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Nord. J. Bot. Section of structural botany

A structural investigation of the ovule in sugar beetBeta vulgaris: Integuments and micropyleP. Olesen and Lone Bruun

Olesen, P. Bruun, L. 1990. A structural investigation of the ovule in sugar beet,Beta vulgaris: Integuments and micropyle. Nord. J. Bot. 9: 499 506. Copenhagen.ISSN 0107-055X.The micropyle and the integuments of sugar beet Beta vulgaris) ovules have beeninvestigated by light and electron microscopy during differentiation and maturationof the ovule. The micropyle itself is formed by the inner integument which issurrounded by th e o uter integument at its base. Th e micropyle containts a fibrillarPAS+ substance and is often covered by a thin she et or hymen. Both integuments arecuticle-covered thin sheets, each 2-few cell layers in thickness. In the outer in-tegument an increase in starch accumulation occurs during ovule maturation andprobably functions as nutrient storage for embryo development. T he inner epidermisof the inner integument differentiates as the most conspicuous cell layer of the beetovule. During growth and maturation of the ovule a system of small perinuclearvacuoles con taining dense material increases steadily in these cells. At maturity thissystem fills up more than half of each cell and very den se material has accum ulated ineach vacuole. This vacuole content is highly refractive and contains tannins andforpolyphenols.P. Olesen, Biotechnology Research Division, DA NIS CO A IS, P.O. Box 17 DK-1001Copenhagen K , Denmark. L . Bruun, Botanical Lab., Univ. ofCopenhagen, Goth-ersgade 140, DK-1123 Copenhagen K , Denmark.

IntroductionEven though the integuments and the micropyle aresome of the most distinct features of the angiospermovule, less attention has been paid to the ultrastructureand function in the reproduction processes of theseparts of the ovule. As regards the integuments, onlytheir number, initiation, testa development (ex. BorBouman 1974; Bosewinkel 1980) and the integu mentarytapetum has been treated in some detail (Kapil Ti-wari 1978). A detailed study on the inner epidermis ofthe inner integument (th e fringe-layer) of cotton ovulesand seeds has been made (Ryser et al. 1988). Tilton(1980) and Tilton Lersten (1981) have described theovule development in Ornithogalum and presented areview on selected papers on the micropyle and theintegume nts, respectively. In spina ch, a species closelyrelated to beets, Wilms (1980) made a comprehensive

study of ovule structure and development and includeda description of the integuments.Although sugar beet is an importa nt socio-economiccrop plant which has been subject to intensive breedingactivities, not much information is available about itsreproduction biology. Such da ta are highly needed for asuccessful implementation of biotechnological methodsin the breeding of new bee t varieties. Early light micro-scopical descriptions of the ovule in sugar beet havebeen m ade with little a ttention to the integuments andthe micropyle (H eel 1925; Artschw ager 1927). Recentlya few studies have been published on structural aspectsof sugar beet ovules, i.e., in vitro cultured ovules (Hos e-mans Bossoutrot 1986) and some observations onfertilization (Shen et al. 1986). Again, these studiescontain no new data related to th e integuments and themicropyle of sugar beet.In this study the integuments and the micropyle fromsugar beet ovules have been investigated by light-, scan-

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Figs 1 4 : Eera vulgaris. Fig. 1. Light microscopical overview showing oute r and inner integuments and micropyle ~ 2 5 0bar =50 pm . Fig. 2. Cross section showing outer and inner integuments at the level of the egg cell ~ 3 6 0bar = 50 pm). Fig. 3.Scanning electron m icrograph of the micropyle showing the different lengths of the inner and outer integuments ~ 7 5 0bar =10 pm). Fig. 4. Scanning electron micrograph of the micropyle covered with a sheet X950 (bar = 10pm).5 N o d J Bot 9 5 ) 1990)


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Figs 5-8: Bern vulgaris. Fig. 5 . Cross section of the micropyle containing a f ibri lar substan ce x 200 (bar = 1 pm). Fig. 6.Cuticles covering the inner and outer in teguments ~ 2 9 5 0 0bar = 1 pm). Fig. 7. Cross section of the border between the innerintegumen t and the micropyle at the level of the apex of the micropylar nucellus, note the accu mulation of black tannin deposits inthe epidermis of the inner integument. ~ 3 0 0 0bar = 1 pm). Fig. 8. Details of outer integumen t, plast id with starch grains andouter cuticle ~ 2 9 5 0 0 bar = 1 pm).


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Figs 9-10: Beta vulgaris. Fig. 9. Cuticle staining with Auramine 0:Slight staining of integument cuticles but strong reaction atnucellar surface ~4.50bar = 50 pm). Fig. 10. Collapsed cell layers in the apex of the outer integument X7 3 0 0 (bar = 1 pm).

ning- SEM), and transmission electron microscopy(TE M). T he study is part of a research programme onthe cellular and molecular biology of reproduction insugar beet, Beta vulgaris L. (Bruun 1987; NielsenOlesen 1988; Bruun Olesen 1988, 1989). Special em -phasis is given to the cells of the inn er epidermis of theinner integument and the importance of tannin deposi-tion in this layer early in ovule development.Materials and methodsMale sterile varieties of Beta vulgaris were supplied byMaribo Seed NSDe D anske Sukkerfabrikker). Plantswere grown in greenhouses throughout the year as well502

as outdoors. Conditions and methods for fixation, em -bedding, and sectioning for light- and transmission elec-tron microscopy were as previously described (Bruun1987). 3.0 km sections were stained in periodic acid-Schiff (PAS, Feder OBrien 1968) for 30 minutes andin 1 Aninline Blue B lack (A BB , Fischer 1968) in 7acetic acid for 2 minutes fo r light microscopic detectionof insoluble polysaccharides and proteins, respectively.For a better localization of tannins and polyphenolssections were double stained with AB B and 0.05 To-luidine Blue 0 (TBO) 5 minutes) in citrate-phosphatebuffer (pH 4.4). 3.0 pm sections were also stained in0.005 Auramine 0 in 0.1 M tris-buffer (pH 7.4) forfluorescence microscopy of cuticles. After photography

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Figs 11-14, Development of the vacuole system in the inner epiderm is of the inner integument of Beta vulgaris (all bars = 1 pm) .Fig. 11. Young cell with perinuclear distribution of the vacuoles, note the heavy cuticle at the nucellar side. xY000. Fig. 12.Later stage, but still perinuclear distribution and heavy cuticle at the nuc ellar side. x7500 -Fig . 13. Vacuole at the mature stageof ovule development x 11200. Fig. 14. Vacuole system during early embryo and seed development X7500.


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(excitation 450-490 nm. Kodak Ektachrome 400 hesame sections were rinsed in distilled water and res-tained with PAS and ABB.Material for scanning electron microscopy (SEM) wasfixed and dehydrated as described for TEM (Bruun1987). After dehydration the material was dried in acritical point dryer (CP D, Polaron E 5000 and sputtercoated with gold (Polaron E 3000). Specimens wereviewed with a Jeol JMS - P15 SEM.ResultsThe outer integument (01) consists of 2 cell layers: theouter epidermis (0.e.) and the inner epidermis (i.e.),except near the funiculus where it becomes multilayered(Figs 1,2 . Also in the apical (micropylar) part of theapex, the 01 consists at least during some parts of thematuration process of more than 2 cell layers. In themature stage a strongly PAS+ area occurs in a medianlayer at the apex (Fig. 1) which by TEM is resolved asan area of persistent cell walls forme d by the collapse ofdegenerating cells (Fig. 10). The ultrastructure of the0.e. and i.e. is very similar. The cytoplasm containsnumerous plastids with a conspicuous accumulation ofstarch grains (Figs 2,8), little rough endoplasm ic reticu-lum, many ribosomes and mitochondria and some lipiddrople ts. The cell walls are strongly PAS+ and consistof fibrillar material.Plasmodesmata occur in both periclinal and anticlinalwalls. An increase in starch accumulation occurs duringthe maturation process and reaches its highest amountwhen the embryo sac is mature (Bruun 1987). Afterfertilization the starch gradually disappears, starting inthe micropylar part of the 0.e. In neither outer norinner integuments any traces of vascular tissue appear s.The nner integument (11) consists like the 0 1 of 2 celllayers: the ou ter epidermis ox . ) and the inner epider-mis (i-e.). As for the 0 1 he apex of the I1 contains anarea of PAS+ cell wall remnants from degeneratingcells. The plastids of 11 contain no or only very fewstarch grains in i.e. All cell walls ar e PA S+ and early inthe maturation process they appear curved. Plasmodes-mata are found especially in the anticlinal walls.Th e two epidermal layers of th e 11 are very differentwith the i.e. being by far the most conspicuous cell layerof beet ovules containing cells differing from all othercell types. In the i.e. an unusual vacuole system devel-ops. During growth and maturation of the ovule (Figs11-14), a system of small perinuclear vacuoles contain-ing dense material increases steadily. At maturity thisvacuolar system fills up a large part of each cell anddense material has accumulated in each vacuole (Figs13, 14 . At this stage the vacuoles ar e typically confinedto t he inner part of the cells with the large nucleuscomprising the outer part (Fig. 13). The cytoplasm iswell preserved with many mitochondria and a few plas-tids and dictyosomes (e.g., Fig. 13). The nucleus ap-504

pears prom inent with only little condensed heterochro-matin. A fter fertilization an d in senescing non-fertilizedovules an enlargement of the cells in the i.e . takes placeand the cytoplasm appears more diluted (Fig. 14). Atthis stage some of the peculiar vacuoles are found alsoin the outer part of the cells (Fig. 14). In the lightmicroscope the vacuole co ntent ap pears strongly refrac-tive and stains green with TBO and brownish/yellowwith PAS/ABB which indicates the presence of tanninsandlor polyphenols. Compared to cells of the 01 the0.e. of the 11 consists of strongly vacuolated cells withonly a few m itochondria in the thin parietal cytoplasmiclayer. Shortly after fertilization (i.e. at early embryodevelopment) this cytoplasm disappears followed by acomplete collapse of the 0.e.The micropyle itself is formed by the inner integument(Figs 1, 3 , and the outer integument is clearly seenaround th e basis of the I (Figs 3, 4). Often the micro-pyle entrance appears to be covered by a thin sheet orhymen (Fig. 4). Inside the micropyle a PAS+ substance(cf. Bruun Olesen 1988,1989) is often seen and mostprobably corresponds to fibrillar material observed byTEM (Fig. 5).The characteristic appearance of the i.e. of the I asseen in Fig. 2 changes exactly where the micropylebegins. He re, cells of this layer become less vacuolated,bigger in size, and loose their dense vacuolar contents.This transition is clearly seen in a tran sverse section justtouching the nucellar apex (Fig. 7) whereas at the levelof the egg cell (Fig. 2) the charac teristic vacuolar systemcontaining tannins is still fully developed.

uticles are prominent features of both surfaces of 1and I as well as of the surface of the nucellus (F igs 5-8).In this way the whole surface of the micropylar channelis covered by cuticles (B ruu n Olesen 1989 and Figs 5,7). Although all the cuticles stain positively with Aura-min 0 he cuticle covering the nucellar surface is by farthe most conspicuous (Fig. 9).DiscussionTh e physiology of ovules du ring formation of the femalegametophyte, embryogenesis and seed developmentforms one of the big enigmas of plant developmentalbiology especially when comp ared with the wellknown processes of seed germination. Still, the generalideas on ovule function are mainly derived from ana-tomical and histochemical data. The source of energyfor embryo germination is well known as accumulatednutrients in the endosperm or parts of the emb ryo itselfbut typically very little is known about the nutritionalaspects (source, pathways etc.) of embryo differentia-tion. In sugar beet t he role of the endospe rm in nutrientaccumulation has been taken over by a specialized nu-cellus tissue, the perisperm (Artschwager 1927).As for Quercus (Mogensen 1973) and Spinuciu

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(Wilms 1980) the ou ter integument in the m ature ovuleof sugar beet has a high concentration of stored nutri- ( i .e .) during seed developm ent but nei ther H eel (1925)nor Artschwager (1927) ment ioned the conspicuousents in the form of accumulated s tarch. In their earlyanatomica l studies neither Heel (1925) nor Artschwage r(1927) paid much at tent ion to the s t ructure of the in-teguments and the micropyle of sugar beet ovules.Artschwager (1927) observed starch in the 0 1 of sugarbeet but did not confirm the disappearance of starchafter fertil ization. The decrease in starch after fertil-ization in sugar beet as well as in Q u e r c w (Mogensen1973) and Spinacia (Wilms 1980) may indicate that thestarch is used as nutrient supply for the developingembryo. Contrary to what was observed in Quercus(Mogensen 1973), there seems in sugar beet to be noindication that the de crease of starch should begin at th echalazal end and cont inue progressively towards theapex. H ere, in fact , the decrease of starch is first seen inthe micropylar part . Nonetheless , th e pathway of nutri-ents in the integuments of the sugar beet ovule must befrom the 01 o the chalazal part of the integument andfrom here , crossing the window between the integu-men ts , to the chalazal part of the nucel lus and from hereone way or ano ther to the e mbryo sac or t he embryo .similar pathway has been suggested for Quercus (Mo -gensen 1973) and Spinacia (Wilms 1980) ovules. Thepresence of heavy cut icles around the integuments an dthe nucellus in sugar beet (Heel 1925; Artschwager1927; Br uun Ole sen 1989) supp orts this view, becauseof the ability of cuticles to function as impermeablebarriers. In contrast to Beta, Quercus (Mogensen 1973)and Spinacia (Wilms 1980), the 01 of Agave (TiltonMoge nsen 1979) only store s few nutrients.The endothel ium or integumentary tapetum of t heplant ovule normally originates from th e i.e . of I (KapilTiwari 1978; Tilton Lersten 1981). Th e early differ-entiation of t he i.e. of t he I in sugar beet ovules in-dicates that this layer could be considered an endo-thel ium. O n the othe r hand i t is generally bel ieved thatonly weakly crassinucellate or tenuinucellate ovuleshave an endothelium and tha t this normally has a nutri-tional function (Kapil Tiwari 1978; Boum an 1984).Furthermore in sugar beet there is only lit t le starch orl ipid accumulation in the i .e. of t he I but a high conten tof tannins which in fact are very difficult to metabolize.Therefore, as also suggested for the fringe-layer i n cot-ton (R yser et al. 1988). this layer in sugar beet d oes notrepresent a true endothelium but might well have un-known functions during ovule differentiation and seeddevelopment .Notwithstanding their widesprea d occurrence in planttissues (often in strategic locations), very lit t le inform a-tion excists as to the functions of tannin and polyphenolinclusions. Since these m olecules often a re dep osited incells associated with d efense reactions against preda torsand fungal and bacterial pathogens, they are normallyconsidered as part of protection barriers (Swain 1979).I n sugar beet ovules Artschwager (1927) showed thattannins are deposited in both the 0 1 0 .e . ) and the I

early deposit of tannins as showed in the present work.Ident ical s t ructures are found in Beta rnaritirna ovules(Bruun, unpubl.) which rules out the possibility thattannin ac cumulation in this cell layer might be related tothe intensive inbreeding of sugar beet. In Pelargoniurn xhortorurn a conspicuous tannin deposi t ion also takesplace in the i .e. of the I dur ing embryo sac formation(Tsai et al . 1973), but n o comme nts were made o n i t spotential functions.If the early devel opm ent of tannin cells in the i.e. oft he I1 should only function as a defense barrier againstpathogens and predator s , i t s deep local izat ion inside theovule is puzzling. For opt imal protect ion one shouldexpect such cells to de velop in the 0 1 which also occursin several species (Tilton Ler sten 1981; Bosewink elBouman 1984).Being epidermal by nature, the tannin layer of the Idisplays obvious similarities to epidermal tanniferousinclusions fou nd in many xero-halophytic species. Her e,the tannin inclusions are considered to function in pro-tect ion against both pathogens as wel l as damages tounderlying t issues from strong exposure to UV-l ight .Being a halophytic or at least salt tolerant species, thetannin layer in the inner integument of sugar beetovules might also function in the accumulation of highamounts of sal t taken u p by the plant nd thus protect-ing the vital gynogenic tissue inside the ovule fromexcess salt stress.Taken together, i t seems rather plausible that thespecial tannin layer of t he I in sugar beet may haveseveral, largely unknown functions and that i ts mainfunction may well change from early stages of ovuledifferentiation to later stages of emb ryo and see d devel-opm ent where i t takes part in the seed coat format ion.Marked changes in funct ion are known from ot her s t ra-tegically positioned and tannin-contaning cell layerssuch as the end oderm is in roots an d bundle sheaths inleaves of certain grasses (cf. Bo che r Ole sen 1978).From the present s tudy there ar e no indicat ions thattannin cells of t he I should have anything to d o withnei ther the presence of PAS+ substances in the micro-pyle nor the precise guidance of pollen tubes towardsthe egg app ara tus (Br uun Ole sen 1988, 1989). It isuncertain wh ether the thin sheet or hymen covering themicropyle entrance is re l a t ed to the PAS+ substance,represents a novel s t ructure, or simply is a fixationartifact. However, Tilton (1980) observed a similars t ructure covering the exostome of Ornithogalurnovules and suggested that a function could b e in keepingthe putative chemotropic agents inside the micropyle.This might well be the case in sugar beet alsoAcknowledgements This work was supported partly by A SDe Danske Sukkerfabrikker and partly by the Danish Agricul-tural Veterinary Resear ch Counsil (grant no . 13-3965). Dr P.Steen and H . Olsen of Maribo Seed kindly provided plantmaterial. The authors thank H. Fasting for valuable discussions

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on staining of tannins, Ruth B. Jakobsen and Lis M. Frederik-sen for excellent technical assistance, and H . E. Jensen and G.Vestergaard-Hansen for photographic work.

ReferencesArtschwag er, E . 1927. Developm ent of flowers and seed in thesugar beet. J. Agr. Res. 34: 1-25.Bor, J. Bouma n, F. 1974. Developm ent of ovule and in-teguments in Euphorbia milii and Codiaeu m variegatum.Phytomorphology 24: 280-296.Bouma n, F. 1984. Th e ovule. In: Johri , B. M. (ed.), Em-bryology of angiosperms. Springer, Berlin, Heidelberg, pp.123-157.Bosewinkel, F. D. 1980. Developm ent of ovule and testa ofL in u m usitatissimum L. Acta Bot. Neerl. 29: 17-32.Boum an, F. 1984. Th e seed: S tructure. - I n : Johri , B. M.(ed.), Embryology of angiosperms. Springer, Berlin Hei-delberg, pp. 567-610.Bruun, L. 1987. The m ature embryo sac of the sugar beet, Betavulgaris: A structural investigation. No rd. J. Bot. 7: 543-511.Oles en. P. 1988. A structu ral investigation of the ovule in

sugar beet, Beta vulgaris: The degenerated synergid and themicropylar nucellus. In: Cresti, M. e t al. (eds ), Sexualreproduction in higher plants. Springer, Berlin, p. 462.Ole sen, P. 1989. A structural investigation of the ovule insugar beet, Beta vulgaris: Th e micropylar nucellus. No rd.Bocher. T. W. Ole sen , P. 1978. Structural and ecophysiolog-ical pattern in the xero-halophytic C, grass, Sporobolusrigens (Tr.) Dew. Kgl. Dan. Vid. Selsk. Biol. Skr. 22,3:Feder, N . OBrien, T. P. 1968. Plant microtec hnique: Som eprinciples and new me thod s. Am er. J. Bot. 55: 123-142.Fischer, D . B. 1968. Protein staining of ribboned epo n sectionsfor light microsc opy. Histoche mie 16: 92-96.Heel, Jr ., J. P. D. van. 1925. Onderzoekingen over de ontwik-keling van den z aadkno p en van het zaad bij Beta vulgarisL. Thesis, Naarden. (Unpubl.).Hosemans. D. Bossoutrot, D. 1986. I n vitro culture ofunpollinated beet Beta vulgaris L.) ovules of male sterileand male fertile plants an d induction of haploid plants. - I n :Chapmann, G. P. et al. (eds), Experimental manipulationof ovule tissues, Longman Inc., New York, pp. 79-87.

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Kapil, R. N. Tiwari, S. C. 1978. The integumentary tape-turn. Bot. Rev . 44: 457-490.Mogensen, H. L. 1973. Some histochemical, ultrastructural,and nutritional aspects of the ovule of Quercus gambelii.Amer. J. Bot. 60: 48-54.Nielsen, J. E. Ole sen , P. 1988. Isolation of sperm cells fromtrinucleate pollen of sugar beet Beta vulgaris). In:Wilms, H . J. Keijzer, C. J. (eds), Plant sperm cell astools for biotechnology. Pudoc, Wageningen, pp. 111-123.Ryser, U., Schorderet , M., Jauch, U. Meier, H . 1988.Ultrastructure of the Fringe-layer, the innermost epider-mis of cotton seed coats. Protoplasma 147: 81-90.Shen, J. , Li, H ., Han, X. Wang, P. 1986. Observations onfertilization in suga r beet. Acta Bo t. Sin. 28: 251-255.Swain, T. S. 1979. Tannins an d lignins. n: Rosenthal , G . A .Janzen, D. H. (eds), Herbivores, their interaction withsecondary plant metabolites. Academic Press, New York,pp. 657-675.Tilton, V. R. 1980. The nucellar epidermis and micropyle ofOrnithogalum caudatum (Liliaceae) with a review of thesestruc tures in othe r taxa. Can . J. Bot. 58: 1872-1884.Lenten, N. R. 1981. Ovule development in Ornithoga-lum caudatum (Liliaceae) with a review of selected paperson angiosperm reproduction. I. Integuments, funiculus,and vascular tissue. New Phytol. 88: 439457 .Mogensen, H. L. 1979. Ultrastructural aspects of theovule of Agave parryi before fertilization. Phytomorph ol-ogy 29: 338-350.Tsai, A H., Harney, P. M. Peterson, R. L. 1973. Mega-sporogenesis and megagametogenesis in Pelargonium xhortorum. Can. J. Bot. 51: 607-612.Wilms, H. J. 1980. Development and composition of the spin-ach ovule. Acta Bot. Nearl. 29: 243-260.

AbbreviationsM = micropyle0 1 = outer integumentI1 = inner integumentES = embryo sacF = funiculusMNu = micropylar nucellusSG = starch grainNu = nucellus

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