[Advances in Cellular and Molecular Biology of Plants] Cellular and Molecular Biology of Plant Seed Development Volume 4 || Endosperm Structure and Development

3. Endosperm Structure and Development

DARLEEN A. DEMASONBotany and Plant Sciences , University ofCalifornia , Riverside, CA 92521, USA

ABSTRACT. Endosperm is a seed storage tissue formed within the angiosperm embryo sacfrom a second fertilization of the central cell, Generally, endosperm cells are triploid, rich incellular reserves, and are compactly arranged without intercellular spaces. Reserves are storedin the form of carbohydrates, protein , and lipids, although specific ratios of these compo-nents vary depending on the species. Three general patterns of endosperm development arerecognized: nuclear, cellular, and helobial. In nuclear, the primary endosperm cell enlarges byexpan sion of the central vacuole and many nuclei are formed in the peripheral cytoplasm byfree nuclear divisions. The mechanism of cell wall formation, especially that of the very firstanticlinal (radial) walls, has been controversial for about 90 years. Replication of nuclear DNAwithout subsequent mitosis, or endopolyploidization, has been described in the endosperm ofmany species . The function of DNA amplification is still unresolved. Stored reserves accu-mulate in endosperrns in specific spatial patterns . Most of the endosperm mutations in cropplants, such as maize , originally selected from morphological characteristics have been shownto be involved in various aspects of storage product accumulation , such as starch or protein, orboth. The morphogenetic potential of endosperm in flowering plants is extraordinary and willcontinue to be important in biotechnology. Although some progress has been made to identifyspecific interactions between endosperm and embryos during seed development, it is clearlyquite meager.

Abbreviations: ABA, abscisic acid; DAP, days after pollination; ER, endoplasmic reticulum;kD, kilodaltons; PCD, programmed cell death ; RER, rough endoplasmic reticulum.

Introduction

One of the most critical events during a plant's life cycle is survival at ger-mination. Many types of structural and functional specializations occur inseeds, which increase the probability of survival. An important specializationis the storage of seed reserves. Endosperm is a seed storage tissue formedwithin the embryo sac from a second fertilization within the central cell(Esau, 1977). The fertilization event involves three, usually haploid, nuclei ,two from the central cell and one from the sperm cell. Endosperm is a tis-sue unique to flowering plants but it is not always present in all angiospermseeds. As with other characters, variability in seed structure and developmentis enormous in flowering plants. The seeds of many species have endospermat early stages of seed development, but do not possess endosperm at seed

B.A. Larkins and IX. Vasil (eds .), Cellular and Molecular BiologJ' of Plant Seed Development , 73- 115.© 1997 Kluwer Academic Publishers.

74 Darleen A. DeMason

maturity as it is used up during seed development. Seeds of these species arecalled exalbuminous or nonendospermic. In these seeds, stored reserves arelocated in some portion of the embryo, usually the cotyledons. In addition,some species store reserves in the nucellus, a maternal diploid tissue calledperisperm. There have been many review papers and book chapters writtenon endosperm (Brink and Cooper, 1947; Bhatnagar and Sawhney, 1981; Vija-yaraghavan and Prabhakar, 1984; Lopes and Larkins, 1993). I would like toconcentrate on some of the newer studie s in the areas of structure and devel-opment, discuss the biological capabilities of endosperm cells , and finally,discuss the interaction of endosperm and embryo during seed development.

A. Endo sperm Structure

Endosperm is one of the simplest tissues in flowering plants since it consistsof only one or two cell types and within a species these cells are often veryuniform in structure (Figures 1-3). Generally, endosperm cells are rich in cel-lular reserves and are compactly arranged without intercellular spaces. Theamount of endosperm varies tremendously in mature seeds, ranging frombeing nonexistent (many legumes), consisting of I or 2 peripheral layerstCucurbita, lettuce), to consisting of three quarters or more of the seed vol-ume (cereals, lilies, palms, most primitive dicots) (Martin, 1946). No cleardistinction exists between endospermous seeds and nonendospermous seedsbased on structural criteria (Boesewinkel and Bouman, 1995).Endosperm can consist of uniform , living reserve cells (lilies, most palms,

castor bean), of reserve cells which differ slightly depending on locationwithin the seed (coconut), or of two distinctly different cell types , reservecells and aleurone cells (cereals, Cyperaceae, Bromeliaceae, Ponterderiaceae,and some endospermic legumes such as fenugreek and guar) (Bhatnagar andSawhney, 1981; DeMason and Chandra Sekhar, 1990; Bewley and Black,1994; DeMason, 1994). The aleurone cells surround the reserve cells andconsist of one layer (maize, rice, wheat) or up to four layers (barley). Thesize and shape of the aleurone cells differ depending on their location in thegrain (Bhatnagar and Sawhney, 1981).Typically aleurone cells are thick-walled, living, nucleated cells with abun-

dant protein bodies (called aleurone grains) surrounded by lipid bodies (Fig-ure 3B). The thickened, primary walls of cereal aleurone cells have abundantplasmodesmata between cells and are rich in arabinoxylans and glucans withsmall amounts of glucomannans and cellulo se (Fincher, 1989). The wallshave two ultrastructurally distinct layers , a thin, inner layer and a thicker,outer layer. An important type of modified aleurone cell is the ' transfer aleu-rone cell' which exhibits cell wall ingrowths like other transfer cells. Thesetransfer aleurone cells have been described in many species of grasses asoccurring near the placental vascular bundle , including maize (Kiesselbachand Walker, 1952), rice (Bechtel and Pomeranz, 1977), and wheat (Wang et

Endosperm Structure and Development 75

Fig. I . Light microscope image s of mature endosperm in various species. (A) washingtoniaft li fera, stained with toluid ine blue (from DeMason, 1986). (B) Ricinus communis, stained withbromophenol blue and acidic toluidine blue. (C) Zea mays, stained with periodic acid-Schiffand anil ine blue black (from Harris and DeMason. 1989). AL - aleurone layer, C - proteincrystalloid, IW - inner wall, LB - lipid body, ML - middle lamella, N - nucleus, PH - proteinbody, SE - starchy endosperm, W - wall. Sca le bars = 0.03 mm.

76 Dar/een A. DeMason

Fig. 2. Light and scanning electron microscope images of Zea mays and Washingtoniafiliferaendosperm. (A) Starch granules stained with periodic acid-Schiff 's reagent from Z.mays. Scalebar = 0.02 mm. (B) Starch granules from Z. mays viewed under polarization optics. Scale bar= 0.02 mm. (C) Scanning electron micrograph of W. filifera endosperm. Scale bar = 0.2 rnm.(D) Scanning electron micrograph of cytoplasm and protein bodies in W. filifera after lipidextraction. Scale bar = 2 pm. PB - protein body, PF - primary pit field, W - wall.


Fig. 3. Protein and lipid bodies from mature endosperm in various species. (A) Differentialinterference contrast image of Ricinus communis . Scale bar =0.02 mm. (B) Electron micro-graph of aleurone from Zea mays. Scale bar = I /lm. (C) Electron micrograph of Phoenixdactylifera. Scale bar = 111m. C - protein crystalloid , G - phytic acid globoid , IW - innerwall, LB - lipid body, N - nucleus, OW - outer wall, PB - protein body.


aI., 1995b) . The functional significance of these specialized aleurone cellswill be discussed later under endosperm/embryo interactions.Reserve cells, whether they are accompanied by an aleurone layer or not,

function primarily in storage. Reserves are stored in the form of carbohy-drates, protein, and lipids , although specific ratios of these components varydepending on the specie s. Reserve cells may be living or non-living (Bewleyand Black, 1994). Living reserve cells contain intact membranes, nuclei anda small amount of cytoplasm with few organelles (mitochondria, plastids,etc.) or endomembranes (ER, polysomes, dictyosomes, etc .) and maintainlow levels of respiration. The unusual ultrastructural features and low respi-ration levels result from the low water content in resting seeds. Non-livingreserve cells lack nuclei and the cytoplasm becomes completely occludedat maturity (cereals and some legume species such as fenugreek). A majorsource of variation in reserve cells of flowering plants with endospennic seedsis the type and forms of carbohydrate reserve stored: starch in the form ofstarch grains or cell wall storage polysaccharides in the form of thickened cellwalls. Reserve cells may be essentially identical in structure throughout theendosperrn, or they may differ slightly in size , shape and reserve structuralfeatures depending on their position within the seed. Positional variation isprobably the result of developmental events. The following paragraphs aredevoted to the structure of stored reserves in reserve cells.Starch is a macromolecule consisting of two structurally distinct glucans:

amylose and amylopectin, organized into single or aggregated intracellu-lar granules (Figure 2A). Starch granules vary to such an extent in shape(spherical, lenticular or polygonal), size (3-100 [tm), and surface and inter-nal features as to be taxonomically distinct (Hood and Liboff, 1983) . Starchis semicrystalline and can rotate the plane of polarized light, and thereforeexhibits birefringence (Sivak and Preiss, 1995) (Figure 2B) . Starch grainshave internal concentric 'growth rings ' around a hilum. The relative amountof amylose and amylopectin can also radically affect the form of starch gran-ules.Starch is absent from those species of angiosperms which store cell

wall storage polysaccharides as the carbohydrate reserve. Cell wall stor-age polysaccharides may be mannans, galactomannans, glucomannans, orxyloglucans in different species (Aspinall, 1983; Bewley and Black, 1994;Meier and Reid, 1982; Boesewinkel and Bouman, 1984; DeMason, 1994) .Thebest studied of these polysaccharides are the mannans and galactomannanswhich occur in the endospenns ofpalms and some legumes (carob, fenugreek,guar and honey locust). Both mannans and galactomannans have a (1-4),8-D-mannose backbone with varying degrees of (1-6)a-D-galactose side chainsubstitutions (Meier, 1958; Meier and Reid, 1982). Mannans are very hard,insoluble in aqueous solutions, and have fewer galactose side chains, whilegalactomannans are soft, hydrophilic, and contain abundant galactose sidechains (Meier and Reid, 1982; Aspinall, 1983) . The best studied mannan-

Endosp erm Structure and Development 79

rich endosperm wall s are from date palm, which consist of 92% mannanand 8% cellulose (Meier, 1958 ). It has been hypothesized that a portion ofthese essenti ally linear mannan molecules occurs in microfibril s together withcellulose in the wall (Aspina ll, 1983). The galactose:mannose ratios of stor-age galactomannans differ especially among legume species and affect seedhardness and water holding capacities. Galactomannans are commerciallyimport ant as thickening agents in many foods. Glucomannan s, which differfrom mannans in that a large proportion of backbone mannose moieties arereplaced by glucose, are stored in endosperms of species in the Liliaceaeand Iridaceae (Meier and Reid , 1982). Xyloglucans consist of a (l-4),8-0 -glucan to which are attached short side-chains of xylose and galactose. Thispolysaccharide is known as 'amyloid ' and is found in the endosperm of manylegumes and other dicot families (Meier and Reid , 1982). Very little work hasbeen done on glucomannans and xyloglucans in comparison to mannans andgalactomannans.Cell wall storage poly saccharides occur in distinctly thickened cell walls

(Figures lA, 2C). These range from being only slightly thickened (coconut)to being so extensively thickened that they occupy 65% of the endospermvolume (Chandra Sekhar and OeMason, 1988). In fenugreek , the developingwall completely occludes the cells at seed maturity (Meier and Reid, 1977).The walls might be uniformly thickened (persimmon) or with distinct pit fields(date palm andWashingtoniafilifera ) (OeMason et al., 1983; OeMason, 1986;OeMason and Chandra Sekh ar, 1990) (Figures IA, 2C). The cell wall consistsof three layers which differ in their staining and ultrastructural characteristics;middle lamella, thickened outer wall and thin inner wall (Figures IA, 3C).Cell wall hydro lases have been localized to the inner wall layer (OeMason etal., 1989; Chandra Sekhar and OeMason, 1990; OeMason et al., 1992).Seed oils, or lipid s, are an important commercial commodity.Most oil crops

grown in temperate zones accumulate lipids in cotyledonary tissue (soybean,maize , sunflower, canola) but two members of the palm family rank second(oil palm ) and sixth (coconut) in world vegetabl e oil production and the oilis accumulated in endosperm in these species. Lipids, or triacylglycerol s,are a major storage component of all reserve cells and they accumulate inintracellular, spherical organelle s called lipid bodies (oil bodie s, oleosomes,or spherosomes) which range in size from 0.2 to 2.0 {tm (Herman, 1995)(Figures 20, 3B-C). The lipid bodies consist of a core of triacylglycerols sur-round ed by a monolayer of phospholipids in which are embedded specializedproteins called oleosins (Huang, 1994) (Figure 4). Oleosins are envisionedto be monomeric, tack-shaped molecules with negativel y charged heads cov-ering the phospholipid surface layer and hydrophobic tails penetrating thephospholipid monolayer and internal matrix . Oleosins are restricted to lipidbody membranes in tissues that undergo developmentally regulated dehy-dration and rehydration , as occurs in seed tissues. Therefore, it has beensuggested that their major function is to stabilize lipid bodies during these


(bl

ly~?:~i i this level 0 ••0

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Fig . 4. A model of the maize lipid body structure. The lipid body is bounded by a halfunit membrane which consists of oleosin molecules (in white) embedded in a single layer ofphospholipids (in black) . (a) A whole lipid body with a quarter cut open to show interior. (b) Aportion of half unit membrane showing two oleos in molecules and two phospholipid molecules.(c) Two tangential views of half unit membrane consisting of 17 phospholipid molecules and4 oleosin molecules from surface of oleosin head (top) and surface of phospholipid head(bottom) . (From Huang, 1994.)

processes (Herman, 1995). These lipid bodies essentially 'fill' the cytoplasmof endosperm reserve cells or they distinctly line the protein bodies and plas-malemma which may result from the charged nature of the outer surface ofthe oleosins .The final major reserve substance, seed storage protein, accumulates in

organelles called protein bodies which have a single unit delimiting mem-brane. There is extensive and comprehensive literature on characteristics,regulation, and genetics of seed storage proteins (Miftin and Shewry, 1979;Shotwell and Larkins, 1989; Lopes and Larkins, 1993) which is covered inother chapters of this volume. The structural characteristics of protein bodiesare highly variable and protein bodies contain other proteins (i.e. enzymes,lectins, etc.) in addition to storage proteins. Protein bodies in nonliving reservecells may lose their membrane integrity allowing dispersal of the containedproteins (cereals). Protein bodies range in size from 0.1 to 25 f-J,m and in thesimplest condition contain a homogeneous matrix of protein. Many proteinbodies contain various types of inclusions, including phytic acid-containinggloboids, proteinaceous crystalloids, calcium oxalate-containing crystals orprotein-carbohydrate bodies (Figures 1A-B, 3). Different classes of proteinbodies may occur in the same cell or in different cells within the endosperm.Position dependent structural variation in protein bodies is known in cerealendosperm (Pomeranz and Bechtel, 1978; Lending et aI., 1988; Lending and


Larkins, 1989) and in the cotyledons of some legumes (Citharel and Citharel,1987; Asghar and DeMason , 1990, 1992), but it is especially extreme incoconut endosperm (DeMason and Chandra Sekhar, 1990). Nonhomogeneousdistribution of matrix protein or the presence of distinct protein crystalloidinclu sions within protein bodi es is thought to be indicative of distinct distri-bution of different protein components. For example, spec ific globulins havebeen locali zed to the crystalloid inclu sions (coconut and castor bean) (Youleand Huang , 1976 , 1981; Gifford et al., 1982; DeMason and Chandra Sekhar,1990), distinct segregation of globulins and prolamins occurs within the pro-tein bodies of oat (Lending et aI., 1989), and segregation of different zeinswithin the protein bodies of com endosperm (Lending et al., 1988; Lendingand Larkins, 1989).The most common type ofmineral inclusion within protein bodie s is phytin

in globoids. Phytin, phytate or phytic acid, is a salt of myoinositol hexaphos-phate that binds various cations (K, Mg, Ca, Ba, Fe, Mn) and is considered tobe a form of reserve (Lott et aI., 1995). Phytin is crystall ine and globoids oftenshatter or fall out of sectioned material (Figure 3). The globoids vary in sizeand frequency in protein bodi es of individual cell s and in different specie s(Lott, 1981 , 1982). An interesting observation is that phytin only accumulatesin seed tissues that remain living upon maturation, therefore it is not presentin cereal starchy endosperm but is present in the aleurone cells (Lott et aI.,1995).Endosperm hau storia of various manifestation s are common in angio sperm

seeds (Bhatnagar and Sawhney, 1981). As the name implies these spec ializedce lls are thought to function in absorption of materials from outer tissues inthe ovule. They are commonly present in either the micropylar or chalzal endof the seed and they consist of highly branched , enlarged cells which grow outinto the integuments adjac ent to vascular bundles (Boesewinkel and Bouman,1995). These cell s often have tran sfer cell-like walls , highly polyploid nuclei ,and cytoplasm rich in RNA and protein (Bhatnagar and Sawhney, 1981).

B. Endosperm Dev elopm ent

General Features ofEndosperm DevelopmentSince endosperm is such a simple tissue and its origin can be pin-pointedexactly, it is a convenient tissue for developmental studies. Endo sperm devel-opment starts when a sperm nucleus fuses with the two polar nuclei of thecentral cell. The resulting cell is called the primary endosperm cell. Threegeneral patterns of endosperm development are then recognized: nuclear,cellular, and helobial. In nucl ear, the primary endosperm cell enlarges byexpansion of the central vacuole and many nuclei are formed in the periph-eral cytoplasm by free nucl ear divisions. Cell wall formation mayor maynot occur later in development. Thi s type is the most commonly studied typeand occurs in many dicotyledonous and monocotyledonous species. In eel-


lular endosperm development, cell wall formation accompanies each mitoticdivision beginning with the first mitosis of the primary endosperm cell. Thisendosperm type occurs in some closely related dicot families (Acanthaceae,Lobeliaceae, Scrophulariaceae, etc.) (Bhatnagar and Sawhney, 1981) . In helo-bial endosperm development, the primary endosperm cell divides into twounequal cells, the larger of which (at the chalazal end) usually develops ina noncellular manner, whereas the smaller micropylar cell has various pat-terns of development depending on the taxon. This type of developmentoccurs only in monocotyledonous species. Extensive coverage of helobialendosperm development and variations in different monocotyledonous plantsis presented in a review by Vijayaraghavan and Prabhakar (1984). Haeman-thus endosperm has been a favorite subject ofendosperm development studiesand has helobial development (Hepler and Jackson, 1968; Newcomb, 1978;Bajer and Mole-Bajer, 1986; Smirnova et aI., 1992) .Most of the work on endosperm development has been done with species

with the nuclear pattern of development. The general scheme of developmentis quite similar in all species (Figure 5). Primary endosperm cell expansionand syncytial mitosis without cellularization occurs first. Cellularization anddifferentiation are the next important events. The mechanism of cell wallformation, especially that of the very first anticlinal (radial) walls has beencontroversial for about 90 years . New methods of microtubule visualizationhave been important in understanding the phenomenon.During the end of thisperiod, average DNA levels per nucleus increase due to endopolyploidization.This phenomenon is fairly universal but its function is not clear. Reserveaccumulation occurs next as a result of the appropriate synthetic enzymeactivities. Finally, metabolic slowdown and water loss occur before sheddinginmost species of flowering plants which sets the stage for future germination.Recent studies of some of these events are discussed below.An important method of understanding normal developmental events in

any biological system is to identify, isolate and characterize mutations thatdisrupt specific events. This approach does have its limitations. One mustrely on mutations which are known or can be induced and those that are notlethal. Also, mutations are not available to study many important aspects ofendosperm development. A major foray into this realm of investigation is thatof Neuffer and Sheridan (1980) in which they identified a large number ofEMS-induced defective kernel (dek) mutations in maize. Some of these havebeen and, hopefully, will continue to be important in understanding variousaspects of seed development in maize. Similarly, Bosnes et al. (1987) haveidentified a number of Na-azide induced shrunken endosperm (sex) mutationsin barley. Under each of the categories below I include information availablefrom mutant analyses.


Maize Endosperm Development

s Ie D E Reserve Deposition DessicationMaturation

o 10 20 30

DAP

40 50 60

Fig. 5. Stages of starchy maize endosperm development plotted as a function of days afterpollination (DAP). Divisions between stages are rough since cellular development is notsynchronous but occurs in waves across the kernel. C - cellularization, 0 - differentiation andmitosis, E - endopolyploidization, S - syncytial phase.

Primary Endosperm Cell Expan sion and Syncytial DevelopmentThe initial expansion of the primary endosperm cell is mainly the result ofenlargement of the central vacuole which confines the nuclei to a thin parietallayer of cytoplasm (Figure 7A). The first few rounds of mitoses have beenobserved to be more or less synchronous in several cereals (Fineran et al.,1982; Gustafson and Lukaszewski , 1985; Olsen et al., 1992; Brown et al.,1994) . Studies ofmutant cell sectors in maize by McClintock (1978) revealedthat the first division of the primary endosperm nucleus produces sister nucleiwhich define the left/right halves of the kernel and the second pair of divisionsis perpendicular to the first, defining the dorsal/proximal pole s of the kernel.Some developmental mutants in barley have been described which illustratethat in this species as well, a similar pattern of nuclear lineages occurs (Olsenet al. , 1992) . Only a single half (right or left) develops normally in the N34mutant of barley (Bosnes et al. , 1992) and the dorsal prismatic starchy cellsfail to develop in the B13 mutant (Bo snes et al., 1987).These initial mitotic divisions during the early stages of primary endosperm

cell expansion are associated with typical phragmoplast arrangements ofmicrotubules between sister nuclei although no formation of a cell plate results(van Lammeren, 1988; XuHan and van Lammeren, 1993, 1994; Brown et al.,1994).Within the parietal cytoplasm at an early stage of free nuclear growth inbean, microtubules run throughout the cytoplasm forming a reticulate network(XuHan and van Lammeren, 1994) (Figure 6). At later stages in barley,wheat ,bean , and Ranunculus sceleratus, the free nuclei become distinctl y, regularlyspaced in the thin layer of cytoplasm due to radially-oriented microtubule s


Fig . 6. Barley syncytial endosperm microtubules stained by indirect immunofluorescence.(Nuclei are unstained and appear black.) (A) Phragmoplasts connecting sister nuclei and radialmicrotubles connecting non-sister nuclei. Os indicate positions of nuclei . (B) Nuclear-based,radially-oriented microtubule system (RMS) organize the cytoplasm and maintain the nucleiin an evenly spaced pattern. (C) Details of the interaction of (RMS) of two adjacent nuclei . (D)Unstained zones at the perimeters of RMSs mark the future sites of the first cell walls. Scalebar = 10 J1m. (From Brown et aI., 1994.)


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cFig. 7. Early phases of date endosperm development. (A) Peripheral cytoplasm and nucleiat 7 wk. (B) Early cell wall formation at 7 wk. (C) Centripetal cellularization at 9 wks. (D)Beginnings of wall thickening in central endosperm at 9 wks. CE - cellular endosperm, CV -central vacuole , EN - endosperm nuclei , II - inner integument, 01 - outer integument, PC -peripheral cytoplasm, W - wall. Scale bar = 0.05 mm. (From DeMason, Chandra Sekhar andHarris, 1989.)

(van Lammeren, 1988; XuHan and van Lammeren, 1993, 1994; Brown etal., 1994) . The radially-arranged microtubules emanating from the nuclearenvelope determine the size and shape of the future cells in that position (vanLammeren, 1988, Brown et al., 1994).


Cellularization and DifferentiationMicrotubule arrangement has been shown to be important in cellularizationof the endosperm. At this point the various authors differ in their interpreta-tion of the formation of the initial anticlinal (radial) cell walls which formaround each nucleus dividing the parietal cytoplasm into open ended boxesor 'alveoli' which face the tonoplast (Figure 7B). Fineran et al. (1982), vanLammeren (1988), and XuHan and van Lammeren (1993; 1994) observed andphotographed normal phragmoplasts between sister nuclei which proceededon to form normal cell plates in the species studied (wheat, Ranuneulus see1-eratus , and bean). Morrison and O'Brien (1976) and Brown et al. (1994) didnot report the occurrence of any cell plates formed in this way between freesister nuclei in wheat and barley. Fineran et al. (1982) subsequently hypothe-sized that all the initial anticlinal walls were initiated in this normal process.Van Lammeren (1988) and XuHan and van Lammeren (1993, 1994) alsoobserved phragmoplast and cell plate formation between non-sister nucleiand hypothesized that many of the initial anticlinal walls in the outermostregion of the primary endosperm cell are formed by ' adventitious ' phragmo-plasts and resulting cell plates, associated with the microtubules radiating outfrom the nuclei. In bean they observed that these cell plates are initiated inan equatorial region between adjacent nuclei and grow out in all directions ina process typical of cell plates (XuHan and van Lammeren, 1994). They dis-tinguished three regions of growing wall : (I) growing edge, (2) accumulationregion, and (3) maturing region (XuHan and van Lammeren, 1993). Theseobservations demonstrate that the freely growing ends are similar to freelygrowing ends of cell plate expansion during cytokinesis in all other meris-tematic tissues in a flowering plant. In one direction the growing wall meetsthe outer, tangential wall of the primary endosperm cell, in two directions itmeets other growing walls and in the third direction (toward the tonoplast)it continues to grow centripetally as 'free ends.' These free ends which con-stitute the growing edge are associated with microtubules (van Lammeren,1988; XuHan and van Lammeren, 1993, 1994; Brown et aI., 1994). Otherauthors have hypothesized that all the initial anticlinal walls are formed by'adventitious' phragmoplasts (Morrison and O'Brien, 1976; Brown et aI.,1994). The reasons for the varied interpretations are not completely clear. Insome cases the authors have observed endosperm development in differentplant species, but Morrison and O'Brien (1976), Fineran et al. (1982) andvan Lammeren (1988) all made their observations on wheat. An interestingobservation made by several authors (Morrison and O'Brien, 1976; Brown etaI., 1994) is that these initial anticlinal walls are callose (131-3 glucan)-richwhich is atypical in the body of a flowering plant. Callose-rich cell walls arepresent only in phloem, pollen tubes, and microspores in angiosperms (Esau,1965, 1977).The alveoli are then sealed off by development of the final wall- the inner

tangential wall. Again there is controversy as to how this occurs. One group


Fig . 8. Later stages in date endospenn development of peripheral cells. (A) Cell enlargementand galactomannan depo sition in cell walls near periphery at 13 wks. (B) Protein body forma-tion in outermost cells at 17 wks. (C) Cell wall hardening and protein body maturation at 21wks. EC - elongating cells, 01 - outer integument, PB - protein body, W - wall, WT - wallthickening. Scale bar = 0.05 mm. (From DeMason, Chandra Sekhar and Harri s, 1989.)


of authors reports that branching and fusing of the freely growing ends ofthe anticlinal walls form the peripheral cellular layer (Morrison and O'Brien,1978). Others report that the nuclei in the open-ended alveoli then undergomitosis such that the resulting phragmoplast is oriented perpendicular to theprimary endosperm cell surface (Fineran et aI., 1982; Yeung and Cavey, 1988;van Lammeren, 1988; Dute and Peterson, 1992; XuHan and van Lammeren,1993; 1994; Brown et aI., 1994, 1996a, 1996b). A typical cell plate forms sub-sequently and the alveoli become sealed off forming a peripheral, uniseriatelayer of cells outside an inner layer of alveoli. The free ends of the anticlinalwalls continue to grow centripetally. This process of periclinal cell divisionscontinues until the primary endosperm cell is completely cellular. In somespecies this takes only two rounds of mitotic divisions associated with cellplate formation (wheat, barley) (Morrison and O'Brien, 1978; Fineran et aI.,1982; Brown et aI., 1994, 1996a), whereas considerably more rounds occurin larger seeds resulting in fairly regular radial files of cells, running fromthe periphery of the endosperm inward (Figures 7C-D, 8A-C). In barley,rice, and wheat the resulting thin-walled, vacuolate cells themselves undergoadditional cell divisions (Fineran et al ., 1982; van Lammeren, 1988; Brown etaI., 1994, 1996b). Enlargement of the developing seed accompanies this finalphase of histogenesis. In the hollow seed of coconut, cellularization ceasesbefore the 'liquid' endosperm is ' used up'.It is clear that the pattern of cellularization in the endosperm is basically

centripetal since the first cell walls are formed at the periphery and the last cellsto form walls are in the seed center. In general, differentiation and cessationof mitotic activity occurs in the opposite direction or centrifugally (Figures 7-8). In com, cytokinesis ceases in the seed center 12 days after pollination,while it occurs in the peripheral region for a much longer time (Knowles andPhillips, 1988). In large seeds these processes are not simultaneous across thelength of the seed either. In com, mitotic activity stops first in the basal regionof the kernel, then in the center, and finally the ces sation progresses from thekernel apex basipetally. InRicinus, the progression of cellularization proceedsfrom the micropylar end to the chalazal end and from the periphery towardsthe center of the seed (Greenwood and Bewley, 1982, 1985). Developingendosperm at these early stages is neither physiologically or molecularlyuniform since different cellular activities occur simultaneously in differentregions of the same developing seed.

Endopolyploidizaton and Ploidy Level ConstraintsReplication of DNA without subsequent mitosis, or endopolyploidization, hasbeen described in the endosperm of many species. It is a normal event in celldifferentiation of other tissues as well, e. g. , cotyledon storage parenchymaand tracheids and vessel elements in xylem and root cap cells. It is oftencorrelated with cell expansion and/or the initiation of stored reserve accu-mulation. In maize it occurs mainly between 10 and 16 DAP which is after


mitotic activity ends (Knowles and Phillips, 1988; Knowles et aI., 1992; Lurand Setter, 1993a, 1993b; Doehlert et aI., 1994). During this time the ploidylevel increases and a shift to increasing numbers of nuclei within the higherploidy levels occurs. This phenomenon does not occur uniformly throughoutthe endosperm in com, but occurs mainly in the central cells (Knowles etaI., 1992). The function of DNA amplification is still unresolved. Among thepossibilities are: (l) enhancement of macromolecule synthesis by providingmultiple copies of important genes; and (2) production of deoxynucleotidereserves to be utilized during germination. The latter hypothesis would notprovide an explanation for why endopolyploidization occurs in developingxylary elements and root cap cells although the former would because exten-sive amounts of cell wall macromolecules and fucose-rich polysaccharidesare synthesized in these cell types, respectively. The former possibility hasbeen supported by some authors (Tsai et aI, 1970), but has been discredited byothers (Doehlert et aI., 1994) who have compared the timing ofDNA increasewith transcript abundances or enzyme activities in maize endosperm. Theproblem with these experiments is that since only the central cells in maizeendosperm undergo DNA increase, the timing of transcript and enzyme peaksof only these cells should be used rather than those of the whole kernel to makea valid correlation. This hasn't been adequately controlled . However, the firsthypothesis would seem to be discounted by recent experiments done on peacotyledonary cells. Corke et al. (1990a) observed DNA content and storageprotein (vicilin) content in individual pea cotyledon cells using fluorescencemicroscopy and found no distinct correlation. They (Corke et aI., I990b)also used aphidicolin treatment to inhibit endoreduplication and found thatstorage protein accumulated in cells with lower average DNA content thantypical during normal development. Therefore, they concluded that storageprotein accumulation is concomitant with, but not dependent on, DNA repli-cation in the endosperm. Knowles et aI. (1992) looked at 35 defective kernel(dek) mutants of maize with relatively more normal embryo development andidentified a large number which displayed distinct effects on DNA endoredu-plication. Most lines had both reduced relative amount of DNA per nucleusand fewer nuclei at higher DNA levels. Lur and Setter (l993b) have shownthat several dek mutants have significantly lower IAA levels at 20 days afterpollination and that there is a consistent correspondence between the extentof endoreduplication and auxin level in the endosperm. Further, the timing ofendoreduplication in maize endosperm has been shown to coincide with theperiod of rapid cell growth, both of which may be a consequence of auxinlevel. The function of amplified DNA during endosperm development is yetto be determined conclusively. One potentially relevant observation is that inthe case of xylary element development, root cap cells, and endosperm devel-opment in cereals, the cells have short life spans. Possibly, normal constraintson nuclear size and functioning are not important (Bennett, 1973). Experi-ments with isolated protoplasts from central endosperm cells of specific dek


mutants compared to wild type lines might allow the functional significanceof auxin levels and DNA levels to be determined.It has long been known that crosses between many plants with differing

ploidy levels often leads to seed abortion due to failure of endosperm devel-opment (Brink and Cooper, 1947) . These observations have led to varioushypotheses regarding 'genome balance' between the maternal, endosperm,and embryo of developing seeds. Using the indeterminate gametophyte (ig)mutant of maize, Lin (1978, 1984) has provided some intriguing data withregard to ploidy level constraints of endosperm development. This mutationaffects the number of polar nuclei participating in the fertilization event lead-ing to production of the primary endosperm cell and therefore the ploidy of itsnucleus (Lin, 1978). Endospenns were generated with ploidy levels rangingfrom 2x-9x. Lin (1984) demonstrated that only 3x (2x female; l x male) and6x (4x:2x) ploidy levels allow normal kernel development to proceed. One4x (3x: 1x) combination with a 2x embryo also developed successfully (Lin,1984). The parental source ratio of the ploidy complement is also important.For tetraploid endosperm, two combinations are possible, 3x: Ix and 2x:2xbut only the former leads to a viable seed. Also, two classes of hexaploidendosperms were produced, 4x:2x and 5x: 1x, but only the former combi-nation allows normal endosperm development. The kernels with illegitimategenome complements are indistinguishable from normal triploid kernels until10 DAP or later, which is about the time cellularization of the endosperm isalmost complete but before significant polyploidization has occurred (Lin,1984; Knowles and Philips, 1988). No histological analysis of abortive ordefective kernels has been done with these different ploidy combinations sowe do not know what specific morphological abnormalities result. The mech-anisms by which genome structure affects endosperm development are stillunknown.

Reserve DepositionStored reserves accumulate in endospenns in specific spatial patterns. In datepalm the reserves accumulate first in cells of the seed center and accumulationproceeds centrifugally such that the outermost cells produce reserves last(DeMason et al., 1989). Cellular characteristics of reserve deposition havebeen of interest to many seed biologists. In particular, deposition of cell wallpolysaccharide and phytic acid reserves have been the subject of some recentinvestigations. There is considerable interest in determining the mechanismof lipid body deposition. The involvement of cytoskeletal elements in proteinbody formation has been also proposed recently.Starch is synthesized in plastids. Starch synthesis has been studied mainly

in green tissues and therefore in chloroplasts, but in seeds starch synthesisoccurs in colorless plastids known as proplastids (undifferentiated plastids) oramyloplasts (containing starch). The exact similarities and differences in thebiochemical events leading to starch biosynthesis in chloroplasts and amy-


loplasts are just starting to be elucidated (Sivak and Preiss, 1995). Amongthe enzymes important to starch biosynthesis, several starch synthases (gran-ule bound starch synthases) and starch branching enzymes have been isolatedfrom starch granule preparations, and are thought to be compartmented withinthe amyloplast (Denyer et aI., 1995). Further information on starch biosyn-thesis is presented later in this section under mutation analysis and in anotherchapter in this volume.In date endosperm development, the deposition of the cell wall reserves

occurs in a distinctly separate phase from the initial cellularization of theendosperm. Cellularization of date endosperm occurs centripetally. Cell wallthickening starts in the seed center after cellularization is complete and pro-ceeds centrifugally (Figures 7-8). But since it occurs concurrently with cellenlargement, it is part of, by definition, a primary cell wall and not a sec-ondary wall (DeMason et aI., 1989) (Figure 8A). Within a cell, the depositionof wall materials occurs in concentric layers. Galactomannan deposition infenugreek endosperm cells is accomplished by ER rather than by dictyosomes(Meier and Reid, 1977). DeMason et al. (1992) have demonstrated that thethickened wall in the endosperm of dates is deposited as a highly substitut-ed galactomannan, and that most of the galactose units are enzymaticallyremoved in situ during wall maturation. Edwards et al. (1992) have pro-posed a similar a-galactosidase-mediated post-depositional modification ofthe galactomannan mannose/galactose ratio during seed development in Sen-na occidentalis. This modification in date endosperm cells results in cellwall hardening which occurs centripetally within a cell. The entire process ofdeposition and maturation (hardening) proceeds centrifugally in the seed asa whole (Figures 8B-C).Since the cloning of the lipid body proteins, oleosins, several laborato-

ries have been interested in studying their regulation and insertion into themembrane of the lipid body. Ultrastructural associations between ER andlipid bodies have prompted early hypotheses that lipid bodies originate fromER (Frey-Wsyssling et aI., 1963; Schwartzenbach, 1971). Huang (1994) hasproposed that triacylglycerols and oleos ins are synthesized simultaneously inassociation with ER in the following way: Oleosins are synthesized on polyri-bosomes bound to ER without appreciable co- or post-translational processingwhile the triacylglycerols accumulate between the phospholipid bilayers ofthe ER membrane (Figure 9). Other authors have questioned the validityof this model because oleosins have not been localized to ER membranes(Herman, 1987), oleosins have not always been demonstrated to accumulatesimultaneously with triacylglycerols (Batcheler et aI., 1994), and the struc-ture of oleosins might not be capable of residing within the bilayer of theER membrane (Herman, 1995). Rangel et al. (in press) describe a completelydifferent mechanism for lipid body formation in developing olive fruit. Theyobserved lipid body origin at specific sites in the cytoplasm of mesocarp cells


~ FA(Plastid)

Fig . 9. Model of lipid body formation, FA - fatty acid , RER - rough endoplasmic reticulum ,TAG - triacylglycerol. (From Huang, 1994.)

with no association with ER or ribosomes. The mechanism of lipid bodyformation may differ in different tissues and plant species.The mechanism of storage protein sequestration into protein bodies of

developing seeds has long been an area of interest. Extensive descriptionsof the two basic mechanisms of protein body formation have been presentedby Shotwell and Larkins (1989). Briefly, they are: (I) storage proteins aretransported from the site of synthesis on RER through dictyosomes to thecentral vacuole, which fragments to form many small protein bodies; (2) stor-age proteins are synthesized in RER, which subsequently pinches off to formprotein bodies. The latter pattern occurs in maize and sorghum endosperm(Khoo and Wolf, 1970; Taylor et aI., 1985). Both mechanisms occur in theendosperm of rice (Krishnan et al., 1986). The significance of the two dif-ferent mechanisms is not clear but it may be related to the structure of thestorage proteins themselves (Shotwell and Larkins, 1989) . It is also evidentfrom the few studies done that the different storage proteins have differenttemporal patterns of accumulation within developing protein bodies of anindividual endosperm cell. Greenwood and Bewley (1985) have shown thataccumulation of the crystalloid protein component (globulin) precedes thatof the matrix in castor bean protein bodies . Lending and Larkins (1989) haveproposed a very elaborate model of protein body filling during development ofmaize endosperm cells. They propose that the (3-and -y-zeins accumulate firstand as the o -zeins accumulate they 'pass through' the peripherally located (3-and , -zeins to fuse into a centrally located core. Such an elaborate sequenceof timing and positioning of storage protein sequestering within the protein


bodies requires careful developmental controls, the mechanism of which areunknown.Very little is known about the mechanism of accumulation of materials

present in other protein body inclusions. There has been some work on accu-mulation of phytic acid in globoids. Greenwood and Bewley (1984) haveshown by elegant microscopic studies that phytic acid is synthesized in thecytoplasm of developing castor bean endosperm cells, possibly in associationwith ER, and is then transported to and sequestered in the developing pro-tein bodies. The transportation mechanism may be vesicles produced directlyfrom the ER or through dictyosome vesicles like storage globlulins into pro-tein bodies. Similar studies have not been done with other species.The mechanism of vesicular movement to the protein bodies, pinching

off of ER to form protein bodies and division of vacuoles into protein bod-ies are not known. In recent studies of developing protein bodies of maizeendosperm, actin has been demonstrated to be co-localized with polysomes,ER, and protein bodies (Davies et al., 1993; Stankovic et al. , 1993; Clore, etal., 1996) . Additional studies, especially in situ, of cytological structure indeveloping endosperm cells would help elucidate the mechanisms of reservebody formation.Most of the endosperm mutations in crop plants such as maize original-

ly selected from morphological characteristics have been shown to involvevarious aspects of storage product accumulation such as starch or protein, orboth . Among the many mutations that have been characterized and mappedin maize are those which affect starch accumulation during kernel develop-ment such as amylose extender, brittle, shrunken, sugary, waxy, and sugaryenhancer. The mutation known as sugary (suI) has a distinctive glassy, wrin-kled and irregular kernel phenotype when dried but the immature endosperm,before drying, is distended and cohesive (Coe et al., 1988). Com homozy-gous for the sui mutation is known as sweet com and this mutation is usedextensively in commercial sweet com varieties (Boyer and Shannon, 1983;Marshall, 1987). The sui mutation has reduced levels of a starch debranchingenzyme (pullulanase) creating a highly branched and soluble starch known asphytoglycogen (Pan and Nelson, 1984; Doeh1ert et al., 1993). The SuI genehas recently been cloned and is reported to be a starch debranching enzyme,which is consistent with previous enzyme data (James et al., 1995). Mature ,dry sh2 kernels contain approximately twice the total sugar content, 1/3 to 1/2the starch levels , and only trace levels of phytoglycogen in comparison to suikernels (Douglass et al., 1993). The sh2 kernel s have dramatically reducedADPG-pyrophosphorylase, an enzyme that is important in the conversion ofsucrose to substrates for starch synthesis (Tsai and Nelson, 1966; Dickinsonand Preiss, 1969). The gene has been cloned and it is clear that sh2 and bt2represent different mutations in the genes for this enzyme (Bhave et al., 1990;Shaw and Hannah, 1992). Another mutation on which there has been recentwork is sugary enhancer (se). It is thought to be a recessive modifier of the


sul endosperm mutation (Ferguson et aI., 1979). When homozygous, the seallele increases total sugar in sul kernels to levels comparable to those insh2 kernel s without a reduction in phytoglycogen content (Gonzales et al.,1974 , 1976; Douglass et aI., 1993). The amylose extender (ae l) is a mutationin the gene which encodes a branching enzyme (SBEIIb) and productionof amylopectin is severely impaired (Shannon and Garwood , 1984; Stinardet aI., 1993). Conversely, waxy (wx) is a mutation in a granule-bound starchsynthase gene and kernel s contain only amylopectin (Shure et aI., 1983; Shan-non and Garwood , 1984; MacDonald and Prei ss, 1985). The Waxy gene iscommon to other species (wheat and pea) but it is only one of many starchsynthases (Denyer et aI., 1995). These variou s mutation s have provided somedata to piece together the pathways leading to starch biosynthesis which arestill poorly understood and to 'genetically engineer' new varieties of sweetcom with the appropriate levels of sugar, amylose, amylopectin, and phy-toglycogen (Creech, 1965, Hannah et aI., 1993; Boyer and Hannah, 1994).More details of starch and carbohydrate synthesis are presented in anotherchapter in this volume .Many of these sweet com mutations have very similar phenotypes; the

maturing kernel is distended and balloon-like and upon drying, it shrinks andcollapses into an angular structure with marked concavities and a brittle tex-ture (Coe et aI., 1988). The explanation for this has been that these mutation sare thought to affect starch synthesis during kernel development resulting inelevated levels of sugars, primarily sucrose . High levels of sucrose wouldresult in a lower osmotic potenti al, causing greater water uptake into the seed.During seed maturation and desiccat ion , there is thus greater water loss overa longer period of time and the endosperm collapses, creating a shrunken,shriveled phenotype. Many other common pleiotropic effects have also beennoted , including altered levels of storage proteins (Tsai et aI., 1970; Girouxet aI., 1994), depressed levels of a -amylase activity (Sanwo and DeMason,1992, 1993, 1994), altered ultrastructural features of aleurone cells (Sanwoand DeMason, 1992, 1994), severe problems with fungal infections, and poorgermination (Douglass et aI., 1993). High cotyledon sugar content has beenimplicated in causing many pleiotropic effects in the wrinkl ed seed mutantseries of peas (Pisum sativum) . These mutations of pea consist of a seriesof defects in enzymes important in the starch synthesis pathway analogousto those available in maize (Wang and Hedley, 1991, 1993). The phenotypesof wrinkled genes , or rugosus loci , are remarkably similar to the sugaryand shrunken phenotypes of com. Common cellular characteristics are highsucrose and/or osmotic levels that may 1) regulate transcription of enzymesaffecting carbohydrate metabolism; 2) result in elevated levels of stress hor-mones; 3) affect membran e conformation within cells; or a combination ofthese various events.A number of kernel mutations of maize have been shown to affect storage

protein accumulation, inciud ing,ftoury2 (jl2), Def ective endosperm B30 (De-


830), Mu cronate (Me), and opaque2. These all have similarities in theirphenotypes in that they impart an opaque look to the kernel. These mutationshave all been shown to affect synthesis of specific zeins in different ways.The ft2 mutant plants show a reduction in total zein synthesis, accumulationof a novel 24 kD zein , altered protein body morphology and overproductionof a 70 kD ER protein known as globulin-binding prote in (Bip) (Fonteset aI., 1991) . Similar proteins are known to participate in protein foldingor conformational modification utili zing energy released by ATP hydrolysis(Lodish et aI., 1995). Although Fl2 is not the gene for the 70 kD Bip (b-70), it affects its synthesis at the mRNA level (Fontes et aI., 1991). De-830 and Me also result in reduced accumulation of zein and increased b-70production (Boston et aI., 1991; Marocco et aI., 1991). The 02 gene has beencloned and is known to possess the characteristic feature s of the 'leucinezipper' class of transcription factors (Schmidt et aI., 1987). The 02 gene hasbeen demonstrated to activate transcription of the 22-kD class of zein genes(Schmidt et aI., 1992) and of a ribosomal-inactivating protein (RIP) (Lohmeret aI., 1991; Bas s et aI., 1992). It is clear that the opaque phenotype is causedby reductions in total amount of zeins, but the cascade of event s leading tothis end is still being elucidated.

Variations in Endosperm Developm entIn most species of flowering plants the second fertilizat ion event results inendosperm development, however, in many specie s the endosperm is com-pletely absorbed by the developing embryo by seed maturity. Seed devel-opment in three nonendospermic species has been followed in detail: pea(Pisum sativum), bean (Phaseolus vulgaris), and sunflower tHelianthus annu-us). These species actually demonstrate a sequence of stages at which theendosperm is absorbed. In pea , the primary endosperm cell enlarges rapidlyby an increase in vacuole volume, accompanied by synchronous free nucleardivisions (Marinos, 1970). The developing embryo absorbs the endospermbefore it becomes cellular. In bean , the embryo absorbs the endosperm duringthe phase in which the endosperm is becoming cellular, and in sunflower itis absorbed after cellularization is complete (Newcomb, 1973; Yeung andCavey, 1988). It is clear that the normal sequence of event s takes place in thedeveloping endosperm but the endosperm/embryo interactions are such thatembryo overtakes the endosperm at early stages during seed developmentand the stage at which this occurs is different in different species. There is,therefore, a complete continuum with endospermic seeds, since even in theseseeds some endosperm digestion , especially in the later stages of endospermfilling , occurs adjacent to the embryo. There is no clear distinction betweenendospermic and nonendospermic seeds based on developmental criteria.The genetics and physiological dynamics of this interaction are completelyunknown.


Another variation on the theme is that of recalcitrant seeds . Seeds of mostflowering plants undergo maturation dry ing after reserve deposition to watercontents of 10% or below (Bewley and Black, 1994). These seeds remainviable for varying periods of time, depending on other conditions, and aretherefore considered desiccat ion tolerant. Thi s is known as orthodox viabilitybehavior (Roberts, 1973). During the pha se of reserve depo sition orthodoxseeds undergo slight water loss and become desiccation tolerant (Kermode,1990). During the final phase, metabolic activity declines and ribo somes andcytoplasmic membranes such as ER and dictyosomes disappear. In someplant species, however, the seeds do not develop desiccation tolerance asthey mature and are known as recalcitrant seeds (Roberts , 1973). Recalcitrantseeds are very sensitive to drying, which reduces viability. The reason forthis sensitivity to desiccation is unknown. There has been comparativelylittle work on the development of recalcitrant seed s (Farrant et a\., 1992;Vertucci and Farrant, 1995). There is little or no water loss during the finalmaturation stages as compared to orthodox seeds (Berj ak et a\. , 1990; Fu eta\., 1990; Farrant et a\., 1992) and there mayor may not be normal levelsof reserves accumulated (Farrant et aI., 1992, 1993). The mechanism ofdesiccation tolerance/intolerance is still unknown. It has been suggested thatdesiccation intoleran ce is the result of the inability to produce ABA responsivelea/dehydrin/rab proteins (Black, 1991; Bradford and Chandler, 1992; Farrantet a\. , 1993). If the latter hypothesis is the case, this could be due to ABAdeficiency, ABA insensitivity, or to the absence of lea/dehydrin/rab genes.In the first test of this hypothesis the data were not very supportive. Finch-Salvage et aI. (1994) used both western and north ern blot anal ysis to identifydehydrins in five desiccation sensitive species (Quercus robur, Castaneasativa, Aescu/us hippocastanum, AceI' psuedoplatanus and A . saccharinum).They detected the presence of deh ydrin proteins immunologically during seeddevelopment. They also detected mRNA for a lea (0 11) in the developingembryo of Quercus robur which was induced by limited desiccation and byABA application (Finch-Savage et a\., 1994) . More recentl y, however, in alarge survey of recalcitrant spec ies from different regions of the world, Farrantet a\. (1996) found that the presence of dehydrin-like proteins in the seedsmay be related to habitat. They found that species from tropical, wetlandhabitats lacked both dehydrin-like proteins and ABA whereas, those fromhigh altitude or temperate areas had both.Although it is well known that starchy endosperm cells of cereals are

nonlivin g at maturity, the characteristics and regulation of the dying processduring kernel development has not been studied. The concept, developmentalcharacteristics and control s of gene-directed programmed cell death (PCD)are well-characterized in animal systems and are becoming areas of activeresearch in plant systems. There are many other examples of developmental-ly or environmentally controlled death of cell s and tissues in higher plants,including xylogenesis, cork formation , root cap cell ontogeny, tapetum cell

A

Endosperm Structure and Develooment 97

B

Days after pollination8 12 1(, 211 24 28 32 36 40 44 S

-1630

-520-400-300-220-150

Fig. 10 . Developmental progression of cell death in Zea mays. Figures A- C. Fresh hand sec-tions of developing kernel s stained with the viability stain, Evans Blue which stains nonviablecells. (A) At 16 days only a few cells in the center of the endosperm (arrow) are dead. (B)At 28 days cell death has progressed centrifugally towards the kernel cap. (C) At 40 days theentire starchy endosperm is dead. (D) DNA fragmentation during endosperm development.Size (bp) of molecular weight markers (lane S) are indicated. Scale bar = 5 mm. (From Younget al. [in press)).

development , senescence, and response to pathogens which may show sim-ilarities to PCD in anima l ce lls (Dangl et a!., 1996; Greenberg, 1996). Thedevelopmental progression of ce ll death in maize endosperm follows a sirn-


ilar pattern to that of endopolyploidization and reserve deposition whichsuggests that it is developmentally controlled. In addition, maize endospermcell death also shares some common features , including intemucleosomalDNA fragmentation and presence of Ca++-dependent endonucleases withPCD of animal cells (Young et aI., submitted) (Figure 10). Whether cell deathin maize endosperm shares common cytological features and controls withthose of other cell death events in plants is yet to be determined.

C. Biological Capabilitie s of Endosperm

Because endosperm cells perform many cellular functions nece ssary to theirrole in storage product production and sequestration, and have specializedgene functions associated with these processes, many workers have beeninterested in either making callus cultures or protoplasts of endosperm cellsto study them in isolation . In addition, the role cereal aleurone cells playof reserve mobilization during germination have been of interest to thosestudying hormone regulation of protein transcription and protein secretion.Much of this work has been carried out on barley aleurone protoplasts. Manyof these studies have provided direct or indirect evidence of the cellularcapabilities of isolated endosperm cells.The first endosperm culture attempts were made with developing maize

endosperm but no callus was obtained (La Rue , 1947). La Rue was eventuallyable to establish long-term endosperm callus cultures of several lines (LaRue, 1949; Strau s and La Rue, 1954). These were initiated from developingendosperm 10-12 DAP.Tabata and Motoyoshi (1965) established the fact thatgenetic variation for succe ss in tissue culture is evident in different maize lines.No successful attempts to culture starchy (dent) genotypes occurred untilmuch later (Shannon and Batey, 1973). Since this time maize endosperm cellsuspension cultures have been used as a standard in vitro system for studyinggene regulation in maize endosperm (Chu and Shannon, 1975; Shannon,1982). Although these cultures do retain abilities to accumulate tissue- andgenotype-specific reserve starch and proteins (Manzocchi, 1991; Saravitzand Boyer, 1987; Quayle et aI., 1991; Veda and Mes sing , 1991), they do soat reduced levels compared to developing endosperm cell s in vivo (Lyznikand Tsai, 1989; Manzocchi et aI., 1989) . In addition, these in vitro cellpopulations have been shown to be highly variable and comprised of cellswith dense cytoplasm and small vacuoles, large vacuolated cells , and cellswhich accumulated storage products in the form of starch grains, proteinbodies, or lipid bodies (Felker, 1987).Along similar lines, callu s cultures of developing coconut endosperm cells

were established to study fatty acid biosynthesis (Ceniza et aI, 1992). Theseauthors found that highly meristematic cell s at the micropylar end of 7.5month-old fruits were most respon sive to successful callus culture initiation.After six months of culture, fatty acid composition was similar to young in


vivo endosperm cells, but that of callus cultured for 18 months showed quitedifferent fatty acid composition (Ceniza et al., 1992). They concluded thatlong term culture conditions probably selected for callus which was mainlycomposed of highly proliferative cells, producing little storage lipids.Since immature endosperm of maize was first cultured, endosperm from

over 40 species of angiosperms has been cultured to produce callus, plantletsfrom organogenesis and embryos from somatic embryogenesis (Srivastava,1982; Srivastava and Johri, 1992). This demonstrates the extraordinary mor-phogenetic potential of endosperm in flowering plants. Although only devel-oping endosperm can be cultured in some species, mature endosperm is notonly capable of callus production, but also plantlet formation in many species(Srivastava and Johri, 1992). No free-nuclear endosperm has been shown tobe amenable to culture techniques. Plantlet regeneration from endosperm tis-sue provides opportunities for studying gene regulation and morphogeneticpotential of triploid plants and a mechanism for plant breeders to developseedless fruit crop cultivars .And finally, endosperm cells have been used for protoplast isolation. Many

laboratories have used cereal aleurone protoplasts for biochemical or tran-sient transformational studies (see Gallie and Young, 1994 for references).In addition, protoplasts have been made from developing starchy endospermcells in several cereals including wheat (Keeling et al., 1989), barley (Lee etal., 1991; Diaz and Carbonero, 1992), and maize (Schwall and Feix, 1988;Giovinazzo et al., 1992; Gallie and Young, 1994). Selmar et al. (1989) havedemonstrated protoplast isolation from endosperm cells of Hevea brasilien -sis. Protoplasts have been made from maize endosperm suspension cultures(Schwall and Feix, 1988; Manzocchi, 1991; Ueda and Messing, 1991; Giov-inazzo et al., 1992; Quayle et al., 1991; Faranda et al., 1994). Protoplasts arerelatively easy to transform and can be used subsequently for short-term geneexpression studies (transient transformation) or for establishing permanenttransformed callus lines (i.e. Faranda et al., 1994). Such techniques furtherexpand the types ofgene expression and morphogenesis experiments, and arepotentially useful for developing new plant cultivars.

D. Function ofEndosperm and Endosperm/Embryo Interactions

The general perception for many years is that the major function of endospermis to provide nutritional support for the developing embryo (endospermic andnonendospermic seeds) and for the germinating seedling (endospermic seeds).Some authors have questioned the idea, however, that the endosperm playsan important nutritional role in early stages of embryogenesis (Vijayaragha-van and Prabhakar, 1984; Murray, 1988; Steeves and Sussex, 1989). It hasalso often been postulated that the endosperm provides the correct physical,chemical, and hormonal environment and therefore may playa role in differ-entiation of the embryo. In fact, coconut milk (liquid, syncytial endosperm


of the coconut palm) was commonly used for tissue culture of develop-ing embryos as well as other tissues before defined media were established.Finally, it is well known that failure of endosperm development is the primarycause of seed abortion in many hybrids (Brink and Cooper, 1947), and youngembryos can be 'rescued' by culture or with 'nurse endosperm' (Raghavan,1986; Raghavan and Srivastava, 1982) . The results of structural, tissue culturestudies of developing embryos and genetic studies provide data to considerthe relationship between the endosperm and embryo of the developing seed .Authors carrying out structural studies on seed development in which they

have addressed issues of endosperm/embryo interactions have provided adiversity of opinions with regard to the importance of the endosperm's nutri-tional role. This diversity may be related to the diversity of species studied,differing criteria used as evidence by different authors and the lack of defini-tive studies of actual uptake and transport in most species. During late stagesof seed development in species with nuclear endosperm developing to thecellular stage (in both endospermic and non-endospermic seeds), there isno doubt that the endosperm provides nutritive substances to the developingembryo, because of the presence of structural evidence of cellular digestion.The question that is not settled is whether the liquid, syncytial endosperm,or some other portion of the developing seed, is a more important source ofnutrition for the young embryo. The other structures that have been consid-ered as possibly surpassing the endosperm in this role are the synergids, thesuspensor, and the integuments. I would like to review the literature with theidea of identifying common features from the many systems studied.A universal fact in all angiosperm seeds is that the embryo sac in the devel-

oping seed has no vascular tissue continuity with the maternal tissues and is,therefore, symplastically isolated. Similarly, the embryo and the endospermshare no vascular system or symplastic continuity. Active uptake by theembryo sac as well as by the embryo must be important and transfer cells ortransfer-cell type wall ingrowths have been used as evidence of such activity.Plasmodesmatal connections have been used as evidence of symplastic con-tinuity. The synergids have transfer cell type walls at their micropylar ends.They are adjacent to the egg cell and subsequently the zygote. One degener-ates rapidly after double fertilization and the other more slowly. Newcomb(1973) has suggested that the persistent synergid may function in transloca-tion of soluble metabolites from the extra embryo sac sources to the growingembryo. Schulz and Jensen (1969) supported the idea that the suspensor alongwith its basal cell functions in the absorption and translocation of nutrientsfrom the surrounding integuments to the developing embryo in Capsella andpossibly other angiosperms. Their evidence for this included: (1) wall pro-jections present in the basal cell increase the absorptive surface area; (2)numerous plasmodesmata in the end wall of the basal cell and the end wallsof all suspensor cells give evidence of symplastic continuity between the basalcell and the embryo; and (3) the active appearance of the cytoplasm and the


presence of starch and lipid reserves in the basal and suspensor cells supporta nutritive role. They also demonstrated that wall projections occur on thelateral walls of the suspensor cells (adjacent to developing endosperm), butthey felt that their function is not necessarily interpretable since the majorflow could be in the reverse direction (towards the endosperm). The suspen-sor is short-lived and undergoes degeneration and becomes crushed after theheart stage in Capsella (Schulz and Jensen, 1969). In Phaseolus coccineusthe suspensor is larger and remains intact for a much longer time (Yeung andClutter, 1978). Since these authors saw wall ingrowths in the suspensor cellsboth adjacent to the integumentary tapetum and adjacent to the endosperm,they hypothesize that both the suspensor and the endosperm play importantroles in nutrient regulation during early embryogenesis (Yeung and Clutter,1978). Yeung (1980) tested this further by labeling Phaseolus pods and seedswith heart-shaped embryos and found substantial label subsequently in thesuspensor and root pole of the embryo. He proposed that at the earliest stagesof embryogenesis (proembryo, globular, and heart stages) the nutrient flowis: parent plant -+ seed coat -+ suspensor -+ embryo-proper.However, in a recent study of metabolite accumulation during almond fruit

and seed development, Hawker and Buttrose (1980) estimated movementrates necessary in the suspensor if materials accumulating in the embryo allpassed through it and concluded that 'the metabolites entering the embryo donot pass exclusively (if at all) via the suspensor cells.' Instead, they suggestthat metabolites must move from the testa through the nucellus, endosperm,and finally into the embryo.The structural basis for this theory has been described in various species.

Commonly the primary endosperm cell expands and undergoes mitoses beforethe zygote (Esau, 1965). Transfer cell like wall ingrowths have been observedin the primary endosperm cell or outermost layer of endosperm in manyspecies such as Pisum sativum (Marinos, 1970), Phaseolus coccineus (Yeungand Clutter, 1978), Phaseolus vulgaris (Yeung and Cavey, 1988), Helianthusannuus (Newcomb, 1973), Euphorbia dulcis (Gori, 1987), barley (Brown etaI., 1994), maize (Schel et al., 1984) and wheat (Morrison and O'Brien, 1976;Fineran et aI., 1982; Wang et al, 1995b). In a study of the liquid endospermof Pisum sativum, Marinos (1970) made some interesting observations withregard to nutrient transfer. He observed that an early expansion of liquidendosperm and the subsequent growth of the embryo correlated with its con-current disappearance. He also noted that not only were extensive cell wallprojections present around the endosperm boundary, but they were particu-larly elaborate in the vicinity of the developing embryo. The cytoplasm ofthe liquid endosperm in pea is dense with mitochondria, free ribosomes, plas-tids, and rough and smooth ER. ER was particularly abundant adjacent to thesuspensor. He speculated that materials were accumulating in the endospermand were subsequently being made available to the developing embryo.


Schel et al. (1984) studied structural aspects of endosperm-embryo inter-actions in maize. They found enhancement of previously existing cell wallprojections in the placentochalaza1 region after endosperm initiation. In addi-tion, they found that abundant endomembranes, especially RER and vesicles ,are present in endosperm cells surrounding the base of the young embryo.They suggested that the suspensor may function to absorb nutrients synthe-sized by these metabolically active endosperm cells . In addition, hydrolysisof endosperm cells themselves is visible as early as 5 DAP around the maizeembryo. They hypothesized that a combination of autolysis and digestionvia embryo-derived hydrolytic enzymes may be involved. These observationssuggest that the endosperm is the major source of nutritional materials for themaize and pea embryos from the earliest stages of embryogenesis.In later stages of endosperm maturation large amounts of storage reserves

are accumulated. The transport mechanism responsible for providing the rawmaterials for these processes has long been of interest to plant biologists. In aseries of papers containing an elegant combination of structural observations,careful analyses, and confirming experiments, Wang et al. (1995a,b) eluci-date the mechanism in developing wheat grains. They provide considerableevidence that photosynthetic transfer is symplastic from the sieve element-companion cell complexes of the inner integument in the ventral crea se areaalong the grain through parenchyma to the nucellar projection transfer cells,where sucrose is released into the endosperm cavity (Wang et al., 1995a).Uptake by endosperm cells could follow a syrn- or apoplastic route. Thesymplastic route, which involves active uptake via modified a1eurone/sub-aleurone complex cells containing extensive wall ingrows and a putativesucrose-proton symporter, is the principal mechanism during later stages ofgrain filling (Wang et al., 1995b). Similar mechanisms probably occur in othercereal grains and other seeds.A couple of genes are thought to be important in transport into the maize

endosperm during development. A maize kernel mutation with relevance tophotosynthate movement into endosperm is miniaturel (mnl). This singlegene recessive mutation results in kernels with only 1/5th normal weightand is characterized by cell death and withdrawal of maternal tissues at thebase of the kernel (Miller and Chourey, 1992). It has been demonstratedthat invertase activity is greatly reduced in the basal endosperm cells andadjacent maternal tissues and that the mutation is inherited strictly as anendosperm trait (Miller and Chourey, 1992; Chourey et al., 1995). Althoughit has been speculated that the mutation is probably in the structural gene forthis enzyme (Chourey et al., 1995), these pleiotropic effects still need to beexplained. One possibility is that all these various manifestations are due to a'transient osmotic imbalance due to impaired movement of photosynthate tothe endosperm ' (Miller and Chourey, 1992). Further work, especially detailedanatomical and ultrastructural studies would be very useful in understandingthis specific mutation and its effects on the mechanism ofgrain filling in maize.


Recently, a gene known as BETI has been isolated from the basal endospermtransfer layer in maize (Hueros et aI., 1995). Based on its temporal expressionand distribution the authors have proposed that it plays a role in transport intothe endosperm.Elucidating the facto rs that control the progressive and orderly develop-

ment of embryos has been a major concern of plant developmental biologistsfor many years. One avenue for determining the nutrit ive and hormonal needsduring this major phase of plant development has been to define tissue cul-ture media which allow norm al embryogenesis to proceed. A comparisonof the con stituents of these med ia and the chemical make-up of developingendosperm in several species has also been done. The literature on the cul-ture of mature embryos is extensive and dates back to the work of Hannig(1904) and Brown (1906). Raghavan and Srivastava (1982) have extensive-ly reviewed the literature on the culture requirements of mature angiospermembryos. They have come to the conclusion that since, in general, theseembryos require only simple media containing mineral salts, a carbohydratesource, and vitamins, they are autotrophic and their subsequent developmentis largely under their own control. Culture of progre ssively younger devel-oping embryos is more complex and necessitate s more extensive additives tothe medi a (Raghavan and Srivastava , 1982; Johri and Rao, 1984; Raghavan,1986; Murray, 1988 ). To successfully culture younger stages of embryos itwas common in early studies for authors to use casein hydrol ysate, coconutmilk , yeas t extract, tomato juice and other undefined additives. Some work-ers thought that an important 'embryo factor ' was present in coconut milk(Raghavan and Srivastava, 1982). In a pivotal study of Datura stramoniumembryo culture requirements at different stages of development, Rietsema etal. (1953) demonstrated that progressively younger embryos require greatersucrose concentrations and higher osmolarity in the medium for optimalgrowth. Rijven (1952) also tested media with differing osmotic potential andobserved that lowering the osmoti c potential reduced premature cell expan-sion and germination of Capsella embryos and was therefore important inmaintaining more norm al growth and development. Raghavan and Torrey(1963) showed that use of plant hormones (auxin , kinetin , and adenine sul-fate ) could substitute for 12-1 8% sucrose or lOX salts for culturing youngCapsella embryos.Chemical analyses of liquid endosperm of several species with nuclear

endosperm, including coconut, have been made as well as analyses at differ-ent stages of seed development within a species (see references in Raghavanand Srivastava , 1982; Murray, 1988). Osmolarity has been shown to be highin liquid endosperm of all species tested (Kerr and Ander son, 1944; Rijven,1952; Ryczkowski , 1960, 1969; Mauney 1961; Smith , 1973) which sup-ports the culture experiments described above. Smith (1973) reported thatmost of the sugar in the liquid endosperm of Phaseolus vulgaris could beaccounted for as sucrose, glucose, and fructose . The high osmolarity in the


liqu id endos perm of this spec ies co uld be accounted for by ammonia, sug-ars (especially reducing suga rs), and organic acids (mainly malate) whichall decreased durin g seed development. The funct ional significance of theapparent necessity for young embryos to be emerged in a medium of highosmolarity (whether natural or artificial) is not specifically known . It hasbeen suggested that this arrangement may be important for effect ive flow ofmetabolit es into the embryo which may be most critica l at the ea rlies t stages,but this has not been tested experimentally (Raghava n and Srivastiva, 1982).Growth hormones have been identifi ed in endosperm of a few species.

The liquid endosperm of Pisum sativum has been shown to contain physio-logically active quantities of gibberellin and auxin (Eeuwe ns and Schwabe,1975 ). Auxin and cytokinin (zeatin and zeatin riboside) production has beendemonstrated in maize endos perm starting approximately 5 DAP (Lur andSetter, 1993a). It is clear that endosperm is capable of hormone productionwhich may be important for embryo development.The final approach to investigating endosperm-embryo interactions is a

genet ic one. Chang and Neuffer ( 1994) used the B-A translocation techniquewith a number of def ective kerne l (dek) mutations of maize to determineif normal endosperm could ' resc ue' embryos with leth al mut ations duringkernel development and vice versa . Their hypothesis was that the embryoor endos perm mutant phenotype might be corrected by interaction with itsnormal (wild-type) counterpart resulting in normal kern els. They were ableto demonstrate four types of interact ions: (1) a unidirectional interactionfrom normal endosperm to mutan t embryo (4 mut ants); (2) a unidirectionalinhibition from mutant endosperm to a normal embryo ( I mut ant ); (3) a unidi-rectional interaction from norma l embryo to mutant endos perm (6 mutants);and (4) bi-d irectional interactions (two mut ant s). These results clearl y demon-strate that important interactions between the endosperm and embryo in maizeare present during kernel development. The mut ation resulting in an inhibito-ry effect on the embryo (2 above) was dek 26. The endosperm in this mutantwas one which Lur and Setter (1993b) have demonstrated to produce signif-icantly less auxin than normal wild -type maize with the same background.These result s suggest that auxin production by the endosperm is necessary fornormal embryo development in mai ze. Nutritional , osmoticum, or hormonalproduction by one component of the kernel could explain the interactionsdescribed by Chang and Neuffer (1994) as well as gene products necessaryfor photo synthate tran sport . Particul arly intriguing are the interactions (num-ber 3 above) in which gene products produced in the embryo unidirectionallyaffect endosperm development. Identifi cation of the spec ific dek mutationsin any of the interactions demonstrated above could provide ex tremely valu-able information on endosperm-embryo interact ions which are still poorlyunderstood .Although some progress has been made in the last hundred years with

respect to identi fying spec ific interactions between endos perm and embryos


during seed development, it is clearly quite meager compared to most oth-er aspects of seed development. Sugar and osmotic differences between thetwo parts of the developing seed and gene expression necessary for transportare important. Hormone production by the endosperm and possibly by theembryo probably playa role in coordinated development. But the most impor-tant developmental question, that of what regulates the timing of endospermconsumption by the embryo, is still a total mystery. Continued characteri-zation of the dek mutants, or similar seed developmental mutants of otherspecies , such as Arabidopsis, is the most likely avenue for improving ourunderstanding of endosperm-embryo interactions.

Summary and Questions for Future Research

A large amount of information about endosperm structure, development, andfunction has been amassed over the years , some ofwhich has been highlightedin this review. Most of the se studies have been ' rnonodisciplinary' in nature.They provide data from a single discipline or group of techniques: structural,physiological, molecular, or genetic. These types of studies have provided thebasic framework of information we know about the structure , development,and function of endosperm, as well as considerable specific details. In mostcases the ' whys ' have still not been answered.It is clear that there is no distinct structural or developmental criterion

that di stinguishes endospermic from nonendospermic seeds, so what controlsthe timing of endosperm dig estion by the embryo? There are three majormodes of endosperm development: nuclear, cellular, and helobial , but verylittle detail is known about the characteristics of the latter two. We also don 'tknow the significance of these major developmental variations. Finally, wedon 't know the developmental mechanism which controls them. Althoughwe know more about nuclear endosperm development, the earliest phasesare still poorly understood. Why does cytokinesis not accompany mitosisin the early phase of nuclear endosperm development? What controls thetermination of this phase and initiation of cell wall formation? The geneticaspects of endosperm development have long been of interest to breeders aswell as developmental biologists. Endopolyploidization is a common featureof seed development. What is its significance? Is it simply a consequence ofcell enlargement or does it have a real developmental function? What is thesignificance of 'genome balance' in endosperm development? Why is theresuch a clear distinction between tho se that allow subsequent development andtho se that do not ?Reserve accumulation is of prime commercial importance to plant breed-

ers. We know many of the details of protein accumulation, but we know verylittle about accumulation of oth er types of reserves. Why do some seed tis-sues store starch and oth ers store cell wall polysaccharides? What controls

106 Darleen A . DeMason

the balance between the types of reserves accumulated and what signals aparticular cell to accumulate reserves? What controls the specific, spatial pat-terns of reserve accumulation within the endosperm? Does one cell signalthe adjacent cell to proceed? How does the whole process start? What is thefundamental difference between orthodox and recalcitrant seeds? Does theexistence of this distinction demonstrate that there is some overall controllingmechanism which affects reserve accumulation, membrane stability, dessica-tion tolerance, and hormone levels? What is the signal transduction pathwaycontrolling cell death in the starchy endosperm of cereals and how is it relatedto other developmental events?It is the overall controlling mechanisms which still need to be identified.

Many of the questions listed above have been asked in previous reviews onendosperm structure and development through the decades. These are, ofcourse, the most difficult questions, and can only be addressed with multidis-ciplinary approaches.

Acknowledgments

I thank several colleagues who have graciously provided figures for thisreview: Roy Brown (Figure 6), John Greenwood (Figures IB and 3A), andTony Huang (Figures 4 and 9). I thank Todd Young for many helpful dis-cussions on maize endosperm development and the following members ofthe laboratory who helped with the manuscript preparations: Stacey Novak,Tracy Kahn, Phil Villani, and Todd Young.

References

Asghar, R., and DeMason, D.A. (1990) Developmental changes in the cotyledons of Lupinusluteus L. during and after germination. Amer. J. Bot. 77: 1342-1353.

Asghar, R., and DeMason, D.A. (1992) Differential activities of acid phosphatase from adaxialand abaxial regions of Lupinus luteus (Fabaceae) cotyledons. Amer. J. Bot. 79: 1134-1144.

Aspinall , G.O. (1983) The Polysaccharides, Vol. 2. Academic Press, New York.Bajer, A.S., and Mole-Bajer, J. (1986) Reorganization of microtubules in endosperm cells andcell fragments of the higher plant Haemanthus in vivo. J. Cell BioI. 102: 263-281.

Bass, H.W., Webster, C., O'Brian , G.R., Roberts , J.K.M., and Boston , R.S. (1992) A maizeribosome-inactivating protein is controlled by the transcriptional activatorOpaque-2 . PlantCell 4: 225-234.

Batcheler, c.,Ross, J.H.E. , and Murphy, 0.1 . (1994) Synthesis and targeting of Brassica napusoleosin in transgenic tobacco. Plant Sci. 104: 39-47.

Bechtel, D.B., and Pomeranz, Y. (1977) Ultrastructure of the mature ungerminated rice (Oryzasativa) caryopsis . The caryopsis coat and the aleurone cells. Amer. J. Bot. 64: 966-973.

Bennett, M.D. (1973) Nuclear characters in plants . Brookhaven Symp . BioI. 25: 344-366.Berjak, P., Farrant, J.M., Mycock, 0.1 ., and Pammenter, N.W. (1990) Recalcitrant (homoio-hydrous) seeds: the enigma of their desiccation-sensitivity. Seed Sci. & Technol. 18:197-310.


Bewley, J.D., and Black, M. (1994) Seeds: Physiology of Development and Germination.Second Edition. Plenum Press.

Bhatnagar, S.P., and Sawhney, V. (198 1) Endosperm - Its morphology, ultrastructure, andhistochem istry. Int. Rev. Cytol, 73: 55-1 02.

Bhave, M.R., Lawrence, S., Barton , C; and Hannah, L.c. (1990) Identification and molecularcharac terization of shrunken-Z cDNA clones of maize. Plant Cell 2: 58 1-588 .

Black, M. (199 1) Involvement of ABA in the physiology of developing and mature seeds.In: Davies, W.J., and Jones, H.G. (eds) Abscisic Acid: Physiology and Biochemistry, pp.99-124, Bios. Scient ific Publishers, Oxford, U.K.

Boesewinkel , ED., and Boum an, E ( 1984) The seed: Structure. In: Johri, B.M. (ed) Embryologyof Angiosperms, pp. 567-610, Springer-Verlag, Berlin ..

Boesewinkel , E D., and Bouman, E (1995) Seed morphology and development. In: Kigel, J.,and Galili, G. (eds) Seed Development and Germin ation. pp. 1-24, Marcel Dekker, Inc.,New York.

Bosnes, M., Harris, E., Aigeltinger, L., and Olsen, a.-A. (1987) Morpbology and ultrastructureof II barley shrunken endosperm mutant s. Theor. and App!. Gen. 74: 177-1 87.

Bosnes, M., Weideman, E , and Olsen, Or-A. (1992) Endosperm differentiation in barley wild-type and sex-mutants. Plant Journ al 2: 66 1-674.

Boston, R.S., Fonte s, E.B.P., Shank , B.B., and Wrobel, R.L. ( 199 1) Increased expression of themaize immunoglobulin binding protein homolog b--70 in three zein regulatory mutants.Plant Cell 3: 497-505.

Boyer, C.D., and Hannah, LiC, ( 1994) Kernel mutants of com. In: Hallauer, A R. (ed) SpecialtyCo rns , pp. 1-28, CRC Press, Boca Raton, FL.

Boyer, C.D., and Shannon , J .c. (1983) The use of endosperm genes for swee t com improve-men t. Plant Breeding Reviews I : 139-161.

Bradford, K.J ., and Chandler, P.M. (1992) Expression of 'dehydrin-likc' proteins in embryosand seedlings of Zizania palustri s and Oryza sativa during dehydration. Plant Physio!. 99:488--494.

Brink, R.A. , and Cooper, D.C. (1947) The endosperm in seed development. Bot. Rev. 13:423-541.

Brown, H.T. (1906) On the culture of the excised embryos of barley on nutrient solutionscontai ning nitrogen in different forms. Trans . Guinnes Res. Lab. I: 288-299.

Brown, R.C., Lemmon, B.E., and Olsen, a .-A. (1994) Endospe rm development in barley:Microtubule involvement in the morphogenetic pathway. Plant Cell 6: 1241-1 252.

Brown, R.C., Lemmon , B.E., and Olsen , a .-A. (1996a) Polarization predicts the pattern ofce llularizat ion in cerea l endosperm. Protoplasma 192: 168-177.

Brown, R.C. , Lemmon, B.E., and Olsen , a .-A. (l996b). Development of the endosperm inrice (Ory za sativa L.) : ce llularization. J. Plant Res. 109:301- 313.

Ceniza, M.S., Ueda, S., and Sugimu ra, Y. ( 1992) In vitro culture of coconut endosperm: callusinduction and its fatty acid s. Plant Ce ll Rep . II , 546--549.

Chandra Sekhar, K.N., and DeMason, D.A. (1988 ) Quantitative ultrastructure and proteincompos ition of date palm (Phoenix dactylifera) seeds: a comparative study of endospermvs embryo. Amer. J. Bot. 75: 323- 329.

Chandra Sekh ar, K.N., and DeMason, D.A. (1990 ) Identification and immunocytochemicallocali zat ion of a -ga lactos idase in resting and germinating date palm (Phoenix dactylif eraL.) seeds . Planta 181: 53-61.

Chang, MT., and M.G. Neuffer, M.G. (1994) Endosperm-embryo interactions in maize. May-dica 39 : 9-18.

Chourey, P.S ., Cheng, W.-H., Taliercio, E.w., and 1m, K.H. (1995) Genetic aspects of sucrose-metaboli zing enzymes in developing maize seed. In: Madore, M.A., and Lucas, W.J. (cds)Carbon Parti tioning and Source-S ink Interact ions in Plants, pp. 239-245, Amer. Soc. PlantPhysio!., Rockville, MD.

108 Dar/een A . DeMason

Chu, L.-J., and J.e. Shannon, J.e. (1975) In vitro cultures of maize endosperm - A modelsystem for studying in vivo starch biosynthesis. Crop Sci. 15: 814-8 19.

Citharel, L., and Citharel, J. (1987) Dualite des corps proteiques du mesophylle adaxial etabaxial des cotyledons de quelques especes de la tribu des Genistees (Legumineuses),Can. J. Bot. 65: 1870-1875.

Clore, A.M., Dannenhoffer, J.M., and Larkins, B.A. (1996 ) EF- IQ is associated with acytoskeletal network surrounding protein bodies in maize endosperm cells. Plant Cell8: 2003-2014.

Coe, E.H., Jr., Neuffer, M.G., and Hoisington, D.A. (1988) The genetics of com. In: Sprague,G.E and Dudley, J.w. (eds) Com and Com Improvement, pp. 81-258, Th ird Ed. Amer.Soc. Agronomy, Madison, WI.

Corke, EM. K., Hedley, e.L. , and Wang, T.L. (l990a) An analysis of seed development inPisum sativum L. Cellular development and the deposition of storage protein in immatureembryos grown in vivo and in vitro. Protoplasma 155: 127- 135.

Corke, EM.K.,Hedley, CL, andWang, T.L. (1990b) An analysis of seed development in Pisumsativum L. In vitro manipulation of embryo development using xenobiotic compounds.Protoplasma 155: 136--143.

Creech, R.G. (1965) Genetic control of carbohydrate synthesis in maize endosperm. Genetics52: 1175-1186.

Dangl, J.L., Dietrich, R.A., and Richberg, M.H. (1996). Death don 't have no mercy: cell deathpatterns in plant-microbe interactions. Plant Cell. 8: 1793-1 807.

Davies, E., Comer, E.e., Lionberger, J.M., Stankovic, B., and Abc, S. ( 1993). Cytoskeleton-bound polysomes in plants. III. Polysome-cytoskeleton-membrane interactions in comendosperm. Cell BioI. Inter. 17: 33 1-340.

DeMason, D.A. (1986) Endosperm structure and storage reserve histochemistry in the palm,Washingtoniafilifera. Amer. J. Bot. 73: 1332-1 340.

DeMason, D.A. (1994) Controls of germination in noncereal monocotyledons. Adv. Struct.BioI. 3: 285-310.

DeMason, D.A., and Chandra Sekhar, K.N. (1990) Electrophoretic characteriza tion andimmunological localization of coconut (Cocos nucifera L.) endosperm storage proteins.Bot. Gaz. 151: 302-313.

DeMason, D.A., Chandra Sekhar, K.N., and Harris, M. (1989 ) Endosperm development in thedate palm (Phoenix dactylifera) (Arecaceae). Amer. J. Bot. 76, 1255-1 265.

DeMason, D.A., Sexton, R., and Reid, J.S.G. (1983) Structure, composition and physiologicalstate of the endosperm of Phoenix dactylifera. L. Ann. Bot. 52: 71- 80.

DeMason, D.A., Madore, M.A., Chandra Sekhar, K.N., and Harris, M.J. (1992) Role of Q-galactosidase in cell wall metabolism of date palm (Phoenix dactylifera L.) endosperm.Protoplasma 166: 177- 184.

DeMason, D.A., Stillman, J.I., and Elimore, G.S. (1989 ) Acid phosphatase localization inseedling tissues of the palms, Phoenix dactylifera and Washingtonia fi life ra, and its rele-vance to controls of germination. Can. J. Bot. 67: 1103-1110.

Denyer, K., Hylton, e.M., Jenner, e.E, and Smith, A.M. (1995) Identi fication of multipleisoforms of soluble and granule-bound starch synthase in developing wheat endosperm.Planta 196: 256--265.

Diaz, I., and P. Carbonero, (1992) Isolation of protoplasts from developing barley endosperm:a tool for transient expression studies. Plant Cell Rep. 10: 595-598.

Dickinson, D.B., and Preiss, J. (1969) Presence of ADP-glucose pyrophosphorylase inshrunken- 2 and brittle- 2 mutants of maize endosperm. Plant Physiol. 44: 1058-1062.

Doehlert , D.e., Kuo, T.M., Juvik, J.A., Beers, E.P., and Duke, S.H. (1993) Characteristics ofcarbohydrate metabolism in sweet com (sugary- i) endosperms. J. Amer. Soc. Hort. Sci.11 8: 661--666.

Doehlert, D.e., Smith, L.J., and Duke, E.R. (1994) Gene expression during maize kerneldevelopment. Seed Sci. Res. 4: 299-305.


Douglass, S.K., Juvik, J.A. , and Splittstoesser, WE. (1993) Sweet com seedling emergenceand variation in kernel carbohydrate reserves. Seed Science Technol. 21: 433-445 .

Dute, R.R., and Peterson, C.M. (1992) Early endosperm development in ovules of soybean,Glycine max (L.) Merr. (Fabaceae) . Ann. Bot. 69: 263-271.

Edwards, M., Scott, e., Gidley, MJ., and Reid, J.S.G. (1992) Control of mannose/galactoseratio during galactomannan formation in developing legume seeds. Planta 187: 67-74.

Eeuwens , CJ., and Schwabe , W.W. (1975) Seed and pod wall development inPisum sativumL. in relation to extracted and applied hormones. J. Exp. Bot. 26: 1-14.

Esau, K. (1965) Plant Anatomy. Wiley and Sons, Inc.Esau, K. (1977) Anatomy of Seed Plants. Wiley and Sons, Inc.Faranda, S., Genga, A., Viotti, A., and Manzocchi, L.A. (1994) Stably transformed cell linesfrom protoplasts of maize endosperm suspension cultures. Plant Cell, Tissue and OrganCulture 37: 39-46.

Farrant, J.M., Pammenter, N.W., and Berjak, P. (1992) Development of the recalcitrant(homoiohydrous) seeds of Avicennia marina: anatomical, ultrastructural and biochemi-cal events associated with develoment from histodifferentiation to maturation. Ann. Bot.70: 75-86.

Farrant, J.M., Pammener, N.W., and Berjak, P. (1993) Seed development in relation to des-iccation tolerance: A comparison between desiccation-sensitive (recalcitrant) seeds ofAvicennia marina and desiccation-tolerant types. Seed Sci. Res. 3: 1-13 .

Farrant, J.M., Pammener, N.W, Berjak, P., Farnsworth, EJ. , and Vertucci, e.W (1996) Pres-ence of dehydrin-like proteins and levels of abscisic acid in recalcitrant (dessiccationsensitive) seeds may be related to habitat. Seed Sci. Res. 6: 175-182 .

Felker, F.e. (1987) Ultrastructure of maize endosperm cultures. Amer. J. Bot. 74: 1912-1920.Ferguson , J.E., Dickinson , D.B., and Rhodes, A.M. (1979) Analysis of endosperm sugars ina sweet com inbred (Illinois 677a) which contains the sugary enhan cer (se) gene andcomparison of se with other com genotypes . Plant Physiol. 63: 416-420.

Finch-Savage, W.E., Pramanik, S.K., and Bewley, J.D. (1994) The expression of dehydrinproteins in desiccation-sensitive (recalcitrant) seeds of temperate trees. Planta 193: 478-485.

Fincher, G.B. (1989) Molecular and cellular biology associated with endosperm mobilizationin germinating cereal grains. Ann. Rev. Plant Physiol. Plant Mol. BioI. 40: 305-346.

Fineran, B.A., Wild, DJ.e., and Ingerfeld , M. (1982) Initial wall formation in the endospermof wheat, Triticum aestivum: a reevaluation . Can. J. Bot. 60: 1776-1795.

Fontes, E.B.P., Shank, B.B., Wrobel, R.L., Moose, S.P., 0 Brian, G.R., Wurtzel, E.T., andBoston , R.S. (1991) Characterization of an immunoglobulin binding protein homolog inthe maize jioury-2 endosperm mutant. Plant Cell 3: 483-496.

Frey-Wyssling , A., Grieshaber, E., and Miihlethaler, K. (1963) Origin of spherosomes in plantcells. J. Ultrastruc. Res. 8: 506-516.

Fu, J.R., Zhang , B.Z., Wang, X.P., Qiao, Y.Z., and Huang, X.L. (1990) Physiological studieson desiccation, wet storage and cryopreservation of recalcitrant seeds of three fruit speciesand their excised embryonic axes. Seed Sci. & Technol. 18: 743-754.

Gallie, D.R., and Young, T.E. (1994) The regulation of gene expression in transformed maizealeurone and endosperm protoplasts . Plant Physiol. 106: 929-939.

Gifford, D.J., Greenwood, J.S., and Bewley, J.D. (1982) Deposition of matrix and crystalloidstorage proteins during protein body development in the endosperm of Ricinus communisL. cv. Hale seeds. Plant Physiol. 69: 1471-1478.

Giovinazzo, G., Manzocchi, L.A., Bianchi, M.W., Coraggio, I., and Viotti, A. (1992) Functionalanalysis of the regulatory region of a zein gene in transiently transformed protoplasts. PlantMol. BioI. 19: 257-263.

Giroux, MJ., Boyer, C., Felix, G., and Hannah, LiC. (1994) Coordinated transcriptionalregulation of storage product genes in the maize endosperm. Plant Physiol. 106,713-722.

110 Darleen A . DeMason

Gonzales, J.w., Rhodes, A.M., and Dickinson, D.B. (1974) A new inbred with high sugarcontent in sweet com . HortScience 9: 79-80.

Gori, P. (1987) The fine structure of the developing Euphorbia dulcis endosperm. Ann. Bot.60: 563-569.

Greenberg, J.T. (1996). Programmed cell death : a way of life for plants . Proc . Natl. Acad. Sci.93: 12094-12097.

Greenwood, J.S., and Bewley, J.D. (1982) Seed development in Ricinus communis (castorbean). 1.Descriptive morphology. Can. J. Bot. 60: 1751-1760.

Greenwood, J.S., and Bewley, J.D. (1984) Subcellular distribution of phytin in the endospermof developing castor bean: a possibility for its synthesis in the cytoplasm prior to depositionwithin protein bodies. Planta 160: 113-120.

Greenwood, J.S., and Bewley, J.D. (1985) Seed development in Ricinus communis (castorbean). III. Pattern of storage protein and phytin accumulation in the endosperm. Can. J.Bot. 63: 2121-2128.

Gustafson, J.P. and Lukaszewski, AJ. (1985) Early seed development in Triticum - Secaleamphiploids. Can. J. Gen. and Cytol. 27: 542-548.

Hannah, L.e. , Giroux, M., and e. Boyer, e. (1993) Biotechnological modification of carbo-hydrates for sweet maize and maize improvement. Sci . Hortic . 55: 177-197.

Hannig, E. (1904) Zur Physiologie pflanzlicher Embryoen. 1.Uber die Kultur van Cruciferen-Embryonen ausserhalb des Embryosacks. Bot. Z. 62: 45-80.

Hawker, J.S ., and Buttrose, M.S. (1980) Development of the almond nut tPrunus dulcis (Mill.)D. A. Webb). Anatomy and chemical composition of fruit parts from anthesis to maturity.Ann. Bot. 46: 313-321.

Hepler, P.K., and Jackson, W.T. (1968) Microtubules and early stages of cell-plate formationin the endosperm of Haemanthus katherinae Baker. J. Cell BioI. 38: 437--466.

Herman, E.M. (1987) Immunogold-Iocalization and synthesis of an oil-body membrane proteinin developing soybean seeds. Planta 172: 336-345.

Herman, E.M. (1995) Cell and molecular biology of seed oil bodies . In: Kigel, J., and Galili, G.(eds) Seed Development and Germination, pp. 195-214, Marcel Dekker, Inc., New York.

Hood, L.E, and Liboff, M. (1983) Starch ultrastruture. In: Bechtel , D.B. (ed) New Frontiers inFood Microstructure , pp. 341-370, Amer. Assoc. Cereal Chemists. St. Paul, MN.

Huang, A.H.C. (1994) Structure of plant seed oil bodies. Current Opinion in Structural Biology4: 493-498.

Hueros, G., Varotto, S., Salamini, E, And Thompson, R.D. (1995) Molecular characterizationof BETl , a gene expressed in the endosperm transfer cells of maize . Plant Cell 7: 747-757.

James, M.G., Robertson, D.S., and Myers, A.M. (1995) Characterization of the maize genesugary ], a determinant of starch composition in kernels . Plant Cell 7: 417--429.

Johri, B.M., and Rao, P.S. (1984) Experimental embryogenesis. In: Johri , B.M. (ed) Emryologyof Angiosperms, pp. 735-802, Springer-Verlag, Berlin.

Keeling, PL., Baird, S., and Tyson, R.H. (1989) Isolation and properties of protoplasts fromendosperm of developing wheat grain. Plant Sci . 65: 55-62.

Kerr, T., and Anderson, D.B. (1944) Osmotic qualities in growing cotton bolls . Plant Physiol.19: 338-349.

Khoo, D., and Wolfe, M.J. (1970) Origin and development of protein granules in maizeendosperm . Amer. J. Bot. 57: 1042-1050.

Kiesselbach, T.A., and Walker, E.R. (1952) . Structure of certain specialized tissues in thekernel of com. Amer. J. Bot. 39: 561-569.

Knowles, R.V., McMullen, M., Yerk, G., Phillips , R.L., Kraemer, S., and Srienc , E (1992)Endosperm mitotic activity and endoreduplication in maize affected by defective kernelmutations. Genome 35: 68-77.

Knowles, R.V., and Phillips, R.L. (1988) Endosperm development in maize . Int. Rev. Cytol.112: 97-136.


Kerrnode, A.R. (1990) Regulatory mechanisms involved in the transition from seed develop-ment to germination, Critical Rev. Plant Sci. 9: 155-1 95.

Krishnan, H.B., Franceschi, V.R., and Okita, T.W. (1986) Immunochemical studies on the roleof the Golgi complex in prote in-body formation in rice seeds. Planta 169: 471-480.

La Rue, C.D. (1947) Growth and regeneration of the endosperm of maize in culture. Amer. J.Bot. 34: 585-586 (abstr).

La Rue, C.D. (1949) Cultures of the endosperm of maize. Amer. J. Bot. 36, 798 (abstr).Lee, B.T., Murdoch, K., Topping, J., Jones, M.G.K., and Kreis, M. (1991)Transient expressionof foreign genes introduced into barley endosperm protoplasts by PEG-mediated transferor into intact endosperm tissue by microprojectile bombardment. Plant Sci. 78: 237-246.

Lending, C.R., Chesnut, R.S., Shaw, K.L., and Larkins, B.A. (1989) Immunolocalization ofavenin and globulin storage proteins in developing endosperm of Avena sativa L. Planta178: 315-324.

Lending, C.R., and Larkins, B.A. (1989) Changes in the zein composition of protein bodiesduring maize endosperm development. Plant Cell I: 1011-1023.

Lending, C.R., Kriz, A.L., Larkins, B.A., and Bracker, C.E. (1988) Structure of maize proteinbodies and immunol ocytochemicallocalization of zeins. Protoplasma 143: 51-62.

Lin, B.-Y. ( 1978) Structural modifications of the female gametophyte associated with theindeterminate gam etophyte (ig) mutant in maize. Can. J. Genet. Cytol. 29: 249-257.

Lin, B.-Y. (1984) Ploidy barrier to endosperm development in maize. Genetics 107: 103-115.Lodish, H., Baltimore, D., Berk, A., Zipursky, S.L., Matsudaira, P., and Darnell, J. (1995)Molecular Cell Biology, 3rd Edit ion. W.H. Freeman and Co., New York.

Lohmer, S., Naddaloni, M., Motto, M., Di Fonzo, N., Hartings, H., Salamini, E, and Thompson,R.D. ( 1991) The maize regulatory locusOpaque-2 encodes a DNA-binding protein whichactivates the transcription of the b-32 gene. The EMBO J. 10: 617-624.

Lopes, M.A., and Larkins, B.A. (1993) Endosperm origin, development, and function. PlantCell. 5: 1383-1 399.

Lott, J.N.A. (198 1) Protein bodies in seeds. Nordic J. Bot. I: 421-432.Lott , J.N.A. (1982 ) Microanalysis of seed tissue. In: Bechtel, D.B. (ed.) New Frontiers in FoodMicros tructure, pp. 317-338, Amer. Assoc. Cereal Chemists. St. Paul, MN..

Lott , J.N.A., Greenwood, J.S., and Batten, G.D. (1995) Mechanisms and regulation of min-erai nutrient storage during seed development. In: Kigel, J. and Galili, G. (eds) SeedDevelopment and Germination, pp. 215-235 , Marcel Dekker, Inc., New York.

Lur, H.-S., and Setter, T.L. (1993a) Role of auxin in maize endosperm development. PlantPhysiol. 103: 273-280.

Lur, H.-S., and Setter, T.L. (1993b) Endosperm development of maize defective kernel (dek)mutants. Auxin and cytokinin levels. Ann. Bot. 72: 1-6.

Lyznik, L.A., and Tsai, C.Y. (1989) Protein synthesis in endosperm cell cultures of maize (Zeamays L.). Plant Sci. 63: 105-114.

Manzocchi , L.A. (1991) Stable endosperm cell suspension cultures from wild-type andopaque-2 maize. Plant Cell Rep. 9: 555-558.

Manzocch i, L.A ., Bianch i, M.W., and Viotti, A. (1989) Expression of zein in long termculturesof wildtype and opaque-Z maize endospenns . Plant Cell Rep. 7: 639-643.

Marinos, N.G. (1970) Embryogenesis of the pea (Pisum sativumi . I. The cytological environ-ment of the developing embryo. Protoplasma 70: 261-279.

Marocco, A .,Santucci, A.,Cerioli, S., Motto,M., Di Fonzo, N.,Thompson, R., andSalamini, E( 1991) Three high-lysine mutations control the level of ATP-binding HSP70-like proteinsin the maize endosperm. Plant Cell 3: 507-51 5.

Marshall, S.w. (1987) Sweet com. In: Watson, S.A., and Ramstad, P.E. (cds) Com: Chemistryand Technology, pp. 431-445, Amer. Assoc. Cereal Chemists, lnc., St. Paul, MN.

Mart in, A.C. (1946) The comparative internal morphology of seeds. Amer. MidI. Naturalist36: 5 13-660.

Mauney, J.R. (1961) The culture in vitro of immature cotton embryos. Bot. Gaz. 122: 205-209.

11 2 Darleen A . DeMason

MacDonald, ED., and Preiss, J. (1985) Partial purification and character ization of granule-bound starch synthases from normal and waxy maize. Plant Physiol. 78: 849-852.

McClintock, B. (1978) Development of the maize endosperm as revealed by clones. In: Subtel-ny, S., and Sussex, LM. (eds) The Clonal Basis of Development , pp. 2 17-237, AcademicPress, New York.

Meier, H. (1958) On the structure of cell walls and cell wall mannans from ivory nuts and fromdates. Biochim. Biophys. Acta 28: 229-240.

Meier, H., and Reid, J.S.G. (1977) Morphological aspects of galactomannan formatio n in theendosperm of Trigonella foenum-graecum L. (Leguminosae) . Planta 133: 243-248.

Meier, H., and Reid, J.S.G. (1982) Reserve polysaccharides other than starch in higher plants.In: Loewus, EA ., and Tanner, W. (eds) Plant Carbohydrates I: Intracellul ar Carbohydrates,pp. 4 18--471, Springer-Verlag, Berlin.

Millin, B.1., and Shewry, P.R. (1979) The synthesis of proteins in normal and high lysine barleyseeds. In: Laidman, D., and Wyn Jones, R.G. (eds) Recent Advances in the Biochemistryof Cerea ls, pp. 239-273, Academic Press, London.

Miller, M.E., and Chourey, P.S. (1992)The maize invertase-deficient miniature-I seed mutationis associ ated with aberrant pedicel and endosperm development. Plant Cell 4: 297-305.

Morrison, LN., and O'Brien , T.P. (1976) Cytokinesis in the developing wheat grain; Divisionwith and without a phragmoplast. Planta 130: 57-67.

Murray, DR (1988) Nutrition of the Angiosperm Embryo. John Wiley and Sons, Inc., NewYork.

Newcomb, W. (1973) The development of the embryo sac of sunflower Helianthus annuusafter fertilization. Can. J. Bot. 51: 879-890.

Newcomb, W. (1978) The develoment of cells in the coenocytic endosperm of Afr ican bloodlily Haemanthus katherin ae. Can. J. Bot. 56: 483- 501.

Neuffer, M.G., and Sheridan, W.F. (1980) Defective kernel mutants of maize. L Genetics andlethality studies. Genetics 95: 929-944.

Olsen, a .-A., Potter, R.H., and Kalla, R. ( 1992) Histo-different iation and molecular biologyof developing cereal endosperm. Seed Sci. Res. 2: 11 7-1 31.

Pan, D., and Nelson, O.E. (1984) A debranc hing enzyme deficiency in endosperms of sugary- Imutants of maize. Plant Physiol. 74: 324-328.

Pomeranz, Y , and Bechtel, D.B. (1978) Structure of cereal gra ins as related to end useproperties. In: Hultin, H.O., and Milner, M. (eds) Postharvest Biology and Biotechnolgy,pp. 244-266, Food & Nutrition Press, Westport, Conn.

Quayle, T.1.A., Hetz, w., and Feix, G. (199 1) Characterization of a maize endosperm cultureexpressing zein genes and its use in transient transformation assays . Plant Cell Rep. 9:544-548.

Raghavan, V. (1986) Embryogenesis in Angiosperms: A Developmental and ExperimentalStudy. Cambridge University Press, Cambridge, England.

Raghavan, V., and Srivastava, P.S. (1982) Embryo culture. In: Johri, B.M. (ed) ExperimentalEmbryology of Vascular Plants, pp. 195-230, Springer-Verlag, Berl in.

Raghavan, V., and Torrey, J.G. (1963) Growth and morphogenesis of globular and olderembryos of Capse lla in culture. Amer. J. Bot. 50: 540-551 .

Rangel, B., Platt, K.A., and Thomson, W.W. Ultrastructural aspects of the cytoplasmic originand accumulation of oil in olive fruit (O /ea europaea L.) Physiol. Plant. (in press).

Rietsema, J., Satina, S., and Blakeslee, A.F. (1953) The effect of sucrose on the growth ofDatura stramonium embryos in vitro. Amer. J. Bot. 40: 538-545.

Rijven, A.H.G.C. (1952) In vitro studies on the embryo of Capsella bursa -pastoris. Acta Bot.Neerl. 1: 157-200.

Roberts, E.H. (1973) Predicting the storage life of seeds. Seed Sci. and Technol. I : 499-5 14.Ryczkowski, M. (1960) Changes of the osmotic value during development of the ovule. Planta55: 343-356.


Ryczkowski, M. (1969) Changes in osmotic value of the central vacuole and endosperm sapduring the growth of the embryo and ovule . Z. Pflanzenphysiol61 : 422--429.

Sanwo, M.M., and DeMason, D.A. (1992) Characteristics of a-amylase during germinationof two high-sugar sweet com cultivars of Zea mays L. Plant Physiol. 99: 1184-1192.

Sanwo , M.M., and DeMason, D.A. (1993) A comparison of a -amylase isozyme profiles inselected Su and high sugar sweet com (sh-2, su-I , su-I se) lines (Zea mays L.). Int. J.Plant Sci . 154: 395--405.

Sanwo , M.M., and DeMason, D.A. (1994) Gibberellic acid (GA3)-induced enhancement ofa-amylase activity in the aleurone of shurnken-2 maize kernels. Amer. J. Bot. 81:987-996.

Saravitz, e.H., and Boyer,CD, (1987) Starch characteristics in cultures of normal and mutantmaize endosperm. Theor. App\. Genet. 73: 489--495.

Schel , J.H.N., Kieft , H., and Van Lammeren, A.A.M. (1984) Interactions between embryo andendosperm dur ing early developmental stages of maize caryopses (Zea mays).Can. J. Bot.62: 2842-2853.

Schm idt, R.J ., Burr, EA. , and Burr, B. (1987) Transposon tagging and molecular analysis ofthe maize regulatory locus opaque-2 . Science 238: 960-963.

Schmidt, R.J ., Ketudat, m, Aukerman, M.J., and Hoschek, G. (1992) Opaque-2 is a transcrip-tional activator that recognizes a specific target site in 22-kD zein genes . Plant Cell 4:689-700.

Schulz, P., and Jensen, W.A. (1969) Capsella embryogenesis: The suspensor and the basal cell.Protoplasma 67: 139-163.

Schwall, M., and Feix , G. (1988) Zein promoter activity in transiently transformed protoplastsfrom maize . Plant Sci . 56: 161-166.

Schwartzenbach, A.M. (1971) Observations on spherosomal membranes. Cytob iologie 4: 145-147.

Selmar, D., Frehner, M., and Conn , E.E. (1989) Purification and properties of endospermprotoplasts of Hevea brasiliensis L. J. Plant Physiol. 135: 105-109.

Shannon, J.e. (1982) Maize endosperm cultures. In: Sheridan, WE (ed) Maize for BiologicalResearch, pp. 397--400, Plant Mol. BioI. Assoc., Charlottesville , VA.

Shannon, J.e. , and Batey, J.W (1973) Inbred and hybrid effects on establishment of in vitrocultures of Zea mays L. endo sperm . Crop Sci. 13: 491--493.

Shannon, J.e. , and Garwood, D.L. (1984) Genetics and physiology of starch development. In:Whistler, R.L., Bemiller, J.N. , and Paschall, E.E (eds) Starch : Chemistry and Technology,pp. 25-86, Academic Press, Orlando, Fl..

Shaw, J.R., and Hannah , LiC. (1992) Genomic nucleotide sequence of wild-type shrunken- 2allele of Zea mays . Plant Physiol. 98: 1214-1216.

Shotwell, M.A. , and Larkin s, B.A. (1989) The molecular biology and biochemistry of seedstorage proteins. In:Marcus ,A. (ed) The Biochemi stry of Plants, Vol. 15: AComprehens iveTreat ise, pp. 297-345, Academic Press , New York.

Shure, M. , Wessler, S. and Federoff, N. (1983) Molecular identification and isolation of theWaxy locus in maize. Cell 35: 225-233.

Sivak , M.N., and Preiss , J. (1995) Starch synthesis in seeds. In: Kigel, J., and Galili, G. (eds)Seed Development and Germination, pp. 139-168, Marcel Dekker, Inc., New York.

Smimova, E.A., Wawrowsky, K.A. , and Bajer, A.S. (1992) Microtubule nucleating centersreflect microtubule polarity in interphase and mitosis of higher plant Haemanthus. Mol.BioI. Cell 3: 343a .

Smith, J.G. (1973) Embryo development in Phaseolus vulgaris: II. Analysi s of selected inor-ganic ions , ammon ia, organic acids , amino acids and sugars in the endosperm liquid. PlantPhysiol. 51: 454--458.

Srivastava, P.S. (1982) Endo sperm culture. In: Johri, B.M. (ed) Experimental Embryology ofVascular Plants , pp. 175-193, Springer-Verlag, Berlin .

Srivastava, P.S., and Johri, B.M. (1992) Endosperm culture . In: Lindsey, K. (ed) Plant TissueCulture Manual, pp. E3, 1-21 , Kluwer Academic Publishers, Dordrecht, The Netherlands.


Stankovic, B., Abe, S., and Davies, E. (1993) Co-localization of polysomes, cytoskeleton , andmembranes with protein bodies from com endosperm. Protoplasma 177: 66-72.

Steeves, T. A., and Sussex, LM. (1989) Pattern s in Plant Development. Cambridge UniversityPress.

Straus, J., and La Rue, C.D. (1954) Maize endosperm tissue grown in vitro. I. Culture require-ments. Amer. J. Bot. 41: 687-694.

Tabata, M., and Motoyoshi, E (1965) Hereditary control of callus formation in maizeendosperm cultured in vitro. Japan J. Genet. 40: 343-355.

Taylor, J.R.N., Schussler, L., and Liebenberg, N.W. (1985) Protein body format ion in starchyendosperm of developing Sorghum bicolor (L.) Moench . seeds. S. Afr. J. Bot. 51 : 35-40.

Tsai, C.Y., and Nelson, O.E. (1966) Starch deficient mutant lacking adenosine dipho sphateglucose pyrophosphorylase activity. Science 151: 341-343.

Tsai, C.Y.,Salmini, E , and Nelson, O.E. (1970) Enzymes of carbohydrate metabolism in thedeveloping endosperm of maize. Plant Physiol. 46: 299-306.

Veda, T., and Messing, J. (1991) A homologous expression system for cloned zein genes.Theor. Appl. Genet. 82: 93-100.

VanLammeren, A.AM. (1988) Structure and function of the microtubul ar cytoskeleton duringendosperm development in wheat: an immunofluorescence study. Protoplasma 146: 18-27.

Vertucci, c.w., and Farrant, J.M. (1995) Acquisition and loss of desiccation tolerance. In:Kigel, J., and Galili, G. (eds) Seed Development and Germination, pp. 237-271, Marcel,Dekker, Inc., New York.

Vijayaraghavan, M.R., and Prabhakar, K. (1984) The endosperm. In: Johri, B.M. (ed) Embry-ology of Angiosperms. pp, 319-376, Springer-Verlag, Berlin.

XuHan, X., and VanLammeren, A A.M. (1993) Microtubular configurations during the cellu-larization of coenocytic endosperm in Ranunculus sceleratus L. Sexual Plant Reproduc-tion. 6: 127-132 .

XuHan, X., and van Lammeren, A.A.M. (1994) Microtubular configurations dur ing endospermdevelopment in Phaseolus vulgaris. Can. J. Bot. 72: 1489-1495.

Wang, H.L., Offler, C.E., and Patrick, J.w. (1995a) The cellular pathway of photosynthatetransfer in the developing wheat grain. II. A structural analysis and histochemical studiesof the pathway from the crease phloem to the endosperm cavity. Plant, Cell and Environ.18: 373-388.

Wang, H..L. , Patrick, J.w. , Offler, C.E. , and Wang, X.E. (1995b ) The cellular pathway ofphotosynthate transfer in the developing wheat grain. III. A structural analysis and physio-logical studies of the pathway from the endosperm cavity to the starchy endosperm. Plant,Cell and Environ. 18: 389-407.

Wang, T.L., and Hedley, CL. (1991) Seed development in peas: knowing your three ' r's' (orfour, or five). Seed Sci. Res. I: 3- 14.

Wang, TL , and Hedley, C.L. (1993) Genetic and developmental analysis of the seed. In:Casey, R., and Davies, D.R. (eds) Peas: Genetics, Molecul ar Biology and Biotechnology,pp. 83- 120, CAB, International, Wallingford, U.K.

Yeung, E.C. (1980) Embryogeny of Phaseolus :The role of the suspensor. Z. Pflanzenphysiol.96: 17-28.

Yeung, E.C., and Cavey, M.J. (1988) Cellular endosperm formation in Phaseolus vulgaris. LLight and scanning electron microscopy. Can. J. Bot. 66: 1209-1216.

Yeung, E.C., and Clutter, M.E. (1978) Embryogeny of Phaseolus coccineus: Growth andmicroanatomy. Protoplasma 94: 19-40.

YouIe, R.I., and Huang, A.H.C. (1976) Protein bodies from the endosperm of castor bean:subfractions, protein components, lectins and changes during germ ination . Plant Physiol.58: 703-709.

Youle, R.J., and Huang, AH.C. (1981) Occurrence of low molecular weight and high cysteinecontaining albumin storage proteins in oilseeds of diverse species. Am. J. Bot. 68: 44-48.

Endosperm Structure and Development L15

Young, T.Y., Gallie, D.R., and DeMason , D.A. Ethylene mediated programmed cell deathduring maize endosperm development of Su and sh2 genotypes. Plant Physiol., in press.

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[Advances in Cellular and Molecular Biology of Plants] Cellular and Molecular Biology of Plant Seed Development Volume 4 || Endosperm Structure and Development