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Obendorf, R.L., M. Horbowicz, and D.P. Taylor. 1993. Structure and chemical composition of developing buckwheat seed. p. 244-251. In: J. Janick and J.E. Simon (eds.), New crops. Wiley, New York.

Structure and Chemical Composition of Developing Buckwheat Seed*

Ralph L. Obendorf, Marcin Horbowicz, and Douglas P. Taylor


  1. REPRODUCTIVE BIOLOGY
  2. SUGARS AND ORGANIC ACIDS
  3. CHEMICAL COMPOSITION
    1. Free Amino Acids
    2. Sterols and Fatty Acids
  4. REFERENCES
  5. Fig. 1
  6. Fig. 2
  7. Fig. 3
  8. Fig. 4
  9. Fig. 5
  10. Fig. 6
  11. Fig. 7
  12. Fig. 8

Buckwheat (Fagopyrum esculentum Moench) grain is a basic food item in porridges, soups, and the preparation of "kasha" in Central and Eastern Europe. In Japan, buckwheat is used mostly for manufacturing a noodle, soba, which is prepared from a mixture of buckwheat and wheat flours (Udesky 1988). Considered a healthy food, Japan's total buckwheat consumption is about 100,000 tons/year (Komeichi et al. 1992). About 80% of Japan's consumption is imported, largely from the United States and Canada. Buckwheat produces a low grain yield because of a low incidence of seed set. Although the plants usually produce an abundant number of flowers, only 10 to 20% of the flowers develop into mature grain (Ruszkowski 1986). Yields vary from 150 to 1,200 kg/ha depending on the soil, climate, and other factors. Demand from export markets has increased the need for stabilization of yields and prompted basic research on factors regulating seed set and cessation of seed growth in buckwheat.

The structure and composition of mature buckwheat fruits has been reviewed by Pomeranz (1983), but little information is available on the changes in the fresh and dry weight and the composition of parts of the developing seeds. In this paper, we summarize our research on the reproductive biology of developing buckwheat seeds and the changes in content of sugars, organic acids, amino acids, sterols, and fatty acids in developing seeds.

REPRODUCTIVE BIOLOGY

Buckwheat flowers are perfect but incomplete. They have no petals, but the calyx has the appearance of petals. Flowers occur in compact racemes, either terminally on the main stem or on branches from the axil of leaves. Common buckwheat plants are dimorphic and heterostylous. One-half of the plants have pin-type flowers with long styles and short stamens, and one-half of the plants have thrum-type flowers with short styles and long stamens (Marshall and Pomeranz 1982). Each type is self-incompatible and cross-incompatible among plants with the same flower type. Seed set requires legitimate cross pollination, pin by thrum and thrum by pin, by insects under field conditions (Namai 1990) or by hand pollination in the greenhouse as in the present study (Horbowicz and Obendorf 1992). Temperature in the greenhouse was controlled at 24° day and 18°C night. Natural sunlight was supplemented 14 h daily with high intensity incandescent light. Buckwheat plants were watered as needed and supplemented weekly with fertilizer.

The mature buckwheat seed is an achene. The mature ovule or groat, the dehulled achene (with pericarp removed), is used for food. Seed set is defined as producing a seed that the miller can use. Seeds usually set if not aborted by 10 days after pollination (DAP). Oil, starch, and protein storage reserves in the embryo and endosperm tissues accumulate rapidly after 10 DAP, the period of seed fill. The cessation of seed growth at full development is called physiological maturity and corresponds to the time of maximum dry weight. The loss of water from the seed is desiccation and leads to harvest maturity and safe storage when sufficiently dry. A number of conditions must be met for seed set to occur.

The female parts of the buckwheat flower, an ovary with styles and stigmas receptive to pollen are illustrated in Fig. 1. Within the ovary (or developing achene), a complete and functional ovule (developing groat) must be present. The outer and inner integuments (each two cells thick) form the seed coat of the dehulled groat at maturity. At the upper end of the ovule, the integuments form an opening, the micropyle, through which the pollen tube enters. The nucellus is positioned inside the integuments. One cell of the nucellus becomes the megaspore mother cell, divides by meiosis (2n to 1n) forming four haploid (1n) megaspores. One of the four divides sequentially to form an egg sac with eight-nuclei (Mahony 1935). One forms the egg cell (future embryo) bordered by two synergids, two fuse to form the nucleus of the central cell (future endosperm), and three migrate to the bottom in close proximity to the hypostase, a structure analogous to the nucellar projection in wheat (Frazier and Appalanaidu 1965) and barley (Cochrane and Duffus 1980).

Viable pollen must be delivered to the stigma by legitimate cross-pollination to achieve seed set. The pollen must germinate rapidly, have vigorous growth of the pollen tube through the style and into the micropyle. The pollen tube must penetrate the nucellus and the egg sac, delivering two sperm nuclei (each 1n) to the egg sac. One unites with the egg (1n) to form the zygote (2n) which develops into the embryo; the other unites with the fused (2n) nucleus of the central cell forming 3n nuclei which develop into endosperm cells. This double fertilization of the egg and central cells is essential for seed set.

The nucellus and embryo sac in the ovule and the tapetum and sporogenous cells of the anther are distinctly different than other tissues in the ovary and stamen. A monoclonal antibody which specifically recognizes an L-arabinose containing arabinogalactan-protein in the plasmalemma of cells reacts with all plant cells except those of the nucellus, egg sac, and embryos during early embryogenesis (Pennell and Roberts 1990). The pattern is the same in pea, maize, and Arabidopsis, and presumably also in buckwheat. Pennell and Roberts (1990) suggest that absence of the arabinogalactan-protein may be an essential prelude to the expression in the gametes themselves of determinants involved with fertilization. Another epitope of a plasma membrane arabinogalactan protein occurs in association with the sperm cells of pollen grains late in anther development in rapeseed flowers (Pennell et al. 1991). In ovular tissues, this epitope appears first in the nucellar epidermis near the micropylar end of the ovule and subsequently in the egg cell and both synergids. Since this epitope is not found in the central cell, Pennell et al. (1991) proposed that it is related to gamete recognition. Upon entry of the pollen tube, the sperm cells are released in a synergid before fertilization of the egg and central cell nuclei (Knox et al. 1986).

Following double fertilization, the embryo develops rapidly during the first day (Stevens 1912; Mahony, 1935), the ovary doubles in size from 1 to 2 mm in length (Fig. 2), and the ovule increases from 0.5 to 1 mm in length. Volume of the endosperm increases rapidly, remains clear and fluid, and contains multiple nuclei. By 3 DAP, the embryo forms cotyledons, the upper endosperm forms cells, and the basal endosperm remains as a thin layer of cytoplasm containing multiple nuclei and a very large vacuole (Stevens 1912). By 6 DAP, the endosperm starts to solidify along the margins (Stevens 1912), and the liquid endosperm becomes milky.

Vascular tissues (for transporting nutrients and water) branch from the stem into the rachis of the raceme (inflorescence), branch into the pedicel and then into the sepals and stamens (Fig. 1). Vascular tissues enter the funiculus and form a saucer-shaped "Y" at the base of the ovule (Fig. 1). Vascular tissues do not enter the integuments, nucellus, nor egg sac. Therefore, nutrients going to the developing egg sac, embryo, and endosperm must exit the symplast (cell cytoplasm), enter the apoplast ("free-space" in cell walls and between cells), and be reloaded into the symplast (cytoplasm) of the developing cells of the egg sac, embryo, and endosperm. At the basal interface between the integuments and the nucellus, and positioned across the apparent path of nutrient flow to the egg sac and developing embryo and endosperm, a group of cells accumulate an osmiophilic substance which may include polyphenols or flavonoids. This deposit may limit transport through these cells. Characterization of the basal tissues of the ovule is lacking in early reports (Mahony 1935; Stevens 1912).

Ovaries of both pin and thrum flower types are about 1 mm in length at anthesis, and the developing achene rapidly increases to maximum length at 5 or 6 DAP (Fig. 2). Width of developing achenes continues to increase until 12 to 14 DAP during the seed-filling period. The ovule undergoes characteristic changes in morphology during the development (Fig. 3). Ovules are about 0.5 mm in length at anthesis, and by 2 DAP, they are about 2 mm in length and have developed a swollen base with expanding endosperm. The vascular attachment is at the base of the bulbous structure while the embryo develops near the micropyle at the apex of the ovule and grows downward into the endosperm. Expanding pericarp tissues rapidly accumulate fresh weight between 2 and 6 DAP (Fig. 4). Ovule tissues, including the endosperm and embryo, increase rapidly in length and width between 6 and 10 DAP with a rapid increase in fresh weight after 6 DAP and a rapid increase in dry weight after 8 DAP. The embryo attains maximum fresh weight at 12 DAP, but the endosperm continues to increase in fresh and dry weights until 16 DAP (Fig. 4A,B). Cessation of achene dry matter accumulation occurs by 20 DAP. Water concentration in the endosperm decreases from 850 g/kg fresh weight at 6 DAP to 220 g/kg fresh weight at 16 DAP (Fig. 4D). Water concentration in embryo tissues declines at a slower rate. The accumulation of storage reserves in the endosperm and embryo contribute to the ovule shape conforming to the shape of the pericarp (Fig. 3). By contrast, when the endosperm does not develop, the pericarp may grow to normal length of the mature achene, but the pericarp walls are folded inward reflecting the lack of endosperm and embryo development.

SUGARS AND ORGANIC ACIDS

Whole or parts of 3 to 20 developing achenes were boiled, homogenized, and extracted in 50% ethanol/water with sedoheptulose as internal standard. Trimethylsilyl (TMS) derivatized sugars and organic acids were analyzed by capillary (DB-1701) gas chromatography (M. Horbowicz, R.L. Obendorf, and W.J. Cox, unpubl.).

Sucrose is the major sugar accumulating in buckwheat achenes. Increases in sucrose accumulation reflect periods of dry weight increases, with the embryo tissues accumulating the largest amount of sucrose (Fig. 5A). Sucrose is abundant in the seed coat and nucellus tissues from anthesis through 6 to 8 DAP and then accumulates in the developing embryo. The high concentration of sucrose in embryo tissues is typical of oilseed tissues (Duffus and Binnie 1990; Kuo et al. 1988). The starchy endosperm accumulates only low transitory levels of sucrose. The ovular maternal tissues (integuments and nucellus) accumulate sucrose before major accumulation of dry matter in the endosperm and embryo, and then, the sucrose level declines consistent with an apparent transport role to the developing endosperm and embryo.

The monosaccharides, glucose and fructose, are mostly in the pericarp and seed coat (Fig. 5B). Levels of glucose and fructose are similar (data not presented). Except in embryo tissues, increasing glucose and fructose levels precede sucrose and starch accumulation (Fig. 5B), a pattern observed in starchy endosperm of maize (Shannon 1968, 1972). Levels of inositol are low in all tissues. Inositol is detected sequentially in the pericarp, seed coat and nucellus, endosperm, and embryo during later developmental stages of each (Fig. 5C). After seed set, faster-growing achenes have higher starch levels and lower sugar levels while slower-growing achenes have lower starch levels and higher sugar levels (Dua et al. 1991), indicating that utilization of sugars may be limiting in slower-growing achenes.

Citric acid accumulates only in green pericarp tissues with peak levels at 6 to 8 DAP (M. Horbowicz, R.L. Obendorf, and W.J. Cox, unpubl.). Malic acid is detected sequentially in pericarp and maternal ovule tissues and then in endosperm and embryo tissues (Fig. 5D) with peak concentrations early in development (4 DAP in endosperm). Malic acid content is much higher than citric acid in cells and amyloplasts of starchy endosperm (Liu and Shannon 1981).

CHEMICAL COMPOSITION

Free Amino Acids

Free amino acids in the ethanol/water, 1:1, extract from endosperm and embryo tissues were diluted 5 to 10 fold with water and analyzed by the ninhydrin method (Rosen 1957). Free amino acids accumulate 4 to 12 DAP primarily in the seed coat and nucellus, maternal tissues of the ovule (Fig. 6). Free amino acids in the endosperm and embryo increase to a constant level at 8 and 10 DAP. The rise in level of free amino acids in the maternal tissues of the ovule at 12 DAP and subsequent depletion by 16 DAP, when dry matter accumulation in the endosperm and embryo has nearly ceased, is indicative of the role of these tissues in transport of amino acids to the developing endosperm and embryo. The concentration of amino acids in all tissues declines during seed development with the accumulation of dry matter (M. Horbowicz, R.L. Obendorf, and W.J. Cox, unpubl.).

The protein content of mature whole grain is 13.8%, dehulled groat 16.4%, pericarp 4%, endosperm 10.1% and embryo 55.9% (Pomeranz and Robbins 1972). The amino acid content of protein in the embryo and endosperm are quite similar. Embryo proteins are enriched slightly in arginine, serine, and glutamine/glutamic acid while endosperm proteins are slightly enriched in lysine, proline, alanine, methionine, and leucine. Amino acid analysis of proteins in mature achenes has been reported (Pomeranz and Robbins 1972; Pomeranz et al. 1975; Pecavar et al. 1990). While protein concentration in the hull declines during seed development, protein concentration in the groat is more constant, and the amino acid composition of groat proteins is very similar at 7, 14, 21, and 28 DAP (Pomeranz et al. 1975). Buckwheat proteins are composed of about 18% albumins, 43% globulins, 1% prolamins, 23% glutelins, and 15% insoluble residue (Javornik et al. 1981). About 30% of the total N is non-protein N and N in molecules of <10,000 molecular mass. A 13 S globulin is 80% of the protein body proteins and 51% of the total protein in cotyledons of the embryo (Elpidina et al. 1990).

Sterols and Fatty Acids

Endosperm and embryo tissues of developing buckwheat seeds were homogenized in methanol containing methyl-heptadecanoate and dihydrocholesterol, which were added as internal standards. Fatty acids and sterols were extracted with potassium hydroxide/methanol solution at 80°C which simultaneously combined extraction and saponification steps (Horbowicz and Obendorf 1992). After addition of saturated sodium chloride solution, the non-saponified fraction containing sterols was extracted with hexane. After evaporation of hexane, extracted sterols were silylated and analyzed by capillary (DB-1701) gas chromatography (Horbowicz and Obendorf 1992). The methanol/water layer containing saponified materials was acidified and the fatty acids were extracted with hexane. After evaporation of hexane, total extracted fatty acids were converted to methyl esters (Metcalfe et al. 1966) and analyzed by capillary (DB-1701) gas chromatography (Horbowicz and Obendorf 1992).

Sterols include ß-sitosterol (70% of total), campesterol, an unknown, and traces of stigmasterol (Fig. 7). Total sterol (2.1 g/kg at 20 DAP; Fig. 7F) and total fatty acid (123 g/kg; Fig. 8F) concentrations are four to five times higher in embryo tissues than in endosperm during development. The amount of each sterol increased continuously during endosperm development (Fig. 7A). In embryo tissues, however, the amount and concentration reached a maximum at 12 DAP (Fig. 7B, E).

At 6 to 10 DAP, 60 to 80% of the fatty acids are saturated (Fig. 8) with palmitic being three to five times higher in concentration than any other fatty acid at 6 DAP (Horbowicz and Obendorf 1992). By contrast, at 12 to 20 DAP, 70 to 80% of the fatty acids are unsaturated (Fig. 8) with linoleic, oleic, and palmitic representing 85% of the total (Horbowicz and Obendorf 1992). The transition at 10 to 12 DAP occurs during rapid growth of the embryo (Fig. 4) and a 10-fold increase in total fatty acids (Fig. 8); 80% of the total fatty acids in embryo tissues accumulate between 10 and 12 DAP. At 12 DAP more than 80% of the total lipids are in the embryo. As the storage lipids begin to accumulate in the achene, sterols and palmitic acid accumulate initially followed by stearic, oleic, linoleic, and linolenic acids (Horbowicz and Obendorf 1992). The long chain fatty acids, eicosenoic, arachidic, and behenic, are synthesized last at 16 DAP. The late appearance of long chain fatty acids precludes them from a role in seed set or abortion. In mature buckwheat achenes, long chain fatty acids are present in higher concentrations in neutral and free lipids but in lower concentrations in phospholipids (Mazza 1988) and milling fractions vary in concentrations of long chain fatty acids (Tauzuki et al. 1991). Lower temperatures during achene maturation result in higher concentrations of linoleic and linolenic acids, slightly lower concentrations of long chain fatty acids and no change in myristic, palmitic, or stearic acids (Taira et al. 1986).

REFERENCES


*Contribution from the Department of Soil, Crop and Atmospheric Sciences, Cornell University Agricultural Experiment Station, Ithaca, NY 14853-1901. Cornell buckwheat research is supported by grants from MINN-DAK Growers Ltd., The Birkett Mills, Japan Buckwheat Millers Association, and Kasho Company Limited.
Fig. 1. Diagram of a buckwheat flower with emphasis on structures of the ovule and embryo sac.


Fig. 2. Schematic development of a buckwheat seed (from M. Horbowicz, R.L. Obendorf, and W.J. Cox, unpubl.).

Fig. 3. Schematic development of a buckwheat ovule (from M. Horbowicz, R.L. Obendorf, and W.J. Cox, unpubl.).
Fig. 4. Changes in fresh weight (A), dry weight (B), length and width (C), and water concentration (D) of developing achene, ovule, pericarp, endosperm, and embryo tissues in relation to days after pollination (from M. Horbowicz, R.L. Obendorf, and W.J. Cox, unpubl.).

Fig. 5. Changes in sucrose (A), glucose plus fructose (B), inositol (C), and malic acid (D) in developing achene, ovule, embryo, and endosperm tissues in relation to days after pollination (from M. Horbowicz, R.L. Obendorf, and W.J. Cox, unpubl.).

Fig. 6. Changes in free amino acids in developing achene, ovule, endosperm, and embryo tissues in relation to days after pollination (from M. Horbowicz, R.L. Obendorf, and W.J. Cox, unpubl.).


Fig. 7. Changes in total content (A, B, C) and specific content (D, E, F) of sterols in endosperm and embryo tissues in relation to days after pollination (from Horbowicz and Obendorf 1992).

Fig. 8. Changes in total content (A, B, C) and specific content (D, E, F) of unsaturated, saturated and total fatty acids in endosperm and embryo tissues in relation to days after pollination (from Horbowicz and Obendorf 1992).
Last update September 10, 1997 aw