Comparative cellular, physiological and transcriptome analyses reveal the potential easy dehulling mechanism of rice-tartary buckwheat (Fagopyrum Tararicum).

BACKGROUND: Tartary buckwheat has gained popularity in the food marketplace due to its abundant nutrients and high bioactive flavonoid content. However, its difficult dehulling process has severely restricted its food processing industry development. Rice-tartary buckwheat, a rare local variety, is very easily dehulled, but the cellular, physiological and molecular mechanisms responsible for this easy dehulling remains largely unclear. RESULTS: In this study, we integrated analyses of the comparative cellular, physiological, transcriptome, and gene coexpression network to insight into the reason that rice-tartary buckwheat is easy to dehull. Compared to normal tartary buckwheat, rice-tartary buckwheat has significantly brittler and thinner hull, and thinner cell wall in hull sclerenchyma cells. Furthermore, the cellulose, hemicellulose, and lignin contents of rice-tartary buckwheat hull were significantly lower than those in all or part of the tested normal tartary buckwheat cultivars, respectively, and the significant difference in cellulose and hemicellulose contents between rice-tartary buckwheat and normal tartary buckwheat began at 10 days after pollination (DAP). Comparative transcriptome analysis identified a total of 9250 differentially expressed genes (DEGs) between the rice- and normal-tartary buckwheat hulls at four different development stages. Weighted gene coexpression network analysis (WGCNA) of all DEGs identified a key module associated with the formation of the hull difference between rice- and normal-tartary buckwheat. In this specific module, many secondary cell wall (SCW) biosynthesis regulatory and structural genes, which involved in cellulose and hemicellulose biosynthesis, were identified as hub genes and displayed coexpression. These identified hub genes of SCW biosynthesis were significantly lower expression in rice-tartary buckwheat hull than in normal tartary buckwheat at the early hull development stages. Among them, the expression of 17 SCW biosynthesis relative-hub genes were further verified by quantitative real-time polymerase chain reaction (qRT-PCR). CONCLUSIONS: Our results showed that the lower expression of SCW biosynthesis regulatory and structural genes in rice-tartary buckwheat hull in the early development stages contributes to its easy dehulling by reducing the content of cell wall chemical components, which further effects the cell wall thickness of hull sclerenchyma cells, and hull thickness and mechanical strength.

Intact endosperm cells in buckwheat flour limit starch gelatinization and digestibility in vitro.

The objective of this study was to determine the biophysical properties of buckwheat (BW) endosperm and their influences on detachment of intact cells, starch gelatinization and digestibility. The intact cells were isolated from BW kernels by dry milling and sieving. The microscopy and texture analysis showed intact endosperm cells could be detached easily due to the fragile structure and low hardness of BW endosperm. More than 70% intact cells were found in commercial light flour. The starch granules entrapped in intact cells exhibited a delay gelatinization and restricted swelling behavior (2-3 â„ƒ higher onset gelatinization temperature than isolated starch). Starch in BW flour had a markedly lower extent of digestion compared to the broken cells and isolated starch. This study provided a new mechanistic understanding of low glycemic index of BW food, and could guide the processing of BW flour to retain slow digestion properties.

In-depth mapping of the seed phosphoproteome and N-glycoproteome of Tartary buckwheat (Fagopyrum tataricum) using off-line high pH RPLC fractionation and nLC-MS/MS.

The seed of Tartary buckwheat (Fagopyrum tataricum) is rich in nutrients and functional ingredients and is recommended as a healthy cereal food. The total proteins of Tartary buckwheat seed (TBS) were extracted and digested; then, the phosphopeptides and glycopeptides were separately enriched and identified by nano liquid chromatography/tandem mass spectrometry. A total of 2613 phosphorylation sites from 1670 phosphoproteins and 404 N-glycosylation sites from 285 N-glycoproteins were identified in TBS. Function and pathway analyses showed that TBS phosphoproteins were significantly enriched in transport, energy metabolism, amino acids biosynthesis/metabolism, and signaling and TBS N-glycoproteins were significantly enriched in modification regulation. The present study reports the first profiles of the phosphoproteome and N-glycoproteome of TBS and provides important post-translational modifications information on the proteins in TBS. The results of this study will aid the understanding of the underlying mechanism of the germination of TBS during cultivation and edible quality changes during storage and processing.

Two MATE Transporters with Different Subcellular Localization are Involved in Al Tolerance in Buckwheat.

Buckwheat (Fagopyrum esculentum) shows high tolerance to aluminum (Al) toxicity, but the molecular mechanisms responsible for this high Al tolerance are still poorly understood. Here, we investigated the involvement of two MATE (multi-drug and toxic compound extrusion) genes in Al tolerance. Both FeMATE1 and FeMATE2 showed efflux transport activity for citrate, but not for oxalate when expressed in Xenopus oocytes. A transient assay with buckwheat leaf protoplasts using green fluorescent protein (GFP) fusion showed that FeMATE1 was mainly localized to the plasma membrane, whereas FeMATE2 was localized to the trans-Golgi and Golgi. The expression of FeMATE1 was induced by Al only in the roots, but that of FeMATE2 was up-regulated in both the roots and leaves. Furthermore, the expression of both genes only responded to Al toxicity, but not to other stresses including low pH, cadmium (Cd) and lanthanum (La). Heterologous expression of FeMATE1 or FeMATE2 in the Arabidopsis mutant atmate partially rescued its Al tolerance. Expression of FeMATE1 also partially recovered the Al-induced secretion of citrate in the transgenic lines, whereas expression of FeMATE2 did not complement the citrate secretion. Further physiological analysis showed that buckwheat roots also secreted citrate in addition to oxalate in response to Al in a dose-responsive manner. Taken together, our results indicate that FeMATE1 is involved in the Al-activated citrate secretion in the roots, while FeMATE2 is probably responsible for transporting citrate into the Golgi system for the internal detoxification of Al in the roots and leaves of buckwheat.

Complete Chloroplast Genome Sequence of Tartary Buckwheat (Fagopyrum tataricum) and Comparative Analysis with Common Buckwheat (F. esculentum).

We report the chloroplast (cp) genome sequence of tartary buckwheat (Fagopyrum tataricum) obtained by next-generation sequencing technology and compared this with the previously reported common buckwheat (F. esculentum ssp. ancestrale) cp genome. The cp genome of F. tataricum has a total sequence length of 159,272 bp, which is 327 bp shorter than the common buckwheat cp genome. The cp gene content, order, and orientation are similar to those of common buckwheat, but with some structural variation at tandem and palindromic repeat frequencies and junction areas. A total of seven InDels (around 100 bp) were found within the intergenic sequences and the ycf1 gene. Copy number variation of the 21-bp tandem repeat varied in F. tataricum (four repeats) and F. esculentum (one repeat), and the InDel of the ycf1 gene was 63 bp long. Nucleotide and amino acid have highly conserved coding sequence with about 98% homology and four genes--rpoC2, ycf3, accD, and clpP--have high synonymous (Ks) value. PCR based InDel markers were applied to diverse genetic resources of F. tataricum and F. esculentum, and the amplicon size was identical to that expected in silico. Therefore, these InDel markers are informative biomarkers to practically distinguish raw or processed buckwheat products derived from F. tataricum and F. esculentum.

[Effect of D,L-buthionine-S,R-sulfoximine on the ratio of glutathione forms and the growth of tatar buckwheat calli].

We studied the intracellular content of reduced (GSH) and oxidized (GSSG) glutathione, glutathione reductase activity, glutathione-S-transferase, and ascorbate peroxidase in morphogenic and nonmorphogenic Tatar buckwheat calli during the culture cycle as well as under the treatment with D,L-buthionine-S,R-sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthase, the first enzyme of glutathione biosynthesis. We found that, during passaging, cultures only slightly differed in total glutathione content; however, the content of GSH was higher in the morphogenic culture, whereas the content of GSSG was higher in the nonmorphogenic culture. In the morphogenic callus, the glutathione-S-transferase activity was 10-20 times higher and the glutathione reductase activity, was 2-2.5 times lower than in the nonmorphogenic callus. Under the treatment with BSO, the decrease in the GSH content in the morphogenic callus was temporary (on day 6-8 of passage), whereas that in the nonmorphogenic callus decreased within a day and remained lower than in the control throughout the entire passage. In the morphogenic callus, BSO did not affect the content of GSSG, whereas it caused GSSG accumulation in the nonmorphogenic callus. These differences are probably due to the fact that, in the BSO-containing medium, glutathione reductase is activated in the morphogenic callus and, conversely, inhibited in the nonmorphogenic callus. Although BSO caused a decrease in the total glutathione content only in the nonmorphogenic culture, the cytostatic effect of BSO was more pronounced in the morphogenic callus. In addition, BSO also had a negative effect on the differentiation ofproembryonic cell complexes in the morphogenic callus. The role of the glutathione redox status in maintaining the embryogenic activity of cultured plant cells is discussed.

Karyotype analysis of buckwheat using atomic force microscopy.

Karyotype analysis and classification of buckwheat chromosomes were performed without chemical banding or staining using atomic force microscopy (AFM). Fagopyrum esculentum (common buckwheat) and Fagopyrum tartaricum (Tartarian buckwheat) chromosomes were isolated from root tissues using an enzymatic maceration technique and spread over a glass substrate. Air-dried chromosomes had a surface with ridges, and the height of common and tartary buckwheat were approximately 350 and 150 nm. Volumes of metaphase sets of buckwheat chromosomes were calculated using three-dimensional AFM measurements. Chromosomes were morphologically characterized by the size, volume, arm lengths, and ratios. The calculated volumes of the F. esculentum and F. tartaricum chromosomes were in the ranges of 1.08-2.09 µm3 and 0.49-0.78 µm3, respectively. The parameters such as the relative arm length, centromere position, and the chromosome volumes measured using AFM provide accurate karyomorphological classification by avoiding the subjective inconsistencies in banding patterns of conventional methods. The karyotype evolutionary trend indicates that F. esculentum is an ancient species compared to F. tartaricum. This is the first report of a cytological karyotype of buckwheat using AFM.

Protecting cell walls from binding aluminum by organic acids contributes to aluminum resistance.

Aluminum-induced secretion of organic acids from the root apex has been demonstrated to be one major Al resistance mechanism in plants. However, whether the organic acid concentration is high enough to detoxify Al in the growth medium is frequently questioned. The genotypes of Al-resistant wheat, Cassia tora L. and buckwheat secrete malate, citrate and oxalate, respectively. In the present study we found that at a 35% inhibition of root elongation, the Al activities in the solution were 10, 20, and 50 muM with the corresponding malate, citrate, and oxalate exudation at the rates of 15, 20 and 21 nmol/cm(2) per 12 h, respectively, for the above three plant species. When exogenous organic acids were added to ameliorate Al toxicity, twofold and eightfold higher oxalate and malate concentrations were required to produce the equal effect by citrate. After the root apical cell walls were isolated and preincubated in 1 mM malate, oxalate or citrate solution overnight, the total amount of Al adsorbed to the cell walls all decreased significantly to a similar level, implying that these organic acids own an equal ability to protect the cell walls from binding Al. These findings suggest that protection of cell walls from binding Al by organic acids may contribute significantly to Al resistance.

Morphology of nectaries and biology of nectar production in the distylous species Fagopyrum esculentum.

BACKGROUND AND AIMS: The mechanisms of floral nectar production in buckwheat (Fagopyrum esculentum, Polygonaceae), a distylous pseudo-cereal, have received relatively little attention, prompting an investigation of the factors that regulate this process. The aim was to perform a refined study of the structures that secrete nectar and of the internal and external parameters influencing nectar volumes and sugar concentrations. METHODS: In order to control environmental parameters, plants were cultivated in growth rooms under controlled conditions. The structure of nectaries was studied based on histological sections from flowers and flower buds. Nectar was extracted using glass micropipettes and the sugar concentration was measured with a hand refractometer. Sugar concentration in the phloem sap was measured using the anthrone method. To test the influence of photosynthesis on nectar production, different light and defoliation treatments were applied. KEY RESULTS: Unicellular trichomes were located in the epidermis at the ventral part of eight nectary glands situated on the flower receptacle alternately with stamens. Vascular bundles consisting of both phloem and xylem were identified at the boundary between a multilayered nectary parenchyma and a sub-nectary parenchyma with chloroplasts. A higher volume of nectar in thrum morphs was observed. No other difference was found in morphology or in sugar supply to inflorescences between morphs. Nectar secretion was strongly influenced by plant age and inflorescence position. Nectar volumes were higher in the upper inflorescences and during the flowering peak. Light had a dual role, (1) acting directly on reproductive structures to trigger flower opening, which conditions nectar secretion, and (2) stimulating photosynthetic activity, which regulates nectar accumulation in open flowers. CONCLUSIONS: In buckwheat, nectar is secreted by trichomes and probably proceeds, at least in part, from phloem sap. Nectar secretion is strongly influenced by floral morph type, plant age, inflorescence position and light.

[Proteins as morphogenetic markers in callus cultures of buckwheat Fagopyrum tataricum (L.) Gaertn with different morphogenetic potential].

The content of soluble proteins and individual polypeptides was studied in calluses of buckwheat Fagopyrum tataricum (L.) Gaertn with different morphogenic potential. The morphogenic callus had a higher content of soluble proteins and cyclic pattern of changes in this index during passaging, which seems to be due to formation of proembryogenic cell complexes. Comparison of the protein patterns of the calluses demonstrated differences in composition and content of individual components. Morphogenic (35 and 73 kDa) and nonmorphogenic callus-specific proteins (16 and 62 kDa) have been revealed.

[Dynamics of mitotic index of buckwheat root meristems, developing from seeds subjected to percussive wave treatment]. / Dinamika mitoticheskogo indeksa kornevykh meristem prorostkov grechikhi, razvivaiushchikhsia iz semian, podvergnutykh impul'snomu davlaneiiu.

A method of percussive wave treatment of seeds, promoting activization of physiological processes in plants, was proposed. The influence of physical treatment on cell nuclei and phytohormones was investigated. We examined dynamics of mitotic indices (MI) of the root meristems of buckwheat seedlings 48-58 h after the treatment. The seeds were treated with pressures of 11, 23, 29 MPa. The synchrony of cell divisions was estimated according to the augmentation of maximum MI versus average MI. The maximum and the average MI of control seedlings was 9.6 and 8.1%, resp. After the treatment with the pressure of 11 MPa the average MI increased by 2.6 times, and synchrony raised by 50%. After the treatment by the pressure of 23 MPa the average value of MI did not change versus the control, but the synchrony enhanced by 2.8 times. After the treatment of the pressure of 29 MPa the average MI decreased by 31%, the synchrony increased by 33%. Examination of phase indexes allowed to reveal the damage of cytotomy, transition to metaphase, and mitotic apparatus formation. The MI of root meristems of the test seedling aged 8 days topped the control by 21, 156 and 20%. So, the pressure of 11 MPa promoted MI increase in 2 days, and those of 23 and 29 MPa in 8 days. The investigation of hormone balance allowed to detect a raising level of abscisic acid. Its influence on the plant growth depends on the content of zeatin. A hypothetical model of plant reaction mechanism was proposed, based on the obtained results. A momentary pressing in the percussive wave was a stressor, promoting the accumulation of ABA and "growth rest", that changed for an active growth induced by zeatin.