ENHANCEMENT OF RUTIN PRODUCTION IN Fagopyrum tataricum HAIRY ROOT CULTURES WITH ITS ENDOPHYTIC FUNGAL ELICITORS

Tartary buckwheat (Fagopyrum tataricum) is a potentially important source of rutin, a natural bioactive flavonoid with antihyperglycemic, antioxidative, antihypertensive, and anti-inflammatory properties. This study examines the effects of endophytic fungi on rutin production in the hairy root cultures of F. tataricum. Without obvious changes in the appearance of the hairy roots, the exogenous fungal mycelia elicitors efficiently stimulated the hairy root growth and rutin biosynthesis, and the stimulation effect was mainly dependent on the mycelia elicitor species, as well as its treatment dose. Two endophytic fungal isolates Fat9 (Fusarium oxysporum) and Fat15 (Alternaria sp.) were screened as promising candidates for promoting F. tataricum hairy root growth and rutin production. With application of polysaccharide (PS) of endophyte Fat9 (200 mg/L), and PS of endophyte Fat15 (100 mg/L) to the hairy root cultures on day 25, the rutin yield was increased to 45.9 mg/L and 47.2 mg/L, respectively. That was about 3.1- to 3.2-fold in comparison with the control level of 14.6 mg/L. Moreover, the present study revealed that the accumulation of rutin resulted from the stimulation of the phenylpropanoid pathway by mycelia PS treatments. This may be an efficient strategy for enhancing rutin production in F. tataricum hairy root culture provided with its endophytic mycelia elicitors.     

UV-B radiation affects flavonoids and fungal colonisation in <Emphasis Type="Italic">Fagopyrum esculentum</Emphasis> and <Emphasis Type="Italic">F. tataricum</Emphasis>

In the present study, we have evaluated the effects of increased UV-B radiation that simulates 17% ozone depletion, on fungal colonisation and concentrations of rutin, catechin and quercetin in common buckwheat (Fagopyrum esculentum) and tartary buckwheat (Fagopyrum tataricum). Induced root growth and reduced shoot:root ratios were seen in both of these buckwheat species after enhanced UV-B radiation. There was specific induction of shoot quercetin concentrations in UV-B-treated common buckwheat, whereas there were no specific responses for flavonoid metabolism in tartary buckwheat. Root colonisation with arbuscular mycorrhizal fungi significantly reduced catechin concentrations in common buckwheat roots, and induced rutin concentrations in tartary buckwheat, but did not affect shoot concentrations of the measured phenolics. Specific UV-B-related reductions in the density of microsclerotia were observed in tartary buckwheat, indicating a mechanism that potentially affects fungus-plant interactions. The data support the hypothesis that responses to enhanced UV-B radiation can be influenced by the plant pre-adaptation properties and related changes in flavonoid metabolism.     

Biosynthesis of cyanidin in buckwheat hypocotyls

Aminooxyacetate (AOA), an inhibitor of phenylalanine transamination and deamination in vitro, inhibits the light-induced formation of chlorogenic acid, leucoanthocyanin, rutin and anthocyanin (cyanidin glycosides) in buckwheat hypocotyls. Anthocyanin production is inhibited 87 ± 4%, when excised hypocotyls are incubated in 0.5 mM AOA in Petri dishes. AOA is also effective when taken up through the roots or sprayed onto seedlings. In the presence of biosynthetic precursors of cyanidin (l-phenylalanine, trans-cinnamic acid, p-coumaric acid, naringenin, eriodictyol, dihydrokaempferol. and dihydroquercetin) the inhibition of anthocyanin formation caused by AOA is completely or partially reversed. The general applicability of a complementation technique involving AOA or a similar inhibitor of phenylpropane synthesis is proposed to investigate the biosynthesis of natural products derived from cinnamic acid.     

日光紫外线B辐射对甜荞苯丙烷代谢的影响

对甜荞(Fagopyrum esculentum Moench)苯丙烷次生代谢受紫外线B辐射的响应进行了研究.结果表明:不仅卢丁、槲皮素等黄酮类化合物含量在紫外线B辐射下显著升高,而且叶片苯丙烷单环酚类化合物,如阿魏酸含量等也大幅度升高,两类化合物的应激提高对甜荞的UV-B胁迫提供了重要的保护作用.     

Identification and characterization of granule bound starch synthase I (GBSSI) gene of tartary buckwheat (Fagopyrum tataricum Gaertn.)

Tartary buckwheat (Fagopyrum tataricum Gaertn.) is increasingly considered as an important functional food material because of its rich nutraceutical compounds. Reserve starch is the major component of tartary buckwheat seed. However, the gene sequences and the molecular mechanism of tartary buckwheat starch synthesis are unknown so far. In this study, the complete genomic sequence and full-size cDNA coding tartary buckwheat granule-bound starch synthase I (FtGBSSI), which is responsible for amylose synthesis, were isolated and analyzed. The genomic sequence of the FtGBSSI contained 3947 nucleotides and was composed of 14 exons and 13 introns. The cDNA coding sequence of FtGBSSI shared 63.3%–75.1% identities with those of dicots and 56.6%–57.5% identities with monocots (Poaceae). In deduced amino acid sequence of FtGBSSI, eight motifs conserved among plant starch synthases were identified. A cleavage at the site IVC↓G of FtGBSSI protein produces the chloroplast transit sequence of 78 amino acids and the mature protein of 527 amino acids. The FtGBSSI mature protein showed an identity of 73.4%–77.8% with dicot plants, and 67.6%–70.4% with monocot plants (Poaceae). The mature protein was composed of 20 α-helixes and 16 β-strands, and folds into two main domains, N- and C-terminal domains. The critical residues which are involved in ADP and sugar binding were predicted. These results will be useful to modulate starch composition of buckwheat kernels with the aim to produce novel improved varieties in future breeding programs.     

Microbial Oxidation of sec-Alcohols to Ketones

The major anthocyanin compound in buckwheat sprouts was determined to be cyanidin 3-O-rutinoside (C3R), based on HPLC data and MS/MS spectra. Investigation of the content of phenolic compounds in commercial buckwheat sprouts indicated that hypocotyls are abundant in C3R and rutin, whereas all of the detected flavonoids are abundant in cotyledons. The superoxide anion radical-scavenging activities (SOD-like activities) of phenolic compounds in buckwheat sprouts and their contents indicated that rutin, isoorientin, and orientin contributed mainly to the SOD-like activity of the extract from buckwheat sprouts. In contrast, the contribution of C3R was substantially lower than that of flavonoids.     

Chalcone synthase homologous genes cloning and expression pattern in flowering <Emphasis Type="Italic">Fagopyrum tataricum</Emphasis> Gaertn

Tartary buckwheat (Fagopyrum tataricum Gaertn.) is highly nutritious and an excellent dietary source of flavonoid compounds. Chalcone synthase (CHS) is the first key enzyme involved in flavonoid biosynthesis. Here, three putative CHS genes (designated as FtCHS1 (GU172165), FtCHS2 (KT284884), and FtCHS3 (KT284885) were isolated from tartary buckwheat. Nucleotide sequence analysis indicated that FtCHS1 and FtCHS2 each contained one intron of 444 bp and 157 bp, respectively. FtCHS3 included two introns, one of 86 bp and another of 73 bp. The results of quantitative real-time PCR (qRT-PCR) showed the FtCHSs expression presented the same pattern in the stems and flowers, with FtCHS1>FtCHS3>FtCHS2. A different tendency was found in leaves, with FtCHS3>FtCHS2>FtCHS1. However, there was no direct correlation between the three CHS expression and total flavonoids. Furthermore, high-performance liquid chromatography (HPLC) performance reveals rutin is the most abundant flavonoid in all tissues, leaves should be the main location for quercetin storage in tartary buckwheat.     

光周期对苦荞芽菜生长与品质的影响

苦荞(Fagopyrum tataricum)芽菜是一种新兴健康食品,该研究通过不同光周期(0、4、8、12、16和20 h·d~(-1))处理苦荞芽菜,测定其生物量、叶绿素和主要营养成分的含量以及黄酮合成相关基因的表达水平,以明确不同光周期对苦荞芽菜品质的影响机理。结果表明:(1)随着苦荞芽菜生长时间的增加,芽菜生物量总体呈上升趋势,16 h·d~(-1)光周期时芽菜生长状态最好,鲜重最大。(2)不同光周期对苦荞芽菜各营养成分含量的影响有所差异,可溶性糖含量在芽菜萌发第2天开始逐渐下降,10 d后含量仅为第2天的13.5%～14.5%;花青素含量在芽菜萌发第2天光周期为12 h·d~(-1)时最高(2.16 mg/g),不同光周期处理4～10 d后均显示出降低趋势;芦丁是苦荞芽菜中主要的黄酮类化合物,芦丁含量在不同光周期处理后有所变化且适当的光照有利于其合成和积累,并在处理第4天光周期为16 h·d~(-1)时含量最高(59.60 mg/g)。(3)qRT-PCR分析表明,不同光周期处理的苦荞芽菜中各黄酮合成相关基因的表达量在第2天最高,随生长周期其表达量均不同程度降低,但黄酮醇合成支路关键酶基因FtFLS1和花青素支路基因FtDFR2表达量上升、且显示出强烈的光诱导特征。研究发现,光周期对苦荞芽菜的生物量影响较大,黑暗有利于胚轴伸长,长光周期(16 h·d~(-1))有利于鲜重的增加;在苦荞萌发过程中,苦荞芽菜可溶性糖、花青素和芦丁含量整体呈减少趋势,可溶性糖和花青素含量均在光周期16 h·d~(-1)处理第2天时最高,芦丁含量在光周期为16 h·d~(-1)处理第4天时最高,建议苦荞芽菜在光周期为16 h·d~(-1)生长4 d时采食。     

Development of microsatellite markers from tartary buckwheat

A genomic library enriched with (gT)_n repeats from tartary buckwheat (Fagopyrum tataricum) was constructed using 5′-anchored PCR for the development of microsattellite markers. Sequencing analysis of 5 clones from the library showed that they all contained microsatellites (totally 10 loci), and each was unique. An additional locus-specific primer was designed according to flanking sequence. Two of the microsatellite loci of 10 tartary buckwheat varieties were amplified using an anchored primer and a locus-specific primer, which revealed a clear polymorphic pattern. The data confirmed that the degenerate primer was reliably anchoring at the 5′-end of the microsatellite, and the primers developed based on this technology could be used for diversity analysis of tartary buckwheat.