Genome-wide investigation of UDP-Glycosyltransferase family in Tartary buckwheat (Fagopyrum tataricum).

BACKGROUND: Tartary buckwheat (Fagopyrum tataricum) belongs to Polygonaceae family and has attracted increasing attention owing to its high nutritional value. UDP-glycosyltransferases (UGTs) glycosylate a variety of plant secondary metabolites to control many metabolic processes during plant growth and development. However, there have been no systematic reports of UGT superfamily in F. tataricum. RESULTS: We identified 173 FtUGTs in F. tataricum based on their conserved UDPGT domain. Phylogenetic analysis of FtUGTs with 73 Arabidopsis UGTs clustered them into 21 families. FtUGTs from the same family usually had similar gene structure and motif compositions. Most of FtUGTs did not contain introns or had only one intron. Tandem repeats contributed more to FtUGTs amplification than segmental duplications. Expression analysis indicates that FtUGTs are widely expressed in various tissues and likely play important roles in plant growth and development. The gene expression analysis response to different abiotic stresses showed that some FtUGTs were involved in response to drought and cadmium stress. Our study provides useful information on the UGTs in F. tataricum, and will facilitate their further study to better understand their function. CONCLUSIONS: Our results provide a theoretical basis for further exploration of the functional characteristics of FtUGTs and for understanding the growth, development, and metabolic model in F. tataricum.

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.

Anticancer effects of rutin from Fagopyrum tataricum (tartary buckwheat) against osteosarcoma cell line.

BACKGROUND: The present study is analysisof the seeds of buckwheat (Fagopyrum sp.),member of the Polygonaceae family for isolation of rutin and its anticancer property againstOsteosarcoma celllines (SAOS2). The selected plant is traditionally used for diabetes and cancer. It has several biological properties such as antibacterial, antioxidant and anti-aging. PURPOSE: Thirty-five buckwheat cultivars were obtained from Nepal Agriculture Genetic Resources Centre (NAGRC) Khumaltar, Kathmandu, Nepal, and Kumrek Sikkim. These plant varieties are scientifically evaluated their biological properties. METHODS: Rutin wasfractionated from buckwheat seeds using methanol fraction and analysed for quality by HPLC method. The rutin fraction of the cultivar NGRC03731 a tartary buck wheat and standard rutin was used against Osteosarcoma cell lines (SAOS2) and human gingival fibroblast cells (hGFs) for anticancer activity. The cell viability using rutin fraction and standard rutin treated with SAOS2 cells were assessed by MTT assay. For further research, the best doses (IC-50: 20 g/ml) were applied. By using AO/EtBr dual staining, the effects of Rutin fraction on SAOS2 cell death were analysed. The scratch wound healing assay was used to analyse cell migration. Real-time PCR was used to analyse the pro-/anti-apoptotic gene expression. RESULTS: The seeds with the highest rutin content, NGRC03731 seeds, had 433 mg/100 g of rutin.The rutin fraction treatment and standard rutin significantly reduced cell viability in the MTT assay, and osteosarcoma cells were observed on sensitive to the IC-50 dose at a concentration of 20 g/ml after 24 h.The SAOS2 cells exposed to rutin fraction at 20 g/ml and standard rutin at 10 g/ml exhibited significant morphological alterations, cell shrinkage and decreased cell density, which indicate apoptotic cells.Rutin-fraction treated cells stained with acridine orange/ethidium bromide (AO/EtBr) dual staining cells turned yellow, orange, and red which indicatesto measure apoptosis.The anti-migration potential of rutin fraction, results prevented the migration of SAOS2 cancer cells.Rutin-fraction significantly increased the expression of pro-apoptotic proteinsBad, using real-time PCR analysis (mRNA for Bcl-2 family proteins) resulted Bcl-2's expression is negatively regulated. CONCLUSION: Osteosarcoma (SAOS2) cell lines' proliferation, migration, and ability to proliferate were reduced markedly by rutin fraction and it also causes apoptosis of Osteosarcoma cell lines (SAOS2).

Novel mutations in acetolactate synthase confer high levels of resistance to tribenuron-methyl in Fagopyrum tataricum.

Tartary buckwheat (Fagopyrum tataricum) field weeds are rich in species, with many weeds causing reduced quality, yield, and crop failure. The selection of herbicide-resistant Tartary buckwheat varieties, while applying low-toxicity and efficient herbicides as a complementary weed control system, is one way to improve Tartary buckwheat yield and quality. Therefore, the development of herbicide-resistant varieties is important for the breeding of Tartary buckwheat. In this experiment, 50 mM ethyl methyl sulfonate solution was used to treat Tartary buckwheat seeds (M1) and then planted in the field. Harvested seeds (M2) were planted in the experiment field of Guizhou University, and when seedlings had 5-7 leaves, the seedlings were sprayed with 166 mg/L tribenuron-methyl (TBM). A total of 15 resistant plants were obtained, of which three were highly resistant. Using the homologous cloning method, an acetolactate synthase (ALS) gene encoding 547 amino acids was identified in Tartary buckwheat. A GTG (valine) to GGA (glycine) mutation (V409G) occurred at position 409 of the ALS gene in the high tribenuron-methyl resistant mutant sm113. The dm36 mutant harbored a double mutation, a deletion mutation at position 405, and a GTG (valine) to GGA (glycine) mutation (V411G) at position 411. The dm110 mutant underwent a double mutation: an ATG (methionine) to AGG (arginine) mutation (M333R) at position 333 and an insertion mutation at position 372. The synthesis of Chl a, Chl b, total Chl, and Car was significantly inhibited by TBM treatment. TBM was more efficient at suppressing the growth of wild-type plants than that of mutant plants. Antioxidant enzyme activities such as ascorbate peroxidase, peroxidase, and superoxide dismutase were significantly higher in resistant plants than in wild-type after spraying with TBM; malondialdehyde content was significantly lower than in wild-type plants after spraying with TBM. Plants with a single-site mutation in the ALS gene could survive, but their growth was affected by herbicide application. In contrast, plants with dual-site mutations in the ALS gene were not affected, indicating that plants with dual-site mutations in the ALS gene showed higher levels of resistance than plants with a single-site mutation in the ALS gene.

QTL Mapping and Candidate Gene Analysis for Starch-Related Traits in Tartary Buckwheat (Fagopyrum tataricum (L.) Gaertn).

Starch is the main component that determines the yield and quality of Tartary buckwheat. As a quantitative trait, using quantitative trait locus (QTL) mapping to excavate genes associated with starch-related traits is crucial for understanding the genetic mechanisms involved in starch synthesis and molecular breeding of Tartary buckwheat varieties with high-quality starch. Employing a recombinant inbred line population as research material, this study used QTL mapping to investigate the amylose, amylopectin, and total starch contents across four distinct environments. The results identified a total of 20 QTLs spanning six chromosomes, which explained 4.07% to 14.41% of the phenotypic variation. One major QTL cluster containing three stable QTLs governing both amylose and amylopectin content, qClu-4-1, was identified and located in the physical interval of 39.85-43.34 Mbp on chromosome Ft4. Within this cluster, we predicted 239 candidate genes and analyzed their SNP/InDel mutations, expression patterns, and enriched KEGG pathways. Ultimately, five key candidate genes, namely FtPinG0004897100.01, FtPinG0002636200.01, FtPinG0009329200.01, FtPinG0007371600.01, and FtPinG0005109900.01, were highlighted, which are potentially involved in starch synthesis and regulation, paving the way for further investigative studies. This study, for the first time, utilized QTL mapping to detect major QTLs controlling amylose, amylopectin, and total starch contents in Tartary buckwheat. The QTLs and candidate genes would provide valuable insights into the genetic mechanisms underlying starch synthesis and improving starch-related traits of Tartary buckwheat.

Genetic analysis of yield components in buckwheat using high-throughput sequencing analysis and wild resource populations.

Fagopyrum tataricum, an important medicinal and edible crop, possesses significant agricultural and economic value. However, the development of buckwheat varieties and yields has been hindered by the delayed breeding progress despite the abundant material resources in China. Current research indicates that quantitative trait loci (QTLs) play a crucial role in controlling plant seed type and yield. To address these limitations, this study constructed recombinant inbred lines (RILs) utilizing both cultivated species and wild buckwheat as raw materials. In total, 84,521 Single Nucleotide Polymorphism (SNP) markers were identified through Genotyping-by-Sequencing (GBS) technology, and high-resolution and high-density SNP genetic maps were developed, which had significant value for QTL mapping, gene cloning and comparative mapping of buckwheat. In this study, we successfully identified 5 QTLs related to thousand grain weight (TGW), 9 for grain length (GL), and 1 for grain width (GW) by combining seed type and TGW data from 202 RIL populations in four different environments, within which one co-located QTL for TGW were discovered on the first chromosome. Transcriptome analysis during different grain development stages revealed 59 significant expression differences between the two materials, which can serve as candidate genes for further investigation into the regulation of grain weight and yield enhancement. The mapped major loci controlling TGW, GL and GW will be valuable for gene cloning and reveal the mechanism underlying grain development and marker-assisted selection in Tartary buckwheat.

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.

Hybridization with Fagopyrum cymosum Meisn. as a way to make cultivated Tartary buckwheat (F. tataricum Gaertn.) with grain characteristics typical for common buckwheat (F. esculentum Moench.).

Compared to common buckwheat (F. esculentum), Tartary buckwheat (F. tataricum) is very polymorphic in the type of seeds, but a seed type which is typical for F. esculentum, i.e. triangular seeds with flat sides and clear ribs, has been not found among the polymorphism. However, such seed type is typical for wild species F. cymosum which produces fertile hybrids in crosses with F. tataricum. Embryo rescue based interspecific cross F. esculentum × F. cymosum allowed reveal functional allelism of the genes determining the similar morphs of these species' seeds, i.e. the seed type resulted from mutation(s) at same gene. The gene can be assigned as TAN (triangular). Variation for the seed shell thickness among recessives for the TAN carrying about 12% of F. tataricum genome, together with the shell thickness of a seed from the F1 hybrid F. esculentum × F. cymosum compared to ones of the parents, suggests there are some genes influencing seed shell thickness. Also, it was supported by analyses of seeds characteristics of Tartary-based forms with some share of F. cymosum genetic material. In addition, cross F. tataricum × F. cymosum looks like an effective tool to increase 1000-seed weight of Tartary buckwheat-based breeding material.

Genome-Wide Identification, Evolution, and Expression Pattern Analysis of the GATA Gene Family in Tartary Buckwheat (Fagopyrum tataricum).

GATA is a transcription factor that exerts a vital function in plant growth and development, physiological metabolism, and environmental responses. However, the GATA gene family has rarely been studied in Tartary buckwheat since the completion of its genome. This study used bioinformatics methods to identify GATA genes of Tartary buckwheat and to analyze their subfamily classification, structural composition, and developmental evolution, as well as to discuss the expression patterns of FtGATA genes in different subfamilies. The twenty-eight identified FtGATA genes in the Tartary buckwheat genome were divided into four subfamilies and distributed on eight chromosomes. One pair of tandem repeat genes and eight pairs of fragments were found in chromosome mapping. Spatiotemporal expression patterns of eight FtGATA genes in different subfamilies indicated that the FtGATA gene family has regulatory roles in tissue specificity, fruit development, abiotic stress, and hormonal responses. This study creates a theoretical and scientific foundation for further research on the evolutionary relationship and biological function of FtGATA.

FtbZIP12 Positively Regulates Responses to Osmotic Stress in Tartary Buckwheat.

ABFs play a key role in regulating plant osmotic stress. However, in Tartary buckwheat, data on the role of ABF genes in osmotic stress remain limited and its associated mechanism in osmoregulation remain nebulous. Herein, a novel ABF family in Tartary buckwheat, FtbZIP12, was cloned and characterized. FtbZIP12 is a transcriptional activator located in the nucleus; its expression is induced by NaCl, mannitol, and abscisic acid (ABA). Atopic expression of FtbZIP12 in Arabidopsis promoted seed germination, reduced damage to primary roots, and improved the tolerance of seedlings to osmotic stress. The quantitative realtime polymerase chain reaction (RT-qPCR) results showed that the expressions of the typical genes related to stress, the SOS pathway, and the proline synthesis pathway in Arabidopsis were significantly (p < 0.05) upregulated under osmotic stress. FtbZIP12 improved the osmotic pressure resistance by reducing the damage caused by reactive oxygen species to plants and maintained plant homeostasis by upregulating the expression of genes related to stress, osmotic regulation, and ion homeostasis. This study identified a key candidate gene for understanding the mechanism underlying osmotic-stress-regulated function in Tartary buckwheat, thereby providing a theoretical basis for improving its yield and quality.

Streptomyces liangshanensis sp. nov., a novel actinomycete isolated from rhizosphere soil of Fagopyrum tataricum.

A novel actinomycete strain, designated strain QMT-12T, was isolated from the rhizospheric soils of Fagopyrum tataricum and characterized using a polyphasic approach. Strain QMT-12T was found to have morphological features typical of the genus Streptomyces. The predominant fatty acids included C18:1 cis9 (35.9%), Summed feature 6 (C18:2 cis9, 12/C18:0 a or C18:0 anteiso/C18:2 c) (30.6%) and C16:0 (16.3%). The whole-cell sugars were arabinose and glucose. The whole-cell-wall amino acids included alanine, aspartate, glutamic acid, glycine and LL-diaminopimelic acid. The menaquinones were MK-9, MK-9(H2), MK-9(H4), MK-9(H6) and MK-9(H8). The diagnostic phospholipids consisted of diphosphatidyl glycerol, phosphatidylethanolamine, phosphatidyl methyl ethanolamine, phospholipids, phosphotidyl inositol, phosphotidylinositol mannosides, and phospholipids of unknown structure containing glucosamine. The full-length 16S rRNA gene sequence analysis showed that strain QMT-12T belonged to the genus Streptomyces and had 98.2, 98.1, 98.1 and ≤ 98.0% similarities to Streptomyces camponoticapitis 2H-TWYE14T, Streptomyces scopuliridis NRRL B-24574T, Streptomyces inhibens NEAU-D10T and other Streptomyces species with validly published and correct names, respectively. Phylogenetic analysis indicated that strain QMT-12T was closely related to Streptomyces inhibens NEAU-D10T. However, the average nucleotide identity value and the digital DNA-DNA hybridization value between strain QMT-12T and S. inhibens NEAU-D10T were 85.0 and 22.3%, respectively, well below 95-96% and 70% cut-off point recommended for delineating species. Based on its phenotypic and genotypic characteristics, strain QMT-12T (= CICC 11056T = JCM 33963T) represents the type strain of a novel species, for which the name Streptomyces liangshanensis sp. nov. is proposed.

First Report of Alternaria alternata Causing Leaf Spot of Tartary Buckwheat (Fagopyrum tataricum) in China.

Buckwheat (Fagopyrum tataricum) is recognized as a healthy food with abundant nutrients and high levels of rutin. In April and May of 2020, an unknown tartary buckwheat leaf spot distinct from Nigrospora leaf spot (Shen et al. 2020) was observed in Xiangxiang, Hunan, China (27°49'54″N, 112°span style="font-family:'Times New Roman'; color:#0000ff">18'48″E.). Disease incidence was 60-70% within three fields (totally 7, 000 m2). The disease occurred after plants emerged. Initial symptoms began as circular, or ellipsoid, chlorotic, water-soaked spots, mostly on leaf apexes or leaf margins. The small spots gradually enlarged and often coalesced to form large circular or irregular, pale to light brown lesions, and the infected leaves eventually withered and fell off. Thirty 2 × 2 mm infected tissue pieces collected from five locations were sterilized in 70% ethanol for 10 S, in 2% NaClO for 30 S, rinsed in sterile water for three times, dried, and placed on PDA with lactic acid (3 ml/L). After 3-5 days at 28°C in the dark, 17 fungal isolates were purified using single-spore isolation method. Almost all fungal isolates had similar morphology. Colonies were initially olive green with white margin and later turned dark olive or black with profuse sporulation. Conidia were borne in long chains, tawny to brownish green, with 1-3 longitudinal and 1-7 transverse septa, pyriform, and measured 9.5-39.6 µm long, and 5.1-12.6 µm wide (n=50). Based on morphological characteristics, the fungus was identified as Alternaria alternata (Simmons 2007). Partial internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-α(TEF) and Alternaria major allergen (Alt a1) genes of isolate BLS-1 were amplified using ITS1/ITS4 (Mills et al. 1992), EF1-728F/EF1-986R (Carbone and Kohn 1999), Gpd1/Gpd2 and Alt-4for/Alt-4rev (Lawrence et al. 2013), respectively. Sequences were deposited into GenBank with acc. nos MW453091 (ITS), MW480219 (GAPDH), MW480218 (TEF), and MW480220 (Alt a1). BLASTn analysis showed 99.8% (ITS, MH854758.1), 100% (GAPDH, KP124155.1), 99.8% (TEF, KP125073.1) and 100% (Alt a1, KP123847.1) identity with reference strain CBS 106.24 of A. alternata, confirming isolate BSL-1 to be A. alternata. A neighbor-joining phylogenetic tree constructed by MEGA7.0 based on concatenated sequences of the four genes indicated that BSL-1 formed a distinct clade with A. alternata CBS 106.24 with 100% bootstrap values. Pathogenicity test was triplicately performed on healthy leaves. Twenty leaves of five 20-day-old plants (cv. Pinku1) were sprayed with conidial suspension (1×106 conidia/ml) collected from PDA cultures with 0.05% Tween 20. An equal number of control leaves were sprayed with sterile water to serve as the controls. Treated plants were kept in a greenhouse at 28±3 °C with relative humidity of 80±5% for 24 h and transferred to natural conditions (22-30°C, RH 50-60%). After 4 to 6 days, all inoculated leaves developed symptoms similar to those observed in the fields, while the control leaves remained healthy. A. alternata was re-isolated from all infected leaves. Occasionally-isolated Diaporthe isolates were not pathogenic. A. alternata causes leaf spot of oat (Zhao et al. 2020) and leaf blight of F. esculentum (Lu et al. 2019). To our knowledge, this is the first report of A. alternata causing leaf spot on F. tataricum in China and the world. Effective strategies should be developed to manage the disease.

Effect of Light and Dark on the Phenolic Compound Accumulation in Tartary Buckwheat Hairy Roots Overexpressing ZmLC.

Fagopyrum tataricum 'Hokkai T10' is a buckwheat cultivar capable of producing large amounts of phenolic compounds, including flavonoids (anthocyanins), phenolic acids, and catechin, which have antioxidant, anticancer, and anti-inflammatory properties. In the present study, we revealed that the maize transcription factor Lc increased the accumulation of phenolic compounds, including sinapic acid, 4-hydroxybenzonate, t-cinnamic acid, and rutin, in Hokkai T10 hairy roots cultured under long-photoperiod (16 h light and 8 h dark) conditions. The transcription factor upregulated phenylpropanoid and flavonoid biosynthesis pathway genes, yielding total phenolic contents reaching 27.0 ± 3.30 mg g-1 dry weight, 163% greater than the total flavonoid content produced by a GUS-overexpressing line (control). In contrast, when cultured under continuous darkness, the phenolic accumulation was not significantly different between the ZmLC-overexpressing hairy roots and the control. These findings suggest that the transcription factor (ZmLC) activity may be light-responsive in the ZmLC-overexpressing hairy roots of F. tataricum, triggering activation of the phenylpropanoid and flavonoid biosynthesis pathways. Further studies are required on the optimization of light intensity in ZmLC-overexpressing hairy roots of F. tataricum to enhance the production of phenolic compounds.

Identifying the primary meteorological factors affecting the growth and development of Tartary buckwheat and a comprehensive landrace evaluation using a multi-environment phenotypic investigation.

BACKGROUND: Tartary buckwheat (Fagopyrum tataricum) is a traditional edible and medicinal crop and has been praised as one of the green foods for humans in the 21st century. However, its production and promotion are restricted by the low yields of current varieties. The interaction of genotype and environment could lead to inconsistent phenotypic performance of genotypes across different environments. Climate change has intensified these effects and poses a substantial threat to crop production. RESULTS: In the present study, the effects of meteorological factors on the phenotypic traits of 200 Tartary buckwheat landraces across four macro-environments were investigated. Overall, the phenotypic performance of these Tartary buckwheat landraces was markedly varied across the different environments. Also, the average daily temperature and precipitation had relatively higher impacts on phenotypic performance. The results also revealed the negative impacts of relative humidity on the yield-related traits. Twenty-five Tartary buckwheat landraces were ultimately identified as having good overall phenotypic performance and high yield stability. CONCLUSION: Understanding the impacts of meteorological factors on the phenotypic performance of crops can guide appropriate measures and facilitate germplasm selection for yield enhancement in the context of climate change. The landraces selected comprehensively in this study could be used as parents or intermediate materials for breeding high-quality Tartary buckwheat varieties in the future. The methods used could also be extended to other crops for breeding and germplasm innovation. © 2021 Society of Chemical Industry.

Transcriptome profiling of Fagopyrum tataricum leaves in response to lead stress.

BACKGROUND: Lead (Pb) pollution is a widespread environmental problem that is harmful to living organisms. Tartary buckwheat (Fagopyrum tataricum), a member of the family Polygonaceae, exhibits short growth cycles and abundant biomass production, could be an ideal plant for phytoremediation due to its high Pb tolerance. Here, we aimed to explore the molecular basis underlying the responses of this plant to Pb stress. RESULTS: In our study, ultrastructural localization assays revealed that Pb ions primarily accumulate in leaf vacuoles. RNA deep sequencing (RNA-Seq) of tartary buckwheat leaves was performed on two Pb-treated samples, named Pb1 (2000 mg/kg Pb (NO3)2) and Pb2 (10,000 mg/kg Pb (NO3)2), and a control (CK). A total of 88,977 assembled unigenes with 125,203,555 bases were obtained. In total, 2400 up-regulated and 3413 down-regulated differentially expressed genes (DEGs) were identified between CK and Pb1, and 2948 up-regulated DEGs and 3834 down-regulated DEGs were generated between CK and Pb2, respectively. Gene Ontology (GO) and pathway enrichment analyses showed that these DEGs were primarily associated with 'cell wall', 'binding', 'transport', and 'lipid and energy' metabolism. The results of quantitative real-time PCR (qRT-PCR) analyses of 15 randomly selected candidate DEGs and 6 regulated genes were consistent with the results of the transcriptome analysis. Heterologous expression assays in the yeast strain Δycf1 indicated that overexpressing CCCH-type zinc finger protein 14 (ZFP14) enhanced sensitivity to Pb2+, while 5 other genes, namely, metal transporter protein C2 (MTPC2), phytochelatin synthetase-like family protein (PCSL), vacuolar cation/proton exchanger 1a (VCE1a), natural resistance-associated macrophage protein 3 (Nramp3), and phytochelatin synthetase (PCS), enhanced the Pb tolerance of the mutant strain. CONCLUSION: Combining our findings with those of previous studies, we generated a schematic model that shows the metabolic processes of tartary buckwheat under Pb stress. This study provides important data for further genomic analyses of the biological and molecular mechanisms of Pb tolerance and accumulation in tartary buckwheat.

Examining a synchrotron-based approach for in situ analyses of Al speciation in plant roots.

Aluminium (Al) K- and L-edge X-ray absorption near-edge structure (XANES) has been used to examine Al speciation in minerals but it remains unclear whether it is suitable for in situ analyses of Al speciation within plants. The XANES analyses for nine standard compounds and root tissues from soybean (Glycine max), buckwheat (Fagopyrum tataricum), and Arabidopsis (Arabidopsis thaliana) were conducted in situ. It was found that K-edge XANES is suitable for differentiating between tetrahedral coordination (peak of 1566 eV) and octahedral coordination (peak of 1568 to 1571 eV) Al, but not suitable for separating Al binding to some of the common physiologically relevant compounds in plant tissues. The Al L-edge XANES, which is more sensitive to changes in the chemical environment, was then examined. However, the poorer detection limit for analyses prevented differentiation of the Al forms in the plant tissues because of their comparatively low Al concentration. Where forms of Al differ markedly, K-edge analyses are likely to be of value for the examination of Al speciation in plant tissues. However, the apparent inability of Al K-edge XANES to differentiate between some of the physiologically relevant forms of Al may potentially limit its application within plant tissues, as does the poorer sensitivity at the L-edge.

First Report of Nigrospora osmanthi Causing Leaf Spot on Tartary Buckwheat in China.

Buckwheat (Fagopyrum tataricum) is a traditional short-season pseudocereal crop originating in southwest China and is cultivated around the world. Antioxidative substances in buckwheat have been shown to provide many potential cardiovascular health benefits. Between August and November in 2019, a leaf spot was found in several Tartary buckwheat cv. Pinku1 fields in Xiangxiang County, Hunan Province, China. The disease occurred throughout the growth cycle of buckwheat after leaves emerged, and disease incidence was approximately 50 to 60%. Initially infected leaves developed a few round lesions, light yellow to light brown spots. Several days later, lesions began to enlarge with reddish brown borders, and eventually withered and fell off. Thirty lesions (2×2 mm) collected from three locations with ten leaves in each location were sterilized in 70% ethanol for 10 sec, in 2% sodium hypochlorite for 30 sec, rinsed in sterile water for three times, dried on sterilized filter paper, and placed on a potato dextrose PDA with lactic acid (3 ml/L), and incubated at 28°C in the dark for 3 to 5 days. Fungal colonies were initially white and later turned black with the onset ofsporulation. Conidia were single-celled, black, smooth, spherical to subspherical, and measured 9.2 to 15.6 µm long, and 7.1 to 11.6 µm wide (n=30). Each conidium was terminal and borne on a hyaline vesicle at the tip of conidiophores. Morphologically, the fungus was identified as Nigrospora osmanthi (Wang et al. 2017). Identification was confirmed by amplifying and sequencing the ITS region, and translation elongation factor 1-alpha (TEF1-α) and partial beta-tublin (TUB2) genes using primers ITS1/ITS4 (Mills et al. 1992), EF1-728F/EF-2 (Carbone and Kohn 1999; O'Donnell et al. 1998) and Bt-2a/Bt-2b (Glass et al. 1995), respectively. BLAST searches in GenBank indicated the ITS (MT860338), TUB2 (MT882054) and TEF1-α (MT882055) sequences had 99.80%, 99% and 100% similarity to sequences KX986010.1, KY019461.1 and KY019421.1 of Nigrospora osmanthi ex-type strain CGMCC 3.18126, respectively. A neighbor-joining phylogenetic tree constructed using MEGA7.0 with 1,000 bootstraps based on the concatenated nucleotide sequences of the three genes indicated that our isolate was closely related to N. osmanthi. Pathogenicity test was performed using leaves of healthy F. tataricum plants. The conidial suspension (1 × 106 conidia/ml) collected from PDA cultures with 0.05% Tween 20 buffer was used for inoculation by spraying leaves of potted 20-day-old Tartary buckwheat cv. Pinku1. Five leaves of each plant were inoculated with spore suspensions (1 ml per leaf). An equal number of control leaves were sprayed with sterile water to serve as a control. The treated plants were kept in a greenhouse at 28°C and 80% relative humidity for 24 h, and then transferred to natural conditions with temperature ranging from 22 to 30°C and relative humidity ranging from 50 to 60%. Five days later, all N. osmanthi-inoculated leaves developed leaf spot symptoms similar to those observed in the field, whereas control leaves remained healthy. N. osmanthi was re-isolated from twelve infected leaves with frequency of 100%, fulfilling Koch's postulates. The genus Nigrospora has been regarded by many scholars as plant pathogens (Fukushima et al. 1998) and N. osmanthi is a known leaf blight pathogen for Stenotaphrum secundatum (Mei et al. 2019) and Ficus pandurata (Liu et al. 2019) but has not been reported on F. tataricum. Nigrospora sphaerica was also detected in vegetative buds of healthy Fagopyrum esculentum Moench (Jain et al. 2012). To our knowledge, this is the first report of N. osmanthi causing leaf spot on F. tataricum in China and worldwide. Appropriate strategies should be developed to manage this disease.

Characterization of mercury-induced stress biomarkers in Fagopyrum tataricum plants.

The effect of mercury stress on antioxidant enzymes, lipid peroxidation, photosynthetic pigments, hydrogen peroxide content, osmolytes, and growth parameters in Tartary buckwheat were investigated. The effect of Hg-exposure was found to be time (15 and 30 days) and concentration (0, 25, 50, and 75 µM) dependent. Hg was readily absorbed by seedlings with higher content in roots and it resulted in reduction of root and shoot length. The root and shoot Hg uptakes were significantly and directly correlated with each other. However, the fresh mass and biomass increased up to 50 µM Hg-treatment at both time periods. A significant positive correlation was observed between biomass accumulation with relative water content. Hg levels were positively correlated with the production of hydrogen peroxide in leaves as evidenced by 3, 3-diaminobenzidine (DAB)-mediated tissue fingerprinting. The osmolyte levels in general were elevated except for proline and protein which showed a decline at 75 µM Hg-treatment at 30-days. Amongst the photosynthetic pigments, chlorophyll showed a decline while as carotenoid and anthocyanin levels were elevated. The activity of antioxidant enzymes such as ascorbate peroxidase (APX), guaiacol peroxidase (POD), glutathione reductase (GR), Glutathione-s-transferase (GST) and superoxide dismutase (SOD) were positively correlated with Hg-treatment except SOD, which declined at 75 µM Hg-treatment in 30-days old seedlings. Catalase (CAT) activity showed a positive correlation up to 50 µM Hg-treatment but at 75 µM Hg-stress it decreases at both 15 and 30 days.

The Potential Role of Auxin and Abscisic Acid Balance and FtARF2 in the Final Size Determination of Tartary Buckwheat Fruit.

Tartary buckwheat is a type of cultivated medicinal and edible crop with good economic and nutritional value. Knowledge of the final fruit size of buckwheat is critical to its yield increase. In this study, the fruit development of two species of Tartary buckwheat in the Polygonaceae was analyzed. During fruit development, the size/weight, the contents of auxin (AUX)/abscisic acid (ABA), the number of cells, and the changes of embryo were measured and observed; and the two fruit materials were compared to determine the related mechanisms that affected fruit size and the potential factors that regulated the final fruit size. The early events during embryogenesis greatly influenced the final fruit size, and the difference in fruit growth was primarily due to the difference in the number of cells, implicating the effect of cell division rate. Based on our observations and recent reports, the balance of AUX and ABA might be the key factor that regulated the cell division rate. They induced the response of auxin response factor 2 (FtARF2) and downstream small auxin upstream RNA (FtSAURs) through hormone signaling pathway to regulate the fruit size of Tartary buckwheat. Further, through the induction of fruit expansion by exogenous auxin, FtARF2b was significantly downregulated. The FtARF2b is a potential target for molecular breeding or gene editing.

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.