Annals of Botany 74: 373-379, 1994 Amaranthus hypochondriacus: Seed Structure and Localization of Seed Reserves SILVIA COIMBRA and ROBERTO SALEMA Instinao de Botinica * Dr. Gongalo Sampaio’ and Centro de Citologia Experimental, Universidade do Porto, Rua do Campo Alegre 823, 4100 Porto, Portugal Received: 6 July 1993 Accepted: 11 April 1994 ‘The mature sed of Amaranthus hypochondriacs L. consists of a peripheric embryo surrounding the nutritive tissue Uhich isthe perisperm. Eodosperm remnants ate lose othe root ip. Cytecherical analysis revealed that the embryo ind endosperm eels had quite homogenous internat organization, with protein bodies embedded ina lipid rate ‘The embryo, however, appears variable jp lssue organisation, due to the dillreniation of the three primary ‘meristemali sus: the procambium appears asa single bundle in the embryonic axis or as sll bundles throughout the cotyledons length. these provasclar cells are small and elongated and with fewer reserves and more celular brganelles than the large protoderm and ground meristem cell. These latter cells have mote protein bodies, and they ‘hoo’ a higher number of larger globo crystal incisions than the others. The periperm is «starchy ste, and calls have thin walls and are Tull of angular starch gras Key words: Amaranth, Amaranth hypochondriacus, sed structure, seed reserves. INTRODUCTION ‘The role of seeds in the plant life cycle requires that they store materials which can be made available to support the early growth and development of the embryo Tollowing. germination. The presence and type of nutrienis in the seeds ‘make them nutrionally and economically important, Grain Amaranths were one of the basic foods of the new world in pre-Columbian times and were used in religious rites that had human sacrifices. Their cultivation dis- appeared soon after Cortez conquered the area in 1519 but since the 1970s there has been a resurgence due to their nutritional qualities as well as their agricultural potential (National Research Council, 1984). The three species that produce significant amounts of edible grains are Amaranthus hypochondriaeus, A. eruentus and A. caudaus (Amaranthacene). One of the most attractive features of grain amaranth is the high quality amino acid balance present in the seed. The amount of seed proteins is higher than that of most cereals, with an expecially high lysine content, which is the limiting amino acid in all other major cereal crops (Kauffman and Haas, 1984). Amaranths grow vigorously in their nature regions because they utilize CO, more efficiently to produce sugars. by the C, pathway of photosynthesis and they are resistant to drought, heat and pests (Becker er af., 1981). Thus, they readily adapt to new environments, including some that are inhospitable to cereal crops. Their capacity for efficient water use is a quality essential for a modern crop to successfully compete for the limited land and diminishing water supplies found in many areas world-wide Saunders and Becker, 1984). Amaranths have several features that make them at tractive as a crop, $0 most research has concentrated on their agronomic potential. Information shout their re- (0805-7364/94/ 100373 +07 $08.00/0 productive biology is scarce, although data of this type is much needed for the implementation of biotechaological methods The biochemical composition of grain amaranth seeds, including total protein content, is known (Becker et al, 1981; Bressani, SanchezMarroquin and Morales, 1992), but no information is available on seed ultrastructure and ‘on seed protein bodies, The aim ofthe present study was to investigate Amaran- thus hypochondriacas. seed structure and the spatial localization of its food reserves MATERIALS AND METHODS Source of material Soods from Amaramhus hypochondriaeus L. (RRC#1023, zgrain type mercado) were obtained from Rodale Research Center (Kutztown, Pennsylvania, USA), ‘Specimen preparation Seeds were fixed directly or after 10 h of imbibition in water. To facilitate embedding, the seeds were cut with different orientations exposing portions near the arcas which were to be studied, Light microscopy ‘Seeds were fixed in 2:5% glutaraldehyde in PIPES buifer (1:25 w, pH 72) for 2 hat room temperature. Fixed material was rinsed four times with buffer and, after dehydration through an ethanol-propylene oxide series, was embedded © 1994 Annals of Botany Company374 Coimbra and Salema—Amaranthus Seed Structure and Seed Reserves Fic. 1 Light micrograph of a median longitudinal section of an Amaranthus hypochondviacus seed. The embryo su soiyladons (C), the hypocotyl (HH), the radi (R) and the shoot apes (S) ean be distinguished. The remnants of | the eadicle tip (arrow); stained with azure A-azure B. x7. Fic. 2. Scanning eectzon micrograph of an Amaranthus hypochondriacs se. The periphere embryo (E) surrounds the petisperm (P). On one side ofthe seed the micropyle i visible between the tip ofthe cotsleons and the root tip (sow). » 180 sunds the persperm (P). The ‘endosperm appear around F105 3-6, Light micrographs of sections from differen regions of Amaranhus seeds Fic. 3. Longitudinal section of pat of the hypocotyl, stained with Aniline Blue Black (ABB), showing the ground meristem (GM) and protoderm (PD) eels. Large sumbers of protein bodies ace present ia GM cel.» 70,Coimbra and Salema—Amaranthus Seed Structure and Seed Reserves in Spure’s resin (Spurr, 1969), Sections approximately O'S am thick were obtained sith a LKB 8800 Ultrotome III using a glass Knife, The following staining procedures were used: (1) Aniline Blue Black (Fisher, 1968) for total proteins; for a control proteins were removed with acetic anhydride (40%) in pyridine at 60 °C, before staining. (2) Sudan Black B (Bronner, 1975) for total lipids; for a control lipids were extracted with methanol and chloroform (1:1) before staining. (3) Periodic acid-Schiff (PAS) (Feder and O’Brien, 1968) for insoluble polysaccharides; for a control the pretreatment with the periodic acid was omitted. (4) Toluidine Blue O (Feder and O'Brien, 1968) for phytin; for a control sections were placed in acetic acid (0-1 N, 3 min) before staining. (5) Azure A Azure B (Dodge, 1964) for general staining. Photographs were taken using a 35 mm Pan F 50 film. Electron microscopy For transmission electron microscopy (TEM), tissue fixed in glutaraldehyde as described above, was postfixed in O30, (2%) in the same buffer for 1h, dehydrated in a graded ethanol propylene-oxide series and embedded in Spurr's medium. Ultrathin sections, obtained with @ LKB 8800 Ultrotome IIT using a glass knife, and approximately 40-50nm thick, were mounted on grids coated with parlodium (1-5%), stained in uranyl acetate followed by lead citrate, and examined in a Siemens Elmiskop 1A or in a Zeiss 10°CR transmission electron microscope. Images were recorded using Agla-Gevaert 23 D cut film. For scanning electron microscopy (SEM), material was fixed and dehydrated as described for TEM, then dried in a critical point dryer, sputter coated with gold and viewed with s Jeol JSM-35 C scanning electron microscope. RESULTS The overall organization of Amaranihus hypochondriacus seed can be seen in Fig. 1. The seeds are cream to golden coloured, lenticular in shape and approximately | mm in diameter (Fig. 2). The embryo is peripheric, surrounding the perisperm which is @ storage tissue of nucellar origin. During development the embryo utilizes most of the endosperm tissue with the remaining portion present mostly around the radicle tip. The embryo consists of the hypocotyl radicle axis and two cotyledons; the apical meristems of the root and shoot are well differentiated (Fig. 1). Nutrient local tion in the different tissues The nutrient reserves of the seeds are mostly, but net exclusively, laid down as discrete cellular structures and 375 include lipid, protein, carbohydrate, organic phosphate and various inorganie compounds. “The reserves are not stored uniformly throughout the seed tissues. Storage proteins occur in membrane-bound protein bodies in the embryo (Figs 3, 4) and endosperm cells. In addition to the homogenous proteinaceous matrix, these structures contain inclusions which were identified, by staining with Toluidine Blue O, as globoid crystals, indicating the presence of phytin (Fig. 5), a salt of myo- inositol hexaphosphoric acid. These globoids appear elec- teon-dense inthe electron microscope (Fig, 13). In contrast protein is preseat as very small deposits around starch gains in the endosperm (Fig. 6). Lipids and carbobydrates are also present within seeds. Lipid bodies are present in both the embryo and endosperm cells Staining with Sudan Black B shows that almost all of the cytoplasm surrounding the protein bodies is full ofthese lipids (Figs 7, 8, 16), The same staining procedure shows no colour reaction in the perisperm cells (data not shown). Carbohydrates are stored as starch in the plastids of the perispetm cells (Fig. 10) and thereis no starch in theembryo (Fig. 9) nor in the endosperm cells, Carbohydrates also are present throughout all ofthe seed as call wall polysaccharides (i. 9). The cells of this starchy perisperm have very thin cell walls and are ful of stareh grains witha clear angular shape as its easily seen under SEM (Fig, 11). Tissue structure of the embryo ‘The embryo tissues have cells of very different shapes and sizes and they show a high degrce of differentiation, with well developed protoderm, procambium and ground mer- istem (Fig. 9). All cells have a quite homogenous internal organization with protein bodies embedded in a li matrix. The procambium can easily be identified as bundles of small cells along the cotyledons (Figs 9, 14) or asa single bundle in the centre of the embryonic axis (Fig. 12). These cells are typically smaller and more elongated than the large isodiametric ground meristem cells (Figs 13, 14). Besides size, these cells also differ in content: the procambium cells hhave less nutrient reserves and more cellular organelles than the ground meristem cells (Figs 14, 15). A number of ‘organelles, particularly mitochondria’ are observed in the procambium cells (Fig. 15). Protein body numbers and morphology differ from one tissue to the other. They appear in larger numbers and with ‘more inclusions in the ground meristem cells (Figs 13, 16). In contrast to ground meristem cells, procambial calls have fewer protein bodies (Fig. 15) with a reduced number of inclusions (Fig. 14), ‘The position and characteristics of the ground meristem cells in the cotyledons is quite indicative of their fate: the Fic. 4. Higher magnification of Fig. 3 GM cell. Globoid crystals (G) can be seen nse protein bodies (Ph). Most globoid crystals appear as light 1 surrounded by the darker proteinaceous matrix when tained with ABB (arrowhead). This oecurs when the had globod ersals become ‘isdged during sectioning. thereby leaving a hole ir the section, % 2300, Fic. 5, Some ofthe sloboid cry is that were not removes by sectioning en be identifies as phytin deposits rrowhends) stained with Toluidine lie 0. 6 Tn the large persperm cells, proteins eppear as small deposits (arrowheads) around the starch grains. ABB stining. « 1700, 2500,376 Coimbra and Salema—Amaranthus Seed Structure and Seed Reserves Fics 7-10, Light micrographs of sections From different regions ofthe Amaranthus sss Fig. 7. Hypocoty els; most of the eytoplasn surrounding the protein bodies is Fl of lipids which stains very dask with Sudan Black B (BB), ‘<0 Fic, 8. Higher magnifoation of Fig. 7, Protein bodes (Pb), lipids (arrows), N, nucleus.» 1700. Fig. 9 Longitudinal vetion showing part ofa cotyledon and the sed coat (SC), stained with periodic acid Schiff (PAS). The protein bodies show some minor staining due to the presence of some carbohydrates in the protcinacoous matrix. Provascular tisue (PC) appear throughout the ‘cotyledons as bundles of smal cals, when seen in cross section, compared with the large ground-merstem cells (GM). Outer protedermal cells (OP) appear stoccurally dilleren from the inner protodermal eels PD), 910.Coimbra and Salema-—Amaranthus Seed Structure and Seed Reserves future palisade tissue is positioned close to the inner protoderm whereas the future spongy tissue lies next to the outer protoderm (Fig. 9). A diflerence is also observed between these two protoderms; the inner (adaxial) one is very similar to the ground meristem cells and the outer (abaxial) one has cells with fewer protein bodies baving a smaller number of inclusions in their matrix (Fig. 9). There is aso a difference between the ground meristem and the protoderm of the embryonic axis, the former cells stain ‘uch more intensely for proteins than do the protodermal calls (Fig 3). ‘The electron microscope shows large protodermal calls and also very large ground meristem cells with some intercellular spaces between the tissues (Fig, 16 DISCUSSION Amaranthus hypochondriacus seeds ate classified as the peripheric type (Martin, 1946) The localization of the stored reserves inside the seeds shows a marked compartmentation. Seeds of angiosperms usually deposit the major fraction of their storage proteins in highly distinctive structures, termed protein bodies. Some protein bodies are structurally simple and consist only of a proteinaceous matrix, whereas others have inclusions such a globoid crystals, protein erystalloids or calcium oxalate crystals in the matrix (Lott, 1981). Mineral nutrient reserves are usually concentrated in globoid erystals and occur as phytin, Less important and more variable is the presence of hydrolytic enzymes, carbohydrates, ribonucteic acids, oxalic acid salts, lipids and tocopherol (Pernoliet, 1978). 4. hypochondriacus embryo and endosperm cells have globoid crystals inclusions in their protein bodies. The information available for protein body structure in other families is quite limited but considerable differences in protein body structure can occur between different seed regions of the Gramineae, Where protein bodies are observed with inclusions in the aleurone cells but without inclusions in the starchy endosperm (Pernollet, 1978). Protein body structure also varies between different tissues within an organ (Pate et al. 1986; Citharel and Citharel, 1987). Maldonado and Lott, (198i) reported that in Datura stramoniuon seeds, endosperm protein bodies had varying sizes and numbers of globoid crystals, even within the same cell, whereas embryo protein bodies rarely had more than two globold crystals. In seeds of 4. hypochondriacus the protodermal and provascular cells have smaller and structurally simpler protein bodies compared to the large ground meristem cells; inclusions are always globoid crystals but they are more abundant in the latter type of cell. Differences in presence or structure of protein bodies between the abaxial and adaxial protoderms Of the cotyledons might be useful characters for studies in. plant systematics (Lott, 1981). Globoid bodies in glutaralde- 377 hyde-osmium fixed tissues appear to be amorphous electron-opaque and quite frequently shatter during sec- tioning (Vijayaraghavan and Prabakar, 1984) leaving frequently empty spaces so often seen in the protein bodies. The reserve lipids of sceds ae casily secognized as distinet protoplasmic inclusions occurring as uid spherical droplets of various sizes either dispersed in the cytoplasm or aggregated into large masses (Mollenhauer and Totten, 1971). The predominant storage lipids of sceds are the neutral fats oF ois if they are liquid at ‘normal’ tempera tures. These are triglycerides which are laid down as lipid bodies. In seeds with high oil deposits, the bodies pack the cll filling almost all the space left unoccupied by the other organelles (Bewley and Black, 1978), What we observed in A. hypockondriaeus embryo cells, is the same as in cereal aleurone (Bewley and Black, 1978), the lipid. bodies completely surrounded the protein bodies. This is most noticeable in the ground meristem cells, where the number of other celular organelles is much less than in the small, procambjum cells Starch grains arise as single or multiple granules in amyloplasts. These grains have a characterstie appearance for each plant species and may be spherical, angular or elliptical (Bewley and Black, 1978). Interestingly, grain shape depends on the amylose content, the less angular, rounded granules having relatively higher amylose levels (Greenwood, 1976). Grains also display a great range of sizes among different species. 4. hypochondriacus starch grains are small and clearly angular in shape. These observations agree with previous reports (Becker et al. 1981; Okuno and Sakagachi, 1981) where Amaranthus hypochondriaeus starch grains were described as. small sranules, 1-3 jm in diameter and mainly amylopectin in nature Grain Amaranth is regarded as an important alternative {00d source for particular habitats, that needs improvement in seed size, yield, reduced sensitivity to. photopherind, synchronous flowering and better palatability. These goals requite a deeper knowledge of seed structure and re- productive biology of these plants. Thus, a study of the Gillet phases of Amaranthus embryogenesis also. is ‘underway inthis laboratory. ACKNOWLEDGEMENTS. ‘We thank Professor Lone Bruun for helpful reviews of the ‘manuscript and Mrs Andrea Costa for excellent photo: ‘graphic assistance, This work was supported by grants fom, the Instituto Nacional de Investigagio Cientifica (INIC), Portugal. Fic, 10, Perispetm cells filled with starch grains (arrows) after staining with PAS. » 1980 Fic, 11, This canning eeciom micrograph of a persperm eel shows he angulat shape of many smal iach grains. «YEO, Fig, 12, The median part ofthe cotyledon ais, 28 viewed in fe una section, showing one single median procambial bundle (PC). Ground ‘meristem (GM) and protederm (PD) stained with PAS. » 320,378 Coimbra and Salema—Amaranthus Seed Structure and Seed Reserves Figs 13-16, Transmission electron micrographs of embryo celle Fic. 13. Ground meristem cls in one of th cotyledons. Protein bodies are spherical to irogular in shape and contain many globoid crystals {arrow}. The empty areas contained slobeid crystals before they were dislodged during sec ring. Lipid bodies (arrowheads) surround the protein bodies. = 3160, Fic. 14, Bundle of procambia els, Notie the diference between the cellular content ofthese cll and the ground meriser calls. The later ones have many more nutrient reserves Ad few ellular organelles, There also are many fewer inclusions in procambial protein bodies, $100. Fi. 15, High mapniteaton of procambjum bundle showing diferemiating vascular ells.» 600. Fic. 16, Pat ofa cotyledon showing protaderm and ground meristem cells Some large inracellulae spaces (IS) can be noticed. x 3160. GM, ‘Ground meristem; PC, procambium:; PD, protoderm:; N, aucious; PB, protean body; L, lipid body; G. globoid crystal LITERATURE CITED Becker R, Wheeler EL, Loren K,Stalford AE, Cilasean OK, Betschart ‘AA, Saunders RM 1991. 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