The transmitting tract in Gloriosa superba: structure, pistil exudate and pollen tube growth

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The transmitting tract in Gloriosa superba: structure, pistil exudate and pollen tube growth L.G . Ie Roux, P.J. Robbertse* and C.F. van der Merwe "Department of Plant Production and Soil Science, University of Pretoria, Pretoria, 0002 Republic of South Africa Unit of Electron Microscopy, University of Pretoria. Pretoria, 0002 Republic of South Africa Received 9 November 1995; reail'ed 8 May 1996

The structure of the pistil exudate. transmitting tissue and polien tube growth of Gloriosa superba L. were studied. Flowers are protogynous, as the stigmas become receptive one day prior to anther dehiscence. and remain receptive for at least four days. Neutral red and auramine 0 proved to be useful for the indication 01 stigma receptivity. The stigma is wet while the papillae and canal cells form slight interior cell wall in growths reminiscent of transfer ce lls. Pistil exudate is mostly lipidic, confirmed by the prevalence of endoplasmic reticulum in the cytop lasm. The canal exudate includes substances derived from degeneratin.g canal cell walls and lipid·like vesicles breaking down into fine fibrillar matter possibly associated with the pollen lube cell wall. Pollen tubes grow in close association with the stylar secretion, reaching most ovules, even though pollen tube numbers decrease on their way down the stylar canal. Callose deposition in the nucellus of some self-pollinated ovules could be indicative of a parlly pre-zygotic gametophytic self·incompatibllity system. Keywords : Pistil exudate , pollen tube growth, self-incompatibility, transmitting tissue, ultrastructure. "To whom correspondence should be addressed.

Introduction Gloriosa superba L. (Colchicaceae) is a typical cryptophyte wit h ephemeral, climbing stems and a perennial subterranean hypopodial tuber (Ie Roux & Robbense 1994). These plants are potentially of economic importance because of the hi gh concentration of colc hicine contained wi thin the seed (Sarin et al. 1974). Seed set, after artificial self- and cross-pollinations (unpublished data), was disappointingly low and variable, and had to be investigated. This article forms part of an extensive study on the sex ual reproduction of this species. Neither the transmitting tract nor the pollen tu be pathway have been described in Cforiosa superba. Many studies concerning sty lar transmitting ti ssue deal with the morphOlogy, ultrastru cture and secretion of the stigma and style (Rosen & Thomas 1970; Labarca & Loewus 1973 ; Knox 1982; Miki-Hirosige er al. 1987; Mackenzie et al. 1990; Nakanishi et 01. 1991). Among surveys describing the pollen tube pathway in the entire pistil. several include species with sty lar canals, e.g. Lilium longiflorwn (Janson et al. 1994), Trimezia fosteriana (Bystedt & Vennigerholz 1991). Casteria (Willemse & Fransen-Verhijen 1988) aod Strelitzia regi,we (Kronestedt et al. 1986). This pool of information served as a source of reference for comparing structu res found in G. superba and could be indirectly related to G. superba where a similar condition prevails. In this report the st ru cture of the transmitting tissu e and composition of stigmatic and stylar secretion, as well as pollen tube growth was studied, applying light microscopical, scanning and transm ission electron microscopic tec hn iques.

Material and Methods G. superba flowers were obtained from container-grown plants cultivated at the University of Pretoria (24°45'S 28°l4'E). These plants were subjected to prevailing weather conditi ons but were watered at regular intervals and protected from sun. birds, insects and hail by means of a 20% shade netting supported by a wire mesh. Lighl microscopy (LM). Semi-thin sections (-I )..lm) of stigmas. embedded in Quetol 65 1 as for TEM, were stained with Periodic acid Schiff's reagent (PAS) and toluidine blue (O'Brien & McCully 1981). Stigma receptivity was determined using 0.01% neutral red which is a non-specific stain indicating the increased permeability of

the cell walls. The presence of stigma surface lipids, which is
Results The Glorioso pistil is tricarpellate and the stylar base is bent at ri ght angles to the ovarian axis. Styles of flowers at anthesis are 40--80 mm long and terminate in three relatively short styloidia covered with stigma papillae (Figure 1A). Figure IB is a tran sverse section of the hollow style showing the sty lar canal lined with transmitting tissue and the peripheral parenchymatous tissue in which three carpellar vascular strands are embedded. The outer subepidermal, large parenchymatous cells contain chloroplasts with PAS-positive starch granul es. Idioblasts, of which the vacuoles are lined with a thick layer of electron-dense, tanninlike substance, are scattered in the ou ter epidermal and parenchymatous tissue (Figure IB & C). Positive staining with neutral red or auramine 0 and hand pollinations indicated that stigmas became receptive approximately one day before anther dehi scence and stayed receptive for a period of at least four days. Pistils pollinated during the mentioned receptive periods resulted in seed se t.

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During stigma rece ptivity, the cytoplasm of the stigma papi llae and stylar canal cells contain abundant ribosomes and both smooth and rough endoplasmic reticulum (ER) with frequently expanding cisternae with associated vesicles of different sizes (Figure ID). Other organelles such as mitochondria, chloroplasts with PAS-positive starch granules and the golgi bodies, consisting of 5-7 cisternae, also appear in the cytoplasm. The ER fragments and golgi vesicles appear to fuse with the plasmalemma of the inner tangential walls of epidermal cells lining the stylae

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canal, to deposi t their contents, thus possibly playing a role in ex udate secretion. Slight cell wall ingrowths develop in the papillae and cell walls of transmitting cells as these vesic les accumulate (Figure 1D). Thl! nuclei in these cells are large and spheril.:al wilh conspicuously electron-dense nucleoli (Figure 2A). Vac uoles in the stig matic tissue are sizeable and contain highly electron-dense phenolic-like granules while the vacuoles of canal cells tend to be smaller and contain fibrillar material. Cells of the transmitting tissue are mutually connected and to the underlying

Figur e 1 A. One of the three stigma lobes (stylodia) showing stigma papillae (P) and germinating pollen grains (G). B. Transverse sec ti on of the hollow style showing the peripheral parenchymatous ti ssue with carpellary vascular strands and scaltered idioblasts (i = idioblasts. vt = vascu lar tissue, tm = transmitting tissue). C. ldioblast showing electron-dense, tannin-like contents within the vacule (c = chloroplast, e =electrondense substances, V = vacuole, cw = cell wall). D. Ultrastructure of stigma papilla showing abundant free ribosomes (r), mitochondria (m), ch loroplasts (e). golgi body (g). producing golgi vesicles Cd) and endoplasmic reticulum (er) wi th frequently expanding cisternae (e). Cell wall intrusions form ed by golgi vesicles.

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Figure 2 A. Longitudinal section of a receptive stigma papillate cell showing subcuticular secretions (S) . Protoplasm contains nucleus (N) , granu lar vacuoles (V), plaslids (P) and mitochondria (M), B. Part of transmitting tissue cell wall and fibrillar exudate (F) containing lipid-like vesicles (LV) (Sc ::::; stylar canal, Cc::::: canal cell) . C. Part of transmitting tissue cell walls (CW) containing lipid-like vesicles (LV) (Cc :::: canal cell), D. Transverse section of a pollen tube tip (PT) growing in th e stylar canal. Lipid -like vesicles (LV) seem to take part in the formation of dark layer (OL) enveloping the pollen tube. .

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parenchyma cells by pl asmodesmata. The unicellu lar stigma papill ae (Fig ure 2A) arc covered by a thin cuticle which hecomes detached at sti gma rece pti vity. The ex udate covering thc papillae appears 10 be foamy with electron-dense and electron-translucent areas. It gave a negative PAS reaction and seemed to hI! mostly lipidic. producing a di stinct ye ll ow fluorescence wi th aura mine O. Pollen grain s germinated on the cxudalc-cOvCrt.!u papi llae , penetrated the cuticle and proceeded to the mucilaginous secretion lining the styla r canal. At thc time when the transmilting tissue ce ll s lining the cana l start to degenerate and the cuticle becomes detached and fibrill ar (Fig ure 2B), an exudate is produced as a film covering the cuticle. Lipid- like vesicle (LV) found in canal cell plastids were a lso observed in the inner layer of the cana l ce ll wa ll (Figu re 2C). The LVs were distri buted along the sty lar cavity, ami in the vicinity of pollen tubes. the fin e tihrillar materi al seems to derive from the LVs and prohabl y forms part of the film around the pollen tu be wall (Figure 2D ). Amorphous elecl run-t ranslut;cnt exudatic substances also ap· reared between the fibrillar compone nts of thc LV Poll en lube cytoplasm is confined to the subapic al zone and cont ains abu ndant mitocho ndria, lipoidal bodies, rihosomes and rough ER (Fig ure 2D). The te rmi na l e nd or the pu llen tu be is re peatedl y sealed off from the rest or the tube by a ca ll ose plug formin g in the vacuo lar zone, behind the nuclear zone. A large number of call ose plu gs per tube was observed after stain ing with deco lo ri zed aniline blue. The growth rate of the pollen tubes was determined by estimati ng the dista nce that the tubes had grown in the sty le at different time intl!rva ls after pollination. Pollen tubes took abo ut 72 h to reac h the end of the stylar transmitting tract, wit h a growt h rate or ap proxim ately 0.7 mm h· l . Si nce sti gmas we re hand·pollinated, a high percen tage o r pollen g rains germinated, producing a large number of pollen tubes that passed through the stylar canaL The number of pollen tubes decreased, however. as the distance fro m both se lfed and crossed stigmas inc reased. Pollen tubes grew alo ng the ovarian tran smitting tissue where they made a 90° (urn towards each ovule, from whe re they were g uided by an obt urator to the micropyle (Figure 1A). Most ovules were reached by pollen tubes approximately fou r days after poll ination. In the case o f self-pollination, bundled -up

207 pollen tubes wi th di sti nct callose fI!at;tion were obse rved in the nucdlu s close to the egg apparat us (Figun; 3B). Ovaries di sSCt;(cd approximately 29 days after pollination showed many aborted ovules amongst seemingly normal devdoping seed.

Discussion The 90:) bend of the style towards the ovarian axis (Le Ruux er al. 1994 - Figure 1A) is mostly typkal of the tribe Iphigenica of the famil y Colchkaccae, including species like Iphigell;a. Omirlwg/ossllfll and Hexacyrris (Dahlgren ct ai. 1985). The bas ic sty· lar morphology of C . .mperh(f is typkal of monocotyledons (Rosen & Thomas 1970; Clarke et al 1977; Knox 1982; Kronested t et al. 1986; Miki-Hirosigi cl al. 1987; Bystedt & Vennigcr· ho hz 1991). T he idioblasts. with electron-dense tannin- like sub· stances in some epidermal cells and ce lls scattered in the ground ti ss ue, arc likely to contain polypht.::nols. Narain (1976) stat~d that !lowe rs of G. Sl/perhll arc prota n ~ drous. He used the bri g ht anll shi ny sti g ma pap illae as a criterion for receptivity and stalt~lI that stigmas stayed receptive for a period of approximatt! ly 32 h. This is in contradic ti on to our observations. where tlowers were dearly protogynous. The stig· mas became recepti ve approximately onc day prio r to a mher dehisccnce, and continued to be so for at least four days. Seed production following pollination in the estim ated receptive stage. as well as the use or the chem icals neutral red and auramine O. seem to give a better ind icat io n of sti gma recepti vity than on ly the colour of stigmatic papi llae. The stigma can be dassified as 'wet papillate' (Heslop Harrison & Shivannah 1977). The stigma papillae and canal t;ells arc si milar to transfer cells (Pate & G unning 1972) with a dense cytoplasm show ing sli g ht interior ce ll wall ingrowths a'isodatcd with ER and dictyosome vesicles whic h are probably active in exudate production. Miki·Hirosigi et al. (1987) reported that ER and dictyosome vesic les are involved in the production of cell walls of sec retory cell s in the style, and also l:lUllched the idea that the exudate is first stored in the cell wall before secn:tion. Simple wall thickenings in the mature papill a celIs, where granulocrine secretion involved ER and dictyosome vesicles, were descri bed in wate r melo n (Sedg ley 198 1. 1982) and Aprcilia

Figure 3 A. Pollen tube (JYr) penetrating the micropyle (M), nuccllus (N) and a sinergid (S). B. Pollen tube (PT) bundling in the nuccllus (N) instead of entering the embryo sac.

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cordifol;a (Kirsten 1992). Highly irregular cell wall ingrowths occurred in the canal eel! walls of Lilium /ol1gijlortlm (MikiHirosigi i'l al. 1987) and Strelitzia regilltlc (Kronestedt eT al. 1986) but WCf\! absent in the papillae, which was quite rcmarkahie since both r.:elI tYpl!S haw a secretory function. Histochemical tests for light microscopy indicated that the pisti l exudate is mostly li pidic. which is supported by the prevalence of ER in the cy toplasm , characteristic of stigmas wilh a lipophilic nature also referred to as reticu late types, as for example Petunia, Forsythia and Persea (Knox 1982). Various polysacc harides, lipids and prote ins have been identitied in moncolyledons (Clarke et al. 1977; Knox 1982; Miki-Hirosigi et al. 1987; Bystedt & Vennigcrholtz 1991) and dicotyledons (Dulberger 1987; Clifford & Owens 1990; Nakanishi et al. 1991). Mackenzie et "I. (1990) showed that the secretion of So/aIJum tubelVSlIm stigmas is purely lipi<.1ic in nature, containing ndlher carbohydrates nor proteins. Olha essen ti al su bstrates for me tabolism in the pistil ex udate may

be. for aampie, minerals, enzymes and hormones (Knox 1982). At ultrastructurallevd , the stigma exudate consists of a foamy substance with c1ectron~dense and electron-translucent arcas, while the canal exudate includes the degenerating canal ce ll walls, LV breaking down into fine fibrillar substances, and amorphous transparent components. Except for the indispensable source of ourrition that the pistil exudate provides to pollen tuhcs, the fibrillar components originating from the LV, and forming a fi lm around the pollen tube, can play an important role in pollen tu be wall formation. Labarca and Loewus (1973) labelled pistils with D-glucose U_14C after anthcsis and have found that 24 h after pollination the pollen tube wall substance cons isted of about 80% of the content found in the cana l exudate. This supports our theory that some substances of the canal exudare might be active in pollen tube wall formation. The pollen tubes of Glorios{I penetrated between the stigma papillae and grew in c lose assoc iation wi th the stylar canal secretion. Among other monocotyledons like Gladiolus (Clarke et al. 1977) and Crocus (Helsop-Harrison 1977), the pollen tube penetrate the cuticle of the papillae and its further growth in the entire transmitting tract is restricted to a mucous layer beneath an intact cuticle. In Gloriosa, a large number of poll en tubes were found passing through the stylar canal but thei r number decr\!ased with increasing distance from the selfed and crossed stigmas. Decreasing numbers of pollen tubes in the stylar traci were reported fro m soya beans (Tilton et fll. 1984) and Strelitzia regillae (Kroncstedt et al. 1986). Although most of the ovules wen! reached by pollen tubes, heavy callose deposition in the egg apparatus area occurred in some self-pollinated ovu les. The decrease in pollen tube numbers, as well as the callose deposition in the micropylar area cou ld be a manifestation of partially pre-zygotic self-incomp atability. Studies on the pollination mechanism and breeding system of Gloriosa revealed that these plums favour cross-poll ination but our results, together wit h those of Narain (1976), confirm that seed set, however, was achieved after sdfing under controlled conditions. Qui te a number of C. superba pollen tu bes seem to be in hi· bited to grow all the way down the transmitting tract, while the bundling of pollen tubes in the nucellus, followed by heavy ca llose deposits near the sine rgids, is a clear indication of unsuccessful fertilization, especially in combination with aborted ovules. The incidence of low and variabl e seed set in G. superba cou ld possibly be the resull of a partially pre-zygotic selfincompati bility mechanism.

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