVIN/NODAL receptor and SMAD4 indicated that SMAD1 and SMAD2 competed for binding to SMAD4 in the cells of the LPM. Nodal regulation by the high and low levels of BMP signaling was dependent on Cfc up-regulation or down-regulation, respectively. We propose a model for the variable effects of BMP signaling on Nodal expression in which different levels of BMP signaling regulate Nodal expression by a balance between BMP-pSMAD1/4 signaling and NODAL-pSMAD2/4 signaling.

BMP inhibition by DAN in Hensen's node is a critical step for the establishment of left-right asymmetry in the chick embryo.

During left-right (L-R) axis formation, Nodal is expressed in the node and has a central role in the transfer of L-R information in the vertebrate embryo. Bone morphogenetic protein (BMP) signaling also has an important role for maintenance of gene expression around the node. Several members of the Cerberus/Dan family act on L-R patterning by regulating activity of the transforming growth factor-ß (TGF-ß) family. We demonstrate here that chicken Dan plays a critical role in L-R axis formation. Chicken Dan is expressed in the left side of the node shortly after left-handed Shh expression and before the appearance of asymmetrically expressed genes in the lateral plate mesoderm (LPM). In vitro experiments revealed that DAN inhibited BMP signaling but not NODAL signaling. SHH had a positive regulatory effect on Dan expression while BMP4 had a negative effect. Using overexpression and RNA interference-mediated knockdown strategies, we demonstrate that Dan is indispensable for Nodal expression in the LPM and for Lefty-1 expression in the notochord. In the perinodal region, expression of Dan and Nodal was independent of each other. Nodal up-regulation by DAN required NODAL signaling, suggesting that DAN might act synergistically with NODAL. Our data indicate that Dan plays an essential role in the establishment of the L-R axis by inhibiting BMP signaling around the node.

A secreted BMP antagonist, Cer1, fine tunes the spatial organization of the ureteric bud tree during mouse kidney development.

The epithelial ureteric bud is critical for mammalian kidney development as it generates the ureter and the collecting duct system that induces nephrogenesis in dicrete locations in the kidney mesenchyme during its emergence. We show that a secreted Bmp antagonist Cerberus homologue (Cer1) fine tunes the organization of the ureteric tree during organogenesis in the mouse embryo. Both enhanced ureteric expression of Cer1 and Cer1 knock out enlarge kidney size, and these changes are associated with an altered three-dimensional structure of the ureteric tree as revealed by optical projection tomography. Enhanced Cer1 expression changes the ureteric bud branching programme so that more trifid and lateral branches rather than bifid ones develop, as seen in time-lapse organ culture. These changes may be the reasons for the modified spatial arrangement of the ureteric tree in the kidneys of Cer1+ embryos. Cer1 gain of function is associated with moderately elevated expression of Gdnf and Wnt11, which is also induced in the case of Cer1 deficiency, where Bmp4 expression is reduced, indicating the dependence of Bmp expression on Cer1. Cer1 binds at least Bmp2/4 and antagonizes Bmp signalling in cell culture. In line with this, supplementation of Bmp4 restored the ureteric bud tip number, which was reduced by Cer1+ to bring it closer to the normal, consistent with models suggesting that Bmp signalling inhibits ureteric bud development. Genetic reduction of Wnt11 inhibited the Cer1-stimulated kidney development, but Cer1 did not influence Wnt11 signalling in cell culture, although it did inhibit the Wnt3a-induced canonical Top Flash reporter to some extent. We conclude that Cer1 fine tunes the spatial organization of the ureteric tree by coordinating the activities of the growth-promoting ureteric bud signals Gndf and Wnt11 via Bmp-mediated antagonism and to some degree via the canonical Wnt signalling involved in branching.

Ontogeny of angiopoietin-like protein 1, 2, 3, 4, 5, and 7 genes during chick embryonic development.

Angiopoietin-like proteins (ANGPTLs) are secreted proteins possessing an amino-terminal coiled-coil domain and a carboxyl-terminal fibrinogen-like domain and are known as angiogenic factors. Several members of ANGPTLs also regulate lipid metabolism independently of angiogenic effects, but most of their functions during vertebrate development are not demonstrated. To ascertain their developmental functions, we examined the expression patterns of Angptl1, 2, 3, 4, 5, and 7 orthologues during chick development using whole-mount in situ hybridization. Angptl1 was first detected at embryonic day 3 (E3) in the somite. At E4, Angptl1 was expressed in somite-derivatives and limb mesenchyme. Angptl2 was first detected at E3 in the hindbrain. At E4, Angptl2 was expressed in neuroepithelium of forebrain and hindbrain and partly in the heart. Angptl3 was first detected at E3 and continued to be expressed in the liver and yolk sac at E4. Angptl4 was first detected at E3 in the somites and liver. At E4, Angptl4 was also observed in the heart. Angptl5 was not detected in these developmental stages. Angptl7 was first detected at E3 in the ectoderm overlying the lenses of the eyes. At E4, Angptl7 was specifically expressed in cornea. These data suggest that each member of the ANGPTL family could be related to angiogenesis during various organogeneses of the developing chick embryo.

FGF signaling segregates biliary cell-lineage from chick hepatoblasts cooperatively with BMP4 and ECM components in vitro.

Intrahepatic bile ducts (IHBDs) are indispensable for transporting bile secreted from hepatocytes to the hepatic duct. The biliary epithelial cells (BECs) of the IHBD arise from bipotent hepatoblasts around the portal vein, suggesting the portal mesenchyme is essential for their development. However, except for Notch or Activin/TGF-beta signaling molecules, it is not known which molecules regulate IHBD development. Here, we found that FGF receptors and BMP4 are specifically expressed in the developing IHBD and the hepatic mesenchyme, respectively. Using a mesenchyme-free culture of liver bud, we showed that bFGF and FGF7 induce the hepatoblasts to differentiate into BECs, and that BMP4 enhances bFGF-induced BEC differentiation. The extracellular matrix (ECM) components in the hepatic mesenchyme induced BEC differentiation. Forced expression of a constitutively active form of the FGF receptor partially induced BEC differentiation markers in vivo. These data strongly suggest that bFGF and FGF7 promote BEC differentiation cooperatively with BMP4 and ECMs in vivo.

Neurturin-GFRalpha2 signaling controls liver bud migration along the ductus venosus in the chick embryo.

During chick liver development, the liver bud arises from the foregut, invaginates into the septum transversum, and elongates along and envelops the ductus venosus. However, the mechanism of liver bud migration is only poorly understood. Here, we demonstrate that a GDNF family ligand involved in neuronal outgrowth and migration, neurturin (NRTN), and its receptor, GFRalpha2, are essential for liver bud migration. In the chick embryo, we found that GFRalpha2 was expressed in the liver bud and that NRTN was expressed in the endothelial cells of the ductus venosus. Inhibition of GFRalpha2 signaling suppressed liver bud elongation along the ductus venous without affecting cell proliferation and apoptosis. Moreover, ectopic expression of NRTN perturbed the directional migration along the ductus venosus, leading to splitting or ectopic branching of the liver. We showed that liver buds selectively migrated toward an NRTN-soaked bead in vitro. These data represent a new model for liver bud migration: NRTN secreted from endothelial cells functions as a chemoattractant to direct the migration of the GFRalpha2-expressing liver bud in early liver development.

Dan is required for normal morphogenesis and patterning in the developing chick inner ear.

During vertebrate inner ear development, compartmentalization of the auditory and vestibular apparatuses along two axes depends on the patterning of transcription factors expressed in a region-specific manner. Although most of the patterning is regulated by extrinsic signals, it is not known how Nkx5.1 and Msx1 are patterned. We focus on Dan, the founding member of the Cerberus/Dan gene family that encodes BMP antagonists, and describe its function in morphogenesis and patterning. First, we confirmed that Dan is expressed in the dorso-medial region of the otic vesicle that corresponds to the presumptive endolymphatic duct and sac (ed/es). Second, we used siRNA knockdown to demonstrate that depletion of Dan induced both a severe reduction in the size of the ed/es and moderate deformities of the semicircular canals and cochlear duct. Depletion of Dan also caused suppression of Nkx5.1 in the dorso-lateral region, suppression of Msx1 in the dorso-medial region, and ectopic induction of Nkx5.1 and Msx1 in the ventro-medial region. Most of these phenotypes also appeared following misexpression of the constitutively active form of BMP receptor type Ib. Thus, Dan is required for the normal morphogenesis of the inner ear and, by inhibiting BMP signaling, for the patterning of the transcription factors Nkx5.1 and Msx1.

Cyclosporin A inhibits thyroid hormone-induced shortening of the tadpole tail through membrane permeability transition.

Regression of the tadpole tail through muscule cell apoptosis is one of the most spectacular events in amphibian metamorphosis. Accumulated evidence has shown that mitochondrial membrane permeability transition (MPT) plays a crucial role in apoptosis. Previously we reported that cyclosporin A (CsA) suppressed 3,5,3'-triiodothyronine (T(3))-induced mitochondrial swelling, which was coupled with cytochrome c (Cyt.c) release through MPT [Comp. Biochem. Phys. 130 (2001) 411-418]. To further clarify the mechanism of tadpole metamorphosis, the present study investigates the effect of CsA on T(3) induced tadpole tail shortening. A low concentration of T(3) (5 x 10(-8) M) was found to induce a shortening of stage X Rana rugosa tadpole tails, accompanied by an increase in caspase-3- and -9 like protease activity, as well as an increase in DNA-fragmentation and ladder formation, while CsA was seen to suppress the effects of T(3). The stage X tadpole tail was found to express Bax mRNA and this expression was not affected by T(3) treatment. CsA, on the other hand, proved to have a slightly supressive effection on Bax expression. 20 microM T(3) as well as 50 microM Ca(2+) induced swelling in mitochondria isolated from the liver of R. rugosa resulting in the release of apoptosis related substances, and the released fraction activated cytosolic caspase-3 and -9 in the presence of dATP. This result indicated that Cyt.c might be released from mitochondria by treatment with T(3) through both direct and indirect action of T(3). From these results and other data it was concluded that mitochondrial MPT plays an important role in T(3)-induced apoptosis in the tadpole tail, resulting in tail shortening, and CsA was seen to suppress the effects of T(3).