DNA methylation analysis of floral parts revealed dynamic changes during the development of homostylous Fagopyrum tataricum and heterostylous F. esculentum flowers.

BACKGROUND: Proper flower development is essential for plant reproduction, a crucial aspect of the plant life cycle. This process involves precisely coordinating transcription factors, enzymes, and epigenetic modifications. DNA methylation, a ubiquitous and heritable epigenetic mechanism, is pivotal in regulating gene expression and shaping chromatin structure. Fagopyrum esculentum demonstrates anti-hypertensive, anti-diabetic, anti-inflammatory, cardio-protective, hepato-protective, and neuroprotective properties. However, the heteromorphic heterostyly observed in F. esculentum poses a significant challenge in breeding efforts. F. tataricum has better resistance to high altitudes and harsh weather conditions such as drought, frost, UV-B radiation damage, and pests. Moreover, F. tataricum contains significantly higher levels of rutin and other phenolics, more flavonoids, and a balanced amino acid profile compared to common buckwheat, being recognised as functional food, rendering it an excellent candidate for functional food applications. RESULTS: This study aimed to compare the DNA methylation profiles between the Pin and Thrum flower components of F. esculentum, with those of self-fertile species of F. tataricum, to understand the potential role of this epigenetic mechanism in Fagopyrum floral development. Notably, F. tataricum flowers are smaller than those of F. esculentum (Pin and Thrum morphs). The decline in DNA methylation levels in the developed open flower components, such as petals, stigmas and ovules, was consistent across both species, except for the ovule in the Thrum morph. Conversely, Pin and Tartary ovules exhibited a minor decrease in DNA methylation levels. The highest DNA methylation level was observed in Pin stigma from closed flowers, and the most significant decrease was in Pin stigma from open flowers. In opposition, the nectaries of open flowers exhibited higher levels of DNA methylation than those of closed flowers. The decrease in DNA methylation might correspond with the downregulation of genes encoding methyltransferases. CONCLUSIONS: Reduced overall DNA methylation and the expression of genes associated with these epigenetic markers in fully opened flowers of both species may indicate that demethylation is necessary to activate the expression of genes involved in floral development.

APETALA2-like Floral Homeotic Protein Up-Regulating FaesAP1_2 Gene Involved in Floral Development in Long-Homostyle Common Buckwheat.

In the rosid species Arabidopsis thaliana, the AP2-type AP2 transcription factor (TF) is required for specifying the sepals and petals identities and confers a major A-function to antagonize the C-function in the outer floral whorls. In the asterid species Petunia, the AP2-type ROB TFs are required for perianth and pistil development, as well as repressing the B-function together with TOE-type TF BEN. In Long-homostyle (LH) Fagopyrum esculentum, VIGS-silencing showed that FaesAP2 is mainly involved in controlling filament and style length, but FaesTOE is mainly involved in regulating filament length and pollen grain development. Both FaesAP2 (AP2-type) and FaesTOE (TOE-type) are redundantly involved in style and/or filament length determination instead of perianth development. However, neither FaesAP2 nor FaesTOE could directly repress the B and/or C class genes in common buckwheat. Moreover, the FaesAP1_2 silenced flower showed tepal numbers, and filament length decreased obviously. Interestingly, yeast one-hybrid (Y1H) and dual-luciferase reporter (DR) further suggested that FaesTOE directly up-regulates FaesAP1_2 to be involved in filament length determination in LH common buckwheat. Moreover, the knockdown of FaesTOE expression could result in expression down-regulation of the directly target FaesAP1_2 in the FaesTOE-silenced LH plants. Our findings uncover a stamen development pathway in common buckwheat and offer deeper insight into the functional evolution of AP2 orthologs in the early-diverging core eudicots.

Buckwheat: an underutilized crop with attractive sensory qualities and health benefits.

The pseudocereal buckwheat is one of the ancient domesticated crops. The aim of the present review was to outline the potential of buckwheat as an agricultural crop and brings studies on buckwheat into a new larger perspective combining current knowledge in agricultural history and practice, nutritional and sensory properties, as well as possible benefits to human health. Historically, buckwheat was an appreciated crop because of its short growth period, moderate requirements for growth conditions, and high adaptability to adverse environments. Nowadays, interest in buckwheat-based food has increased because of its nutritional composition and many beneficial properties for human health. Buckwheat is a rich course of proteins, dietary fibers, vitamins, minerals, and bioactive compounds, including flavonoids. Moreover, it contains no gluten and can be used in the production of gluten-free foods for individuals diagnosed with celiac disease, non-celiac gluten sensitivity, or wheat protein allergies. Buckwheat is traditionally used in the production of various foods and can be successfully incorporated into various new food formulations with positive effects on their nutritional value and attractive sensory properties. Further research is needed to optimize buckwheat-based food development and understand the mechanism of the health effects of buckwheat consumption on human well-being.

Nectar-Inhabiting Bacteria Affect Olfactory Responses of an Insect Parasitoid by Altering Nectar Odors.

Floral nectar is ubiquitously colonized by a variety of microorganisms among which yeasts and bacteria are the most common. Microorganisms inhabiting floral nectar can alter several nectar traits, including nectar odor by producing microbial volatile organic compounds (mVOCs). Evidence showing that mVOCs can affect the foraging behavior of insect pollinators is increasing in the literature, whereas the role of mVOCs in altering the foraging behavior of third-trophic level organisms such as insect parasitoids is largely overlooked. Parasitoids are frequent visitors of flowers and are well known to feed on nectar. In this study, we isolated bacteria inhabiting floral nectar of buckwheat, Fagopyrum esculentum (Polygonales: Polygonaceae), to test the hypothesis that nectar bacteria affect the foraging behavior of the egg parasitoid Trissolcus basalis (Hymenoptera: Scelionidae) via changes in odors of nectar. In behavioral assays, we found that T. basalis wasps are attracted toward nectar fermented by 4 out of the 14 bacterial strains isolated, which belong to Staphylococcus epidermidis, Terrabacillus saccharophilus (both Firmicutes), Pantoea sp. (Proteobacteria), and Curtobacterium sp. (Actinobacteria). Results of chemical investigations revealed significant differences in the volatile blend composition of nectars fermented by the bacterial isolates. Our results indicate that nectar-inhabiting bacteria play an important role in the interactions between flowering plants and foraging parasitoids. These results are also relevant from an applied perspective as flowering resources, such as buckwheat, are largely used in agriculture to promote conservation biological control of insect pests.

Buckwheat (Fagopyrum esculentum) Hulls Are a Rich Source of Fermentable Dietary Fibre and Bioactive Phytochemicals.

Pseudo-cereals such as buckwheat (Fagopyrum esculentum) are valid candidates to promote diet biodiversity and nutrition security in an era of global climate change. Buckwheat hulls (BHs) are currently an unexplored source of dietary fibre and bioactive phytochemicals. This study assessed the effects of several bioprocessing treatments (using enzymes, yeast, and combinations of both) on BHs' nutrient and phytochemical content, their digestion and metabolism in vitro (using a gastrointestinal digestion model and mixed microbiota from human faeces). The metabolites were measured using targeted LC-MS/MS and GC analysis and 16S rRNA gene sequencing was used to detect the impact on microbiota composition. BHs are rich in insoluble fibre (31.09 ± 0.22% as non-starch polysaccharides), protocatechuic acid (390.71 ± 31.72 mg/kg), and syringaresinol (125.60 ± 6.76 mg/kg). The bioprocessing treatments significantly increased the extractability of gallic acid, vanillic acid, p-hydroxybenzoic acid, syringic acid, vanillin, syringaldehyde, p-coumaric acid, ferulic acid, caffeic acid, and syringaresinol in the alkaline-labile bound form, suggesting the bioaccessibility of these phytochemicals to the colon. Furthermore, one of the treatments, EC_2 treatment, increased significantly the in vitro upper gastrointestinal release of bioactive phytochemicals, especially for protocatechuic acid (p < 0.01). The BH fibre was fermentable, promoting the formation mainly of propionate and, to a lesser extent, butyrate formation. The EM_1 and EC_2 treatments effectively increased the content of insoluble fibre but had no effect on dietary fibre fermentation (p > 0.05). These findings promote the use of buckwheat hulls as a source of dietary fibre and phytochemicals to help meet dietary recommendations and needs.

Adding Modified Buckwheat Sprouts to an Atherogenic Diet - the Effect on Selected Nutritional Parameters in Rats.

The germinated seeds of many plants are a natural source of substances that can be used to supplement food and increase its functionality. The seeds' metabolism may be modified during germination to produce specific health-promoting compounds. Fagopyrum esculentum Moench is a rich source of nutrients. Buckwheat seeds modified during germination may be helpful as an additive to new functional food products with anti-atherogenic properties. However, their effect and safety should be assessed in in vivo studies. The aim of the study was to evaluate the effect that adding modified buckwheat sprouts (Fagopyrum esculentum Moench) to an atherogenic (high-fat) diet has on the morphology and digestibility parameters of rats. Buckwheat seeds were modified by adding the probiotic strain of the yeast Saccharomyces cerevisiae var. boulardii. The study was carried out on 32 Wistar rats, and digestibility and blood counts were assessed during the experiment. There was no evidence of an adverse effect on the animals' weight gain and nutritional efficiency. However, the influence of diets with freeze-dried buckwheat sprouts on digestibility and morphological parameters was noticed. Fat digestibility registered a statistically significant decrease in the groups fed a high-fat diet with the addition of sprouts. The study shows a new direction in the use of buckwheat sprouts.

Plant Ploidy Level and the Presence of Cadmium in the Growing Environment Changes the Content of the Main Components of the Phenolic Complex in Buckwheat Sprouts.

For the first time, the composition and the content of the main components of the phenolic complex of aboveground organs of buckwheat plants (Fagopyrum esculentum Moench) with different levels of ploidy (2n and 4n) at the initial stages of ontogenesis and their changes under the action of cadmium (Cd, 65 µM) were studied by the method of high-performance liquid chromatography. In all variants, phenolic compounds (PCs) were represented by chlorogenic acid (phenylpropanoid), rutin (flavonol), as well as orientin, isoorientin, vitexin, and isovitexin (flavones). The amount of PCs in the cotyledon leaves in most cases exceeded that in the hypocotyls. Buckwheat seedlings of the tetraploid genotype are characterized by a higher accumulation of PCs as compared to the diploid genotype. Under the effect of Cd, the content of their individual representatives changed: in the hypocotyls of the diploid genotype, it decreased in most cases, whereas in the hypocotyls of the tetraploid genotype it increased. In the cotyledon leaves, the tendency was opposite. It was found that seedlings of two F. esculentum genotypes with different ploidy levels differed in the content of the main PCs and in their response to Cd, which is important for breeding this culture for stress resistance.

Identification of Colletotrichum liriopes as the Causative Agent of Anthracnose in Buckwheat (Fagopyrum esculentum) in China.

Buckwheat (Fagopyrum esculentum), belonging to the Polygonaceae family, is one of the most important "functional food" crops in China. In fall 2020, buckwheat plants grown in field exhibiting stem canker symptoms were found in Tongxin county, Ningxia province, China. Symptoms included stem canker, dieback and extensive vascular discoloration. Cankers were bleached, silvery-white to dark gray, slightly sunken, oval to linear with slightly tapered tips, pycnidia formation was also observed within the cankers. Disease incidence was approximately 30% and moderate to high severity across the field. Symptomatic tissues were cut into 1-2 cm pieces, surface sterilized (75% ethanol for 30 s and 0.1% NaClO for 2 min) and washed four times with sterile distilled water, dried in sterile filter paper for 3 times, and placed on potato dextrose agar (PDA) at 25 ℃. Fluffy mycelium was visible for all isolates after 48 h of incubation. Twenty-five single isolates were hyphal-tip purified on PDA. Six representative isolates were used for further study. The fungal colonies on PDA were flat with an entire margin, gray aerial hyphae, light brown pigmentation, appressed slimy mycelium within which numerous brown-black perithecia formed. Colonies on oatmeal agar (OA) were flat, with flocculent mycelium, conidiomata and conidia and the reverse side was black to smoke-grey. Sparse brown-black perithecia were observed within the mycelium. Conidia were hyaline, one-celled, smooth-walled, rarely finely verruculose, aseptate, slightly curved, both sides gradually tapering towards the round to slightly acute apex and truncate base, measured (15.7-23.7) µm (length) × (2.8-5.7) µm (width), (avg. 20.2 µm×4.2 µm, n=100). Genomic DNA was extracted from the same six isolates, the internal transcribed spacer (ITS) region and the genes encoding beta-tubulin (TUB), chitin synthase (CHS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and histone H3 (HIS3) were independently amplified with primers V9G/ITS4, T1/Bt-2b, CHS-354R/CHS-79F, GDF1/GDR1 and CYLH3F/CYLH3R, respectively (Damm et al., 2019). Sequences for all six isolates were identical. The sequences of the representative isolate 9J1 were deposited in GenBank (accession nos. MW819604, MW836580, MW836577, MW836578 and MW836579). The results of BLASTn showed that the ITS, TUB, CHS, GAPDH and HIS3 sequences of 9J1 were greater than 98% (555/557bp, 477/486bp, 258/259bp, 242/248bp and 339/345bp, respectively) identical to those of Colletotrichum liriopes (GenBank: MT645674 (ITS), GU228098 (TUB), MT663546 (CHS), MH291255 (GAPDH) and MH292811 (HIS3)). MrBayes phylogenetic analysis using concatenated sequences of ITS, TUB, CHS, GAPDH and HIS3 showed that the isolate clustered monophyletically with strains of C. liriopes. Based on morphological and molecular characteristics, the isolate was identified as C. liriopes. To fulfill Koch's postulates, spores of the isolate 9J1 grown on OA for 14 days were harvested in 0.01% Tween 20, and the suspension were adjusted to 104 spores/ml. Six one-month-old potted plants of buckwheat were inoculated by spraying the spore suspension until run-off. Plants were kept inside a plastic bag for 3 d to maintain high relative humidity and maintained in the greenhouse. Six control plants were sprayed with sterile deionized water and kept under the same conditions. Two weeks after inoculation, all inoculated plants showed stem canker symptoms as described above, whereas control plants remained healthy. The pathogen was successfully reisolated from leaf and stem symptomatic tissue, and identified as C. liriopes based on morphological features and DNA sequence analysis, thereby fulfilling Koch's postulates. C. liriopes has been reported causing anthracnose on Eria coronaria (Yang et al., 2011), Liriope spicata (Chen et al., 2019) in China, Liriope muscari in Mexico (Damm et al., 2009), Rohdea japonica in Korea (Kwon and Kim, 2013) and in the United States (Trigiano et al., 2018). To our knowledge, this is the first report of C. liriopes causing anthracnose on buckwheat worldwide. Occurrence of this disease may represent a significant impact for buckwheat production because this crop is the major agricultural commodity in some parts of China. More studies are needed to understand the epidemiology of this disease and foster disease management programs in China.

First Report of Leaf Spot Disease on Fagopyrum esculentum Caused by Bipolaris zeae in China.

Common buckwheat (Fagopyrum esculentum Moench), a dicotyledonous plant in family Polygonaceae, is recognized as a valuable nutritional source of fatty acids, phytosterols, phenolic compounds and tocopherols. It has received increased attention as a so-called "functional food" in China. During scouting of common buckwheat in August and September 2018, unfamiliar symptoms were observed on leaves in 20 fields in Yanchi County, Ningxia, China, with 35% incidence and moderate to high severity across the field. Brown spots most commonly occurred on lower leaves of buckwheat beginning in late July. The spots were initially light brown with an irregular border and pale brown center. Older spots were almost dark brown, and often coalesced although spots were restricted by veins. Symptomatic leaf samples were collected in late-August, and washed with flowing water for 2 min. Tissue samples were excised from the margins of the lesions and sterilized with 75% ethanol for 20 s and 0.1% NaClO for 2 min, before being rinsed with sterilized water four times, dried on sterile paper towels, and cultured on Potato Dextrose Agar medium at 20 °C. After 48 to 72 h, hyphae grew from tissue pieces. The obtained isolates were purified using the single-spore isolation technique. A total of thirteen isolates were recovered and characterized. Colonies of all isolates on PDA were identical, and were pale brown to brown, velvety, with irregular borders. Hyphae were hyaline to brown, branched, septate, smooth, sometimes verruculose, 3-7 µm wide. Conidiophores were (83-297) µm×(5.5-9) µm (avg. 205 µm×8 µm, n=30), dark brown, cylindrical, straight to slightly flexuous, geniculate at the upper end, septate, single, smooth. Conidia were rostrate, obclavate, straight to slightly curved, hyaline to pale brown when immature and brown to reddish brown when mature, and measured (38-104) µm×(11-19) µm (avg. 67 µm×16 µm, n=50). End cells were often paler than middle cells and with a thick dark hilum. Based on the cultural and morphological characteristic, these isolates are preliminary identified as Bipolaris zeae (Manamgoda et al., 2014; Sivanesan, 1985). Genomic DNA of the representative isolate qyj-5A was extracted and amplified using GAPDH primers (gpd 1/2) and ITS primers (ITS 1/4) respectively. The sequence of the amplicons was compared with reference sequences. The ITS sequence (GenBank: MT645704) showed 100% (571/571bp, 564/564bp) identity with B. zeae (GenBank: KU356179 and KU571464, respectively). The GAPDH sequence (GenBank: MW426530) also showed 100% (594/594bp) identity values with B. zeae (GenBank: MF415650 and KU571468). Pathogenicity tests were performed on three healthy and asymptomic buckwheat plants, which were surface sterilized with 75% ethanol and rinsed with sterilized distilled water. The leaves were sprayed with 1×105 conidia/ml of the suspensions which contain 0.01% Tween 20 and three control plants sprayed with same volume of sterile distilled water. A strip of parafilm was wrapped around the inoculated leaves for 48 h to maintain high relative humidity. At 6 days postinoculation, all the inoculated leaves showed symptoms identical to those described above. While no symptoms were observed on the control plants. The fungus was reisolated and identified as B. zeae based on morphological features and DNA sequence analysis, it was identical to the original isolate to satisfy Koch's postulates. B. zeae has been reported to be pathogenic on Acer truncatum (Sun et al., 2011), Helianthus tuberosus (Zhao et al., 2017) and Hemarthria altissima (Xue et al., 2016) in China. To our knowledge this is the first report of B. zeae causing leaf spot on F. esculentum in China. This fungal pathogen represents a severe threat and has the potential to cause yield losses of F. esculentum, so further research is required to define effective management strategies.

First Report of Leaf Spot Disease Caused by Stemphylium vesicarium on Fagopyrum esculentum in China.

Common buckwheat (Fagopyrum esculentum) is a widely cultivated non-grass cereal. It has a considerable market value with nutritional qualities and ability to treat or prevent cancers, hypertension and diabetes (Cawoy et al., 2006). In July-August 2018 and 2019, leaves exhibiting unfamiliar symptoms were observed in Huan County and Huachi County, Gansu, China, with 15% incidence and moderate to severe severity across the field. Initial symptoms consisted of small chlorotic, circular to oval leaf spots. As disease progressed, the spots enlarged and turned light brown to brown with chlorotic margins. When the disease was severe, the leaf spots coalesced and the leaves became prematurely chlorotic and senescent. Spots occurred mostly but not exclusively on older foliage. Diseased tissues were surface sterilized with 75% ethanol for 20 s and 0.1% NaClO for 2 min, placed on PDA medium and incubated at 20 °C for 48-72 h. A total 24 isolates were obtained and purified through single-spore cultures, 19 of which were characterized. Colonies on PDA of all 19 isolates were identical, exhibiting a light gray color, with whitish aerial mycelium that later turned light brown on the reverse of the culture plates, and sporulated sparsely. Conidia were brown colored, cylindrical, and borne singly, often had three main transverse septa, at which points there were conspicuous constrictions, and measured (17-37) µm long × (13-21) µm wide (n=30) in V8 juice agar, (22-38) µm long × (11-19) µm wide (n=30) in PCA, (21-41) µm long × (13-20) µm wide (n=30) in SNA, the mean length/width ratio was 1.6 to 2.0 in V8 juice agar, 1.6 to 2.5 in PCA, 1.3 to 2.2 in SNA. Conidiophores were unbranched, 5.1 to 7.9 µm wide. Dictyospores were produced on well-differentiated conidiophores, the apical cell of conidiophore was slightly to distinctly swollen. Based on morphological features, the isolates were tentatively identified as a member of the Stemphylium vesicarium species complex (Koike et al., 2013; Simmons, 1967). Genomic DNA of representative isolate B1 was extracted, and the internal transcribed spacer (ITS) region and calmodulin gene (cmdA) were amplified using ITS1/4, V9G/ITS4, and CALDF1/CALDR1, respectively. The resulting sequences were deposited in GenBank (acc. nos. MT629829, MW406903, MW417122). Nucleotide BLAST similarity analysis of the sequence fragment of ITS and cmdA from isolate B1 resulted in higher than 99% (99.32% for ITS1/4, 100% for V9G/ITS4, and 100% for CALDF1/CALDR1) identity with S. vesicarium strains (GenBank acc. nos. LC512757, MH863402 and MH206181). Based on morphological features and molecular data, the buckwheat isolates were identified as S. vesicarium. To verify pathogenicity, the back side of leaves from six asymptomic plants were inoculated by spraying the spore suspension (104 spores/ml) harvested from isolate B1 grown on V8 juice agar for 20 days. Control plants were sprayed with sterile water. Each plant was covered with a black plastic bag for 48 h and then was kept in a greenhouse. Stemphylium spot symptoms were observed on all inoculated leaves after 14 days, whereas control leaves were symptomless. The pathogen was reisolated from symptomatic leaf spots, micromorphological features and colony characters of the reisolated fungi were identical to the original isolate. To our knowledge, this is the first documentation of leaf spot of buckwheat caused by S. vesicarium in China, and the first characterization of a Stemphylium foliar pathogen on this crop.

Buckwheat conservation as hay or silage: agronomic evaluation, nutritive value, conservation quality, and intake by lactating dairy goats.

Buckwheat was conserved either as silage at two dry matter (DM) levels attained with (wilted silage (WS), 313 g/kg) and without wilting (direct cut silage (DS), 171 g/kg) or as hay (856 g/kg) and was included in the isonitrogenous and isocaloric total mixed ration (TMR) of four groups of six Saanen goats each at the rate of 200 g/kg DM. Ensiling buckwheat at low DM led to a poorly fermented silage with higher ammonia-N and butyric acid contents and extensive growth of enterobacteria and mold-yeast than WS. The DM recovery of DS was lower than that of WS. The total phenol (TP) content of buckwheat was also preserved more efficiently in hay than in DS. The nutritive value of hay was higher than that of the silages because hay had less fiber contents but more non-fiber carbohydrates and total digestible nutrients. The decrease in nutritive value in buckwheat silages, in particular for DS, increased the nutrient intensity in the concentrated feed. The DM intake of goats fed DS TMR tended to be lower (P=0.09) than that of the goats fed WS TMR in the first week of the experiment, but the goats quickly adapted to the poorly fermented silage and did not reduce their overall DM intake. The DS, WS, and hay TMRs did not affect fat corrected milk yield but increased the milk TPs compared to a TMR with legume-grass hay. It was concluded that buckwheat hay and silages are well accepted by dairy goats, but the DM content of buckwheat at ensiling may require over 330 g/kg for producing well-fermented silages.

Perception of and Behavioral Responses to Host Plant Volatiles for Three Adelphocoris Species.

In China, the genus Adelphocoris (Hemiptera: Miridae) includes three dominant pest species (A. suturalis, A. lineolatus and A. fasciaticollis), which cause great damage to cotton, alfalfa and other crops. In this study, we examined the role of the major volatile organic compounds from plants in host location by these three insects. Gas chromatography-electroantennography and gas chromatography/mass spectrometry analyses identified seven electroantennogram (EAG)-active compounds from 11 host plants. Although the insects responded to all of these compounds in EAG trials, some compounds did not elicit behavioral responses in Y-tube olfactometer bioassays. Adelphocoris suturalis adults showed behavioral responses to four EAG-active compounds, n-butyl ether, butyl acrylate, butyl propionate and butyl butyrate. These four compounds, in addition to p-xylene, were also attractive to A. lineolatus adults. However, A. fasciaticollis adults were attracted only by butyl acrylate, butyl propionate and butyl butyrate. In field trials, A. suturalis and A. fasciaticollis were each attracted to five individual compounds (m-xylene, n-butyl ether, butyl acrylate, butyl butyrate and butyl propionate for A. suturalis and m-xylene, butyl acrylate, butyl butyrate, butyl propionate, and 1,8-cineole for A. fasciaticollis). By contrast, A. lineolatus aduts were attracted to six individual compounds, m-xylene, p-xylene, n-butyl ether, butyl acrylate, butyl butyrate, and butyl propionate. These compounds may be important in host plant location by the Adelphocoris species, and may be useful for developing attractants for adults of these species.

Ectopic Expression of a Fagopyrum esculentum APETALA1 Ortholog only Rescues Sepal Development in Arabidopsis ap1 Mutant.

Fagopyrum esculentum (Polygonaceae: Caryophyllales) exhibits an undifferentiated perianth comprising five showy tepals, which does not completely correspond to the perianth differentiated into typical sepals and petals in most core eudicots. In Arabidopsis, the APETALA1 (AP1) gene is involved in specifying sepals and petals development. Here we isolated AP1 ortholog, FaesAP1, and a 2.2kb FaesAP1 promoter (pFaesAP1) from F. esculentum. FaesAP1 expression is mainly detectable in all floral organs and maintains at a high level when tepals elongate rapidly both in pin and thrum flowers. Moreover, the GUS reporter gene driven by pFaesAP1 was activated in flowers where the sepals were intense, but the petals very weak or absent. Additionally, FaesAP1 ectopic expression in Arabidopsis ap1-10 mutant rescues sepal development fully, obviously prompting early flowering, but failing to complement petal development. In this study, evidence was provided that the showy tepals in the F. esculentum are homologs to core eudicots sepals. Furthermore, these findings show a different perianth identity program in Caryophyllales, suggesting that AP1 orthologs involved in petal development may evolve independently across different clades of core eudicots. Our results also suggest that FaesAP1 holds potential for biotechnical engineering to develop early flowering varieties of F. esculentum.

Transcriptome Analysis Reveals the Accumulation Mechanism of Anthocyanins in Buckwheat (Fagopyrum esculentum Moench) Cotyledons and Flowers.

Buckwheat (Fagopyrum esculentum) is a valuable crop which can produce multiple human beneficial secondary metabolites, for example, the anthocyanins in sprouts and flowers. However, as the predominant group of visible polyphenols in pigmentation, little is known about the molecular mechanisms underlying the anthocyanin biosynthesis within buckwheat. In this study, a comparative transcriptome analysis of green and red common buckwheat cultivars was carried out through RNA sequencing. Overall, 3727 and 5323 differently expressed genes (DEGs) were identified in flowers and cotyledons, respectively. Through GO and KEGG analysis, we revealed that DEGs in flowers and cotyledons are predominately involved in biosynthesis of anthocyanin. A total of 42 unigenes encoding 11 structural enzymes of the anthocyanin biosynthesis were identified as DEGs. We also identified some transcription factor families involved in the regulation of anthocyanin biosynthesis. Real-time qPCR validation of candidate genes was performed in flowers and cotyledons, and the results suggested that the high expression level of structural genes involved in anthocyanin biosynthetic pathway promotes anthocyanin accumulation. Our results provide the insight understanding for coloration of red common buckwheat.

Immunomodulatory effects of polysaccharide fraction isolated from Fagopyrum esculentum on innate immune system.

The present study investigates the immunomodulatory activities of buckwheat polysaccharide fraction (BPF) from the seed of Fagopyrum esculentum on RAW 264.7 macrophage cell line and Cyclophosphamide-induced immunosuppressed conditions in mice models. The results of in vitro showed that treatment with 0.5-10 µg/mL of BPF can modulate immune responses. MTT assay and nitric oxide production and immune-related cytokine levels were conducted. Treatment with BPF at a dose of 10 µg/mL of BPF increased immune responses on macrophages. Moreover, natural killer (NK) cell cytotoxicity was conducted. The apoptosis of YAC-1 cells increased as the co-culture ratio between spleen cells and YAC-1 cells increased approximately 4- fold compared to the control group from 12.5:1 to 50.0:1. The in-vivo immunomodulatory effects of BPF were evaluated by cyclophosphamide-induced mice model. The immune response of BPF was determined against cyclophosphamide (100 mg/kg) immunosuppressed mice at doses of 50 mg/kg and 100 mg/kg of BPF as compared to control. The results of this study showed that BPF administration increased spleen and thymus indices as well as the leukocytes count in the blood of immunosuppressed mice. All of results suggested that BPF are potentially acts as immunomodulator for activation of immune responses.

Chemical Composition, Antimicrobial and Antioxidant Activities of the Flower Volatile Oils of Fagopyrum esculentum, Fagopyrum tataricum and Fagopyrum Cymosum.

The purpose of this study was to investigate the chemical composition and biological activity of the volatile oils (VOs) from the flowers of three buckwheat species, Fagopyrum esculentum, Fagopyrum tataricum and Fagopyrum cymosum. The VOs were obtained from the fresh buckwheat flowers by hydrodistillation, and were analyzed for their chemical composition by gas chromatography-mass spectrometry (GC-MS). Nonanoic acid (7.58%), (E)-3-hexen-1-ol (6.52%), and benzothiazole (5.08%) were the major constituents among the 28 identified components which accounted for 92.89% of the total oil of F. esculentum. 2-Pentadecanone (18.61%), eugenol (17.18%), 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester (13.19%), and (E,E)-farnesylacetone (7.15%) were the major compounds among the 14 identified components which accounted for 88.48% of the total oil of F. tataricum. Eugenol (12.22%), (E)-3-hexen-1-yl acetate (8.03%), linalool oxide (7.47%), 1-hexanol (7.07%), and benzothiazole (6.72%) were the main compounds of the 20 identified components which accounted for 90.23% of the total oil of F. cymosum. The three VOs were screened to have broad spectrum antibacterial activity with minimum inhibitory concentration (MIC) values ranged from 100.0 µg/mL to 800.0 µg/mL against the tested bacteria, and their median inhibitory concentration (IC50) values were from 68.32 µg/mL to 452.32 µg/mL. Xanthomonas vesicatoria was the most sensitive bacterium. Moreover, the flower VOs of F. esculentum, F. tataricum and F. cymosum also exhibited noteworthy antioxidant capacity with the IC50 value of 354.15 µg/mL, 210.63 µg/mL, and 264.92 µg/mL for the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay, and the value of 242.06 µg/mL, 184.13 µg/mL, and 206.11 µg/mL respectively for the ß-carotene-linoleic bleaching test. These results suggested the volatile oils of buckwheat flowers could be potential resource of natural antimicrobial and antioxidant agents.

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.

Influence of Indole-3-Acetic Acid and Gibberellic Acid on Phenylpropanoid Accumulation in Common Buckwheat (Fagopyrum esculentum Moench) Sprouts.

We investigated the effects of natural plant hormones, indole-3-acetic (IAA) acid and gibberellic acid (GA), on the growth parameters and production of flavonoids and other phenolic compounds in common buckwheat sprouts. A total of 17 phenolic compounds were identified using liquid chromatography-mass spectrometry (LC-MS) analysis. Among these, seven compounds (4-hydroxybenzoic acid, catechin, chlorogenic acid, caffeic acid, epicatechin, rutin, and quercetin) were quantified by high-performance liquid chromatography (HPLC) after treating the common buckwheat sprouts with different concentrations of the hormones IAA and GA. At a concentration of 0.5 mg/L, both IAA and GA exhibited the highest levels of growth parameters (shoot length, root length, and fresh weight). The HPLC analysis showed that the treatment of sprouts with IAA at concentrations ranging from 0.1 to 1.0 mg/L produced higher or comparable levels of the total phenolic compounds than the control sprout and enhanced the production of rutin. Similarly, the supplementation with 0.1 and 0.5 mg/L GA increased the content of rutin in buckwheat sprouts. Our results suggested that the treatment with optimal concentrations of IAA and GA enhanced the growth parameters and accumulation of flavonoids and other phenolic compounds in buckwheat sprouts.

Purification, molecular cloning and functional characterization of flavonoid C-glucosyltransferases from Fagopyrum esculentum M. (buckwheat) cotyledon.

C-Glycosides are characterized by their C-C bonds in which the anomeric carbon of the sugar moieties is directly bound to the carbon atom of aglycon. C-Glycosides are remarkably stable, as their C-C bonds are resistant to glycosidase or acid hydrolysis. A variety of plant species are known to accumulate C-glycosylflavonoids; however, the genes encoding for enzymes that catalyze C-glycosylation of flavonoids have been identified only from Oryza sativa (rice) and Zea mays (maize), and have not been identified from dicot plants. In this study, we identified the C-glucosyltransferase gene from the dicot plant Fagopyrum esculentum M. (buckwheat). We purified two isozymes from buckwheat seedlings that catalyze C-glucosylation of 2-hydroxyflavanones, which are expressed specifically in the cotyledon during seed germination. Following purification we isolated the cDNA corresponding to each isozyme [FeCGTa (UGT708C1) and FeCGTb (UGT708C2)]. When expressed in Escherichia coli, both proteins demonstrated C-glucosylation activity towards 2-hydroxyflavanones, dihydrochalcone, trihydroxyacetophenones and other related compounds with chemical structures similar to 2',4',6'-trihydroxyacetophenone. Molecular phylogenetic analysis of plant glycosyltransferases shows that flavonoid C-glycosyltransferases form a different clade with other functionally analyzed plant glycosyltransferases.

Rapid genotyping with DNA micro-arrays for high-density linkage mapping and QTL mapping in common buckwheat (Fagopyrum esculentum Moench).

For genetic studies and genomics-assisted breeding, particularly of minor crops, a genotyping system that does not require a priori genomic information is preferable. Here, we demonstrated the potential of a novel array-based genotyping system for the rapid construction of high-density linkage map and quantitative trait loci (QTL) mapping. By using the system, we successfully constructed an accurate, high-density linkage map for common buckwheat (Fagopyrum esculentum Moench); the map was composed of 756 loci and included 8,884 markers. The number of linkage groups converged to eight, which is the basic number of chromosomes in common buckwheat. The sizes of the linkage groups of the P1 and P2 maps were 773.8 and 800.4 cM, respectively. The average interval between adjacent loci was 2.13 cM. The linkage map constructed here will be useful for the analysis of other common buckwheat populations. We also performed QTL mapping for main stem length and detected four QTL. It took 37 days to process 178 samples from DNA extraction to genotyping, indicating the system enables genotyping of genome-wide markers for a few hundred buckwheat plants before the plants mature. The novel system will be useful for genomics-assisted breeding in minor crops without a priori genomic information.