POST-POLLINATION CALLOSE DEVELOPMEN IN T OVULES OF ... · POST-POLLINATION CALLOSE DEVELOPMEN IN T OVULES OF RHODODENDRON AND LEDUM (ERICACEAE): ZYGOTE SPECIAL WALL E. G. WILLIAMS

J. Cell Sci. 69, 127-135 (1984) 127Printed in Great Britain © The Company of Biologists Limited 1984

POST-POLLINATION CALLOSE DEVELOPMENT INOVULES OF RHODODENDRON AND LEDUM

(ERICACEAE): ZYGOTE SPECIAL WALL

E. G. WILLIAMS, R. B. KNOX, V. KAULPlant Cell Biology Research Centre, School of Botany

AND J. L. ROUSESchool of Physics, University of Melbourne, Parkville, Victoria 3052, Australia

SUMMARY

In Rhododendron spp. and Ledum groenlandicum a callose wall is laid down around the zygotein the first 2 days after fertilization. The periodic acid/Schiff-positive, aniline blue-fluorescence-positive callosic wall is initiated adjacent to the degenerating synergid, extends to cover the entirezygote surface, and remains visible during the initiation of embryogeny as the zygote elongatesbefore the first proembryonal division. Unfertilized ovules show eventual callose deposition in theovule wall cells during senescence in undeveloped abscising pistils, but show no development ofcallose within the embryo sac. Possible roles of a zygote special callose wall are discussed.

INTRODUCTION

After fertilization the angiosperm zygote must begin a new programme ofsporophyte development based on the diploid genotype established at the time ofgamete fusion. In close proximity to maternal and endosperm cells of differentgenotypes, this cell must initiate expression of a new genotype in a new developmentalsequence. The process has conceptual similarities to the reciprocalsporophyte—* gametophyte phase change, which occurs at the time of sporogenesisand is accompanied by deposition around the sporocytes of an aniline blue-fluorescence(ABF)-positive wall of callose (De Sloover, 1961; Waterkeyn, 1961,1962, 1964; Heslop-Harrison, 1964, 1966a,6; Rodkiewicz, 1967, 1970; Rodkiewicz& Gorska-Brylass, 1968; Jalouzot, 1970; Knox & Heslop-Harrison, 1970; Russell,1979; and see review by Kapil & Tiwari, 1978). This wall is composed predominantly ofl,3-/3-glucans or mixed 1,3-/3- and 1,4-jS-glucans (Clarke & Stone, 1984). Temporarydeposition of callose special walls around the sporocytes and their products, and theconsequent severance of protoplasmic connections between them, has been suggestedto confer some degree of genetic independence between haploid sibs and diploidparent tissues (Heslop-Harrison, 1964, 1966a,6; Rodkiewicz, 1967, 1970; Knox &Heslop-Harrison, 1970). While enclosed within the callose special wall micro-sporocytes and spores are not readily penetrated by various molecules (Heslop-Harrison & Mackenzie, 1967; Knox & Heslop-Harrison, 1970; Southworth, 1971).Thus the presence of these walls may allow the initiation of a developmental phasechange without macromolecular interference from neighbouring cells of different

128 E. G. Williams, R. B. Knox, V. Kaul andj. L. Rouse

genotype. It is not altogether clear whether this isolating function of the callose special

wall is imposed by virtue of impermeability or physical restraint to uptake by expan-

sion (Knox & Heslop-Harrison, 1970).

Our observations on several species of Rhododendron and Ledum groenlandicum

suggest that a callose special wall is laid down around the zygote at a time that may

be related to the gametophyte—+ sporophyte phase change, and at which a rapid

separation of the living zygote from degenerating synergids may also be required.

MATERIALS AND METHODS

The species investigated belong to the Ericaceae: Rhododendron ellipticum Maxim., R. occiden-tale A. Gray, R. championae Hook., R. macgregoriae F.v.M., R. kawakamii Hay. varflaviflorumLiv et Chuang, R. retusum (Bl.) Benn., R. amamiense Ohwi and Ledum groenlandicum Oeder.Plants growing in the private collection of one of us (J.L.R.) at Toorak, Melbourne, wereemasculated before anthesis and compatibly hand-pollinated. At various intervals in the first 3 weeksafter pollination, pistils were fixed for examination by either squashing or sectioning. Whole pistilsquash preparations were prepared as described by Williams et al. (1982). For sectioning ovaryslices (2 mm thick) were fixed in 4% paraformaldehyde at pH7, 4°C for 24 h, washed for 2h inmultiple changes of 0-1 M-phosphate buffer at 4°C, dehydrated in a graded ethanol series andembedded in JB-4 resin. Longitudinal sections were cut at 4-6/mi, and temporarily mounted indecolourized aniline blue (Merck Anilinblau WS 0-1 % in OIM-K3PO4 in 10% (v/v) glycerol).These mounts were sealed with nail polish, stored overnight in the dark at 4°C, and examined forcallose fluorescence (Smith & McCully, 1978) using a Zeiss epifluorescence microscope with thefilter combination, KP490, KPS00, RF1510, LP528. After examination the coverslips were removedand the aniline blue stain was washed from the slides. The sections were then stained with theperiodic acid/Schiff reaction (PAS: Jensen, 1962, p. 199) followed by toluidine blue (Feder &O'Brien, 1968), and permanently mounted in Eukitt (O. Kindler, W. Germany).

OBSERVATIONS

Before fertilization in the species examined, the only ABF-positive structures

within the ovule are the filiform apparatus, and the hypostase, which lies outside the

chalazal end of the embryo sac (Williams, Knox & Rouse, 1982).

During the first 48 h after compatible pollen-tube entry into an ovule, an ABF-

positive and PAS-positive wall appears around the zygote. This is first deposited most

Fig. 1. Longitudinal section of an unfertilized mature ovule of R. ellipticum showing eggcell (e), synergid (s), filiform apparatus if), micropyle (m) and central cell with polarnucleus (c). PAS—toluidine blue staining; bright-field illumination. X435.

Fig. 2. Longitudinal section of an unfertilized mature ovule of R. occidentale about 1week after the time at which pollination would normally have occurred, showing egg cell(e), synergid beginning to degenerate, (s) and central cell with polar nucleus (c). ABFstaining; epifluorescence illumination. Only a dull orange-yellow autofluorescence isvisible in this paraformaldehyde-fixed material. No specific callose fluorescence is seen inthe egg cell. X435.

Figs 3, 4. Longitudinal sections of R. ellipticum ovules 11 days after compatible pollina-tion, showing development of the special callose wall (w) between the zygote (2) anddegenerating synergid (s). Fig. 3: PAS—toluidine blue staining; bright-field illumination.X435. Fig. 4: ABF staining; epifluorescence illumination. An intense yellow-green specificcallose fluorescence is visible in the zygote special wall. X43S.

Zygote special callose wall in Ericaceae 129

Figs 1-4

130 E. G. Williams, R. B. Knox, V. Kaul andjf. L. Rouse

Figs 5, 6. ABF-stained squash preparations of/?, ellipticum ovules 8 days (Fig. 5) and 10days (Fig. 6) after compatible pollination. Note fluorescence of pollen tube (p) inmicropyle, zygote special wall (w) and hypostase (h). Epifluorescence illumination. X 160.

Fig. 7. Longitudinal section of an ovule of R. kawakamii 10 days after compatible pollina-tion, showing thick zygote wall (tv), degenerating synergid (s) and micropyle (m).PAS-toluidine blue staining; bright-field illumination. X435.

Fig. 8. Longitudinal section of an ovule of R. kawakamii 12 days after compatible pollina-tion, showing elongation of the zygote (2) and thinning in the chalazal region of the zygotespecial wall (w); degenerating synergid (s); micropyl (m). PAS—toluidine blue staining;bright-field illumination. X435.

Fig. 9. ABF-stained squash preparation of an ovule of R. occidentale 14 days after com-patible pollination, showing elongation of the zygote (2) just before the first proembryonaldivision; pollen tube in micropyle (p); hypostase (h). The line of fluorescence between thezygote and hypostase marks the first plane of division in the endosperm that is cellular abirtitio. Epifluorescence illumination. X160.

Fig. 10. ABF-stained squash preparation of ovules of L. groenlandicum 7 days aftercompatible pollination, showing callose special wall (w) around elongating zygotes; pollentube in micropyle (p); hypostase (h). Epifluorescence illumination. X160.

Fig. 11. ABF-stained squash preparation of senescent R. ellipticum ovules (arrows)from an unpollinated pistil abscising approximately 3 weeks after normal pollinationwould have occurred. Note general callose deposition in ovule wall cells. Epifluorescenceillumination. X63.


Figs 8-11


conspicuously between the zygote and the adjacent degenerating synergid (compareFigs 3—5 with Figs 1 and 2). Later the fluorescence is observed as a sphere around theentire zygote wall (Figs 6, 7). Subsequently, as the zygote elongates before the firstproembryonal division, this wall becomes attenuated in the region of expansion distalto the micropyle (Figs 8-10).

Unfertilized ovules show no callose deposition in the region of the egg cell, eitherbefore or after pollination. In undeveloped pistils abscised after 3-4 weeks frompollination, unfertilized senescent ovules show generalized deposits of callose in ovulewall cells, but no callose within the embryo sac (Fig. 11).

DISCUSSION

In previous work (Williams et al. 1982) the fluorescent zygote wall was observedin several incompatible interspecific crosses of Rhododendron, and interpreted at thattime as a possible anomaly of the pollen-tube tip within the embryo sac, or a callosedeposit within the ovum stimulated by incompatible pollen tube/embryo sac inter-action. Subsequent work with a greater range of compatibly pollinated materials hasshown this phenomenon to be characteristic of normal early post-fertilization develop-ment. Embryological studies on compatible and incompatible interspecific crosses inwhich pollen tubes enter the ovules (Kaul, Rouse & Williams, unpublished), haveshown that abortion in incompatible crosses may occur after apparently normalfertilization and early proembryonal development.

Deposition of new wall material around the zygote in the first 2 days after fertiliza-tion has also been observed in ultrastructural studies of cotton embryogenesis byJensen (1968, 1974). As in the present study, this wall was described as thickest at themicropylar end and thinnest at the chalazal end. Changes accompanying walldeposition included the shrinkage of the zygote to half the original egg volume,apparently by decrease in the vacuolar volume; appearance of prolific tubule-containing endoplasmic reticulum; relocation of plastids; starch accumulation in theplastids; formation of giant polyribosomes and the appearance of large numbers ofnew ribosomes. These correlated changes were interpreted to indicate a period ofactivation or conversion in cell function. Synthesis of a new ribosome population hasalso been observed at the sporophyte/gametophyte phase change (Mackenzie,Heslop-Harrison & Dickinson, 1967; Dickinson & Heslop-Harrison, 1970; Williams,Heslop-Harrison & Dickinson, 1973). The presence of a continuous PAS-positivewall around the zygote has also been reported by Olson & Cass (1981) for Papavernudicaule. Since it appears that the egg wall is likely to be discontinuous, some post-fertilization wall synthesis is implied.

A special callose wall around the zygote may function to preserve the geneticisolation of this cell from maternal and endosperm tissues of different genotype duringinitiation of the new sporophyte development phase. In addition, it may functionsimilarly to 'wound callose' to seal off the zygote from the adjacent degeneratingsynergid. Alternatively, as suggested by Jensen (1968), the special wall may be invol-ved in determining the precise shape and volume of the zygote, and in controlling


osmotic balance between this cell, the now developing endosperm, and other adjacentcells. An analogous alternative function for the microsporocyte callose special wall hasbeen proposed by Knox & Heslop-Harrison (1970): apart from possible involvementin direct filtering out of macromolecules by virtue of low callose permeability, the wallmay function to restrain cell expansion physically and therefore limit movement ofmaterials into cells to that possible by exchange only. The assumption of a sphericalshape by microsporocytes after callose wall deposition, and the immediate expansionof young microspores on release from meiotic tetrads, do suggest a degree of physicalrestraint imposed by the callose special wall.

In developing pollen, the generative cell becomes temporarily isolated from thecytoplasm of the vegetative cell by a plasmodesma-free, callose wall (Gorska-Brylass,1967; Heslop-Harrison, 1968; McConchie, 1983). This wall is at first hemispherical,cutting off a lens-shaped generative cell against the vegetative cell wall. Subsequently,as callose deposition continues to cover the entire generative cell surface, the cellassumes a spherical shape and separates from the vegetative cell wall. In parallel withpossible functions suggested for microsporocyte and zygote special walls, thegenerative cell callose wall may isolate the future gamete genome from transientactivating molecules in the highly metabolic vegetative cell cytoplasm (Heslop-Harrison, 1968). A further possible function may also be envisaged: the physicalrestraint imposed by the wall during a period of osmotic expansion of the generativecell may cause it to become spherical and to aid the detachment from the pecto-cellulosic intine, to which it is initially attached.

Of particular relevance to the present study are the observations of Wilms, vanWent, Cresti & Ciampolini (1983) on development of special walls around the nucellar'zygote-resembling' cells, which give rise to adventive embryos in Citrus. These cellslay down thick new walls within the original primary walls, severing plasmodesmatalconnections and isolating themselves from neighbouring nucellar cells, which laterdegenerate. Although cytochemical tests were not applied, the ultrastructural imageof the wall of these zygote-resembling cells is similar to that of other examples ofcallose deposits (e.g. see Dickinson & Lewis, 1975). The severance of plasmodesmatalconnections by deposition of a special wall is analogous to the blockage of cytomicticchannels by deposition of the sporocyte special callose wall during the first meioticprophase in microsporogenesis (Heslop-Harrison, 1966a,b).

Possibly, the special wall of Citrus zygote-resembling, embryogenic cells forms anisolating genetic screen that allows a phase change from the differentiated nucellarcondition to the re-initiation of sporophyte morphogenesis. As suggested for thezygote wall in Rhododendron and Ledum, the special wall of Citrus adventive 'zygotes'may also function to isolate these viable meristematic cells from adjacent degeneratingcells.

The early appearance of a special wall around a proembryonal cell may be a featureextending to certain instances of somatic embryogenesis in vitro. Street & Withers(1974), for example, described and illustrated the isolation of the basal proembryonalcell of induced embryoids of Daucus carota from surrounding cells by a thick,plasmodesma-free wall.


A zygote callose special wall therefore appears to be an important developmentalmarker in embryogenesis, adding a new and significant finding to the list of processesof reproduction in which callose is involved (see Dumas & Knox, 1983).

In plant breeding, callose is also known as a useful marker of ovule viability in certainspecies. Callose deposits become general throughout the ovule when it becomesinviable, presumably because of senescence. This has been demonstrated in cytologi-cal studies of the breeding systems of various stone fruits (see Anvari & Stosser, 1978,1981; Stosser & Anvari, 1982; Martinez-Tellez & Crossa-Raynaud, 1981). Theoccurrence of callose deposits in the unfertilized ovules of abscised pistils ofRhododendron indicates that the phenomenon may be more general, and may providea useful guide for the occurrence of fertilization during breeding programmes.

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(Received 9 January 1984 -Accepted 16 February 1984)

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POST-POLLINATION CALLOSE DEVELOPMEN IN T OVULES OF ... · POST-POLLINATION CALLOSE DEVELOPMEN IN T OVULES OF RHODODENDRON AND LEDUM (ERICACEAE): ZYGOTE SPECIAL WALL E. G. WILLIAMS