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ULTRASTRUCTURAL AND CYTOCHEMICAL STUDIESOF THE EMBRYO SUSPENSOR

OF SEDUM REFLEXUM L. (CRASSULACEAE)

DARIA CZAPLEJEWICZ AND MAŁGORZATA KOZIERADZKA-KISZKURNO*

Department of Plant Cytology and Embryology, University of Gdańsk, Wita Stwosza 59, 80-308 Gdańsk, Poland

Received May 21, 2013; revision accepted October 21, 2013

The Crassulaceae family mainly comprises herbaceous leaf succulents, some of which are used as ornamentals.The development of the embryo suspensor in Sedum reflexum L. was investigated using cytochemical methods,light and electron microscopy. The full development and functioning of the suspensor occurs during the lateglobular and heart-stage embryos. The suspensor consists of a large basal cell and a single row of 6–10 chalazalcells. The basal cell produces a branched haustorium which invades ovular tissues. The walls of the haustori-um and the micropylar part of the basal cell form the wall ingrowths that are typical for transfer cells. The densecytoplasm filling the basal cell is rich in profiles of endoplasmic reticulum, active dictyosomes, mitochondria,plastids, microtubules, bundles of microfilaments, microbodies and lipid droplets. The present work revealsthat the suspensor structure in S. reflexum markedly differs from that found in other representatives ofCrassulaceae. Ultrastructural analysis and cytochemical tests (including proteins, insoluble polysaccharides andlipids) indicate that in S. reflexum the embryo suspensor is involved mainly in absorption and transport ofmetabolites from the ovular tissues to the developing embryo proper via the chalazal suspensor cells.

KKeeyy wwoorrddss:: Embryogenesis, suspensor differentiation, ultrastructure, cytochemistry, Sedum.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 55/2: 76–89, 2013DOI: 10.2478/abcsb-2013-0028

PL ISSN 0001-5296 © Polish Academy of Sciences and Jagiellonian University, Cracow 2013

INTRODUCTION

In the embryogenesis of many angiosperms it is typ-ical for the early embryo to differentiate into twoparts, the suspensor and the embryo proper. Thesuspensor is a specialized structure which functionsmainly to facilitate the continued development of theembryo proper within the seed. There is an enor-mous amount of diversity of suspensor morphologyamong angiosperms. The differentiated suspensorranges from a reduced structure consisting of a sin-gle cell to a massive structure composed of hun-dreds of cells. Morphologically, a simple filamentousmulticellular suspensor is the type described inCapsella bursa-pastoris, Arabidopsis, and mem-bers of the Brassicaceae (Raghavan, 2006). Othersare spheroid masses of large cells and are muchlarger than the young embryo proper. Some suspen-sors develop haustoria that penetrate theendosperm and integuments. Suspensor diversitymost likely reflects the different roles of the suspen-sor in complex interactions between maternal tis-sues and the endosperm in providing nutrition to

the developing embryo proper (for review see Yeungand Meinke, 1993; Shwartz et al., 1997).

Classically, the function assigned to the suspen-sor has been that of holding the embryo in a fixedposition in the seed. Hence this embryonic organwas thought to play a rather passive role in the devel-opment of the embryo. However, an increasing num-ber of microscopy, physiological, biochemical andgenetic studies have produced data suggesting thatthe suspensor plays an active role during earlyembryo development (Schulz and Jensen, 1969;Newcomb and Fowke, 1974; Yeung and Clutter,1978; Singh et al., 1980; Kozieradzka-Kiszkurno andBohdanowicz, 2006; Lee et al., 2006; Kozieradzka-Kiszkurno et al., 2011a,b; Kozieradzka-Kiszkurnoand Płachno, 2013). Recently there has been signifi-cant progress in studies in Arabidopsis and tobaccoin vivo as well as in microspore-derived embryos ofBrassica in vitro (Panitz et al., 1999; Prem et al.,2012). In most species this rapidly developing short-lived organ undergoes programmed cell death (PCD)and is not present in the mature plant (Raghavan2006). The Arabidopsis suspensor is an attractive

Embryo suspensor in Sedum reflexum 77

system for studying the mechanisms of PCD in plants.The Arabidopsis suspensor and embryo propertogether offer a good system for identifying molecularmechanisms involved in specification and mainte-nance of cell identity during plant embryogenesis(Kuriyama and Fukuda, 2002; Kawashima andGoldberg, 2010).

Crassulaceae is one of the families in which thesuspensor develops haustorial branches that invadethe micropyle and adjacent tissues. This family,which includes approximately 1500 species, is oneof the groups of succulents that are widely cultivat-ed as ornamentals. Sedum, the largest genus of thisfamily, is cosmopolitan in its distribution and showsmuch of the morphological diversity present inCrassulaceae as a whole. The ultrastructural andcytochemical aspects of suspensor development inthis family have been described in only two speciesof Sedum: S. acre and S. hispanicum (Kozieradzka-Kiszkurno and Bohdanowicz, 2006; Kozieradzka-Kiszkurno et al., 2011a; Kozieradzka-Kiszkurnoand Płachno, 2013) and three other genera:Sempervivum (S. arachnoideum), Jovibarba (J. sobolifera) and Graptopetalum (G. bellum)(Kozieradzka-Kiszkurno et al., 2011b; Kozieradzka-Kiszkurno and Płachno, 2013). These studies clear-ly confirmed structural and functional differencesbetween the suspensor and the embryo proper. Theyindicated that the suspensor basal cells function asa synthetically active transfer cell which absorbsnutrients from maternal tissue, and then metabo-lizes and translocates them through the chalazalsuspensor cells to the embryo proper.

In this paper we report further research on sus-pensor development in Crassulaceae. We show itssimilarities and structural differences in thisgenus/family, and especially that the suspensor mor-phology of S. reflexum differs from that of two Sedumspecies previously studied. We describe the develop-ment, ultrastructure and cytochemistry of the embryosuspensor in S. reflexum and discuss the possiblerole of this embryonic organ in embryogenesis.

MATERIALS AND METHODS

PLANT MATERIAL

Plants of Sedum reflexum L. at different stages ofdevelopment were obtained from a Polish company(http://kaktusiarnia.pl/index1.html).

ELECTRON MICROSCOPY

For ultrastructural studies, ovules at different stagesof embryo development were fixed in 2.5%formaldehyde and 2.5% glutaraldehyde in 0.05 Mcacodylate buffer (pH 7.0) for 4 h at room tempera-

ture. Then the material was rinsed in the samebuffer and post-fixed in 1% osmium tetroxide incacodylate buffer at 4°C overnight. Specimens weretreated with 1% uranyl acetate in distilled water for1 h, dehydrated in an acetone series and embeddedin Spurr's resin. Serial ultrathin (60–100 nm) sec-tions were cut with a diamond knife on a Sorvall MT2B microtome. Material on the grids was post-stained with a saturated solution of uranyl acetate in50% ethanol and 0.04% lead citrate. Observationswere made using a Phillips CM 100 transmissionelectron microscope operating at 80 kV.

LIGHT MICROSCOPY

For light microscopy, semithin sections (0.5–1.5 μm)were cut with glass knives and mounted on glassslides. Sections were stained with 0.05% ToluidineBlue O in 1% sodium tetraborate for 1 min at 60°C ona hot plate. For cytochemical studies, sections werestained with periodic acid-Schiff (PAS) for localizationof insoluble polysaccharides (Jensen, 1962), withAniline Blue Black for proteins (Jensen, 1962) andwith Sudan Black B for lipids (Bronner, 1975). Freshembryos at different stages of development were dis-sected from living ovules and placed in a drop of flu-orochrome Auramine O solution. Cuticle was local-ized using Auramine O staining (Heslop-Harrison,1977). Sections were examined and photographedwith a Nikon Eclipse E 800 microscope equippedwith a Nikon cooled CCD camera.

RESULTS

Embryo development in Sedum reflexum is of theCaryophyllad type. After fertilization the zygote dividestransversely into two unequal-size cells: a large basalcell (~54 × 24 μm) and a smaller apical one (~11 ×7 μm) (Fig. 1a). The basal cell is anchored to themicropylar end of the embryo sac. This cell produceshaustorial branches that invade the micropyle andadjacent integumentary tissue. At this stage the basalcell is filled with dense cytoplasm. The nucleus is themost conspicuous organelle within the cell and islocated at the chalazal pole (Fig. 1a). The basal cellundergoes no further division and becomes the basalsuspensor cell, while the apical cell develops into theembryo proper and the chalazal suspensor.Differentiation of the suspensor was compared withthe development of the embryo proper.

DIFFERENTIATION OF THE BASAL CELL – GLOBULAR-STAGE EMBRYO

The basal cell is anchored to the micropylar end ofthe embryo sac. The suspensor of the globular-stageembryo is morphologically simple and filamentous,

consisting of a large pear-shaped basal cell (~70 ×33 μm) and a few chalazal cells. The micropylar anu-cleate haustorium of the basal cell is alreadystrongly developed and ramifies in the integumen-tary tissue (Fig. 1b). At this stage of developmentthe basal cell undergoes further differentiation. Asingle nucleus is located in the peripheral cyto-plasm in the middle of the cell (Fig. 1b–d). Thecytoplasm of suspensor cells is highly vacuolated,more so than the embryo proper (Fig. 1b). AnilineBlue Black staining indicates that the protein con-tent of the cytoplasm of the basal cell and of thechalazal suspensor is lower than in the cells of theembryo proper (Fig. 1c). Numerous lipid dropletsare visible in the basal cell at the beginning of theglobular stage. Occasionally lipids are also presentin the chalazal suspensor cells and in the micropy-lar haustorium (Fig. 1d), in an amount comparablewith that in the embryo proper. The cell walls ofthe suspensor and the embryo proper are clearlyPAS-positive. The basal cell wall is visibly thickerthan the chalazal cell wall. Delicate PAS-positiveingrowths are produced in the micropylar part ofthe basal cell and in the micropylar haustoriumcell (Fig. 1e). After staining with Auramine O,microscopy indicated the presence of a fluorescentcuticle on the surface of the embryo proper but noton the suspensor (not shown).

FULL DEVELOPMENT AND FUNCTION OF THE SUSPENSOR – LATE GLOBULAR AND HEART-STAGE

EMBRYOS

The morphology and cytochemistry of the late glob-ular and heart-stage embryos are similar enoughthat these stages can be described together. The fullydeveloped filamentous suspensor contains anenlarged basal cell (~70 × 28 μm) which is attachedto the maternal tissues and a single row of 6–10 cha-lazal cells. The cytoplasm of the basal cell becomesdenser than in the previous stage of development. Asdevelopment progresses the nucleus graduallyenlarges. The chalazal suspensor, linking the basalcell with the embryo proper, consists of a fewstrongly elongated and vacuolated cells (Fig. 2a).Aniline Blue Black staining indicates similar proteincontent in the cytoplasm of the basal suspensor cell,the micropylar haustorium and chalazal suspensorcells (Fig. 2b). Numerous lipid droplets of varioussize are visible throughout the suspensor and themicropylar haustorium (Fig. 2c,d). A fluorescingcuticle envelops the embryo proper but does notextend to the suspensor as in the previous stage ofdevelopment (Fig. 2e). Ultrastructural investigationsindicate that an electron-dense cuticular layer accu-mulates on the surface of the walls of the embryoproper (Fig. 2f).

ULTRASTRUCTURE OF THE SUSPENSOR

The ultrastructure of the fully differentiated suspen-sor was observed in late globular and heart-stageembryos. The dense cytoplasm of the basal cell con-tains an irregularly shaped nucleus. The nucleus isthe most prominent organelle within the cell (Fig. 3a). It grows to a considerable size and theamount of chromatin and the size of the nucleolusalso increase. There are many pores in the foldednuclear envelope and the interior of the nucleus isfilled with patches of condensed chromatin inter-spersed with decondensed chromatin (Fig. 3a). Athigher magnification, bundles of actin filaments areseen in the cytoplasm near the nucleus (Fig. 3 c). Thecytoplasm of the basal cell is rich in differentorganelles: mitochondria, plastids, dictyosomes,profiles of endoplasmic reticulum, microbodies andvacuoles (Figs. 3a, 4a,b). Many mitochondria of dif-ferent size (both spherical and ellipsoidal) are dis-tributed throughout the cytoplasm; they have a fewtubular cristae (Figs. 3a, 4a,b). At the micropylarend of the basal suspensor cell, wall ingrowthsappear as small papillae along the cytoplasmic faceof the cell wall. Mitochondria are abundant and arelocated near the wall ingrowths (Fig. 3b). There arefewer plastids than mitochondria. They are alsogenerally larger than the mitochondria, and some-times are irregular in shape. These organelles aresituated mainly near the nucleus and are poorly dif-ferentiated. The plastids are filled with a low-densi-ty matrix with very small tubules, and sometimes afew internal membranes (Figs. 3a, 4a,b).Occasionally there are also plastids that have a spherical body consisting of intertwined bundlesof tubules (Fig. 5a). During this stage there are a fewdictyosomes which produce different kinds of dic-tyosomal vesicles distributed throughout the basalcell cytoplasm (Fig. 4b). In the transverse cell,which separates the basal cell from the chalazalcells, there are many plasmodesmata. Ribosomedensity is lower in the basal cell than in the chalazalsuspensor cells. A group of vacuoles containing finefibrillar material appears in the chalazal region ofthe cell (Fig. 4a). Free ribosomes are numerous andoccur singly or are aggregated in small polysomeswhich may have a helical arrangement (Fig. 5a). Theprofiles of the endoplasmic reticulum are ratherlong and may be scattered singly throughout thecytoplasm. The ER frequently runs parallel to theplasmalemma (Fig. 3b) and to the surface of theplastids (Fig. 5a). Some rough ER cisternae appearto be stacked in three to four layers parallel to thecell or the nuclear surface (Fig. 4b). At this stage thecytoplasm of the basal cell has a well-developedsmooth endoplasmic reticulum (SER) (Fig. 4b).Microbodies occur less frequently in the cytoplasmthan other organelles. Various types of vesicles are

Czaplejewicz and Kozieradzka-Kiszkurno78


FFiigg.. 11.. Development of the Sedum reflexum L. embryo. Results of cytochemical tests. (aa) Two-celled proembryo.Longitudinal section showing large basal cell and smaller apical cell, (bb––ee) Globular stage embryos: (bb) Semithin sectionshowing basal cell with large uninucleate micropylar haustorium, a few chalazal suspensor cells and embryo proper, (cc) Embryo stained with Aniline Blue Black showing protein distribution, (dd) Section stained with Sudan Black B show-ing lipid distribution, (ee) Embryo stained for polysaccharides. Note PAS-positive wall ingrowths of basal cell andmicropylar haustorium. AC – apical cell; BC – basal cell; CHS – chalazal suspensor cells; EP – embryo proper; MH –micropylar haustorium, WI – wall ingrowths.


FFiigg.. 22. Late globular and heart-stage embryos. Results of cytochemical tests. (aa) Semithin section showing fully devel-oped suspensor consisting of large basal cell with micropylar haustorium, a few chalazal suspensor cells; the embryoproper, (bb) Section stained with Aniline Blue Black showing the location of proteins; basal cell, micropylar haustorium,chalazal suspensor cells, embryo proper, (cc––dd) Sections stained with Sudan Black B showing lipid distribution, (cc) basalcell, chalazal suspensor cells, embryo proper, (dd) basal cell, micropylar haustorium, (ee) Portion of embryo proper.Fluorescing cuticle visible on surface of embryo proper, (ff) Fragment of embryo proper with electron-dense cuticle layer.BC – basal cell; MH – micropylar haustorium; CHS – chalazal suspensor cells; EP – embryo proper; C – cuticle.


FFiigg.. 33.. Suspensor development in late globular and heart-stage embryos. Electron micrographs. (aa) Fragment of basalcell contains large nucleus (N) and nucleolus (NU). Cytoplasm is filled with rough endoplasmic reticulum (RER) cister-nae and dictyosomes (D); plastid (P), small vacuole (V), (bb) Fragment of micropylar part of basal cell. Mitochondria (M)near wall ingrowths (WI); cell wall (W), nucleus (N), (cc) Many nuclear pores (NP) visible in folded nuclear envelope. Notebundles of microfilaments (arrows) near nucleus.


FFiigg.. 44.. Suspensor development in late globular and heart-stage embryos. Electron micrographs. (aa) Part of cell wall (W)between basal cell (BC) and first cell of chalazal suspensor cells (CHS) with plasmodesmata (PD). Ribosome density islower in basal cell than in chalazal suspensor cells. In chalazal region of basal cell, a group of vacuoles (V) containingfibrillar material appears; plastid (P), nucleus (N), (bb) Higher magnification of portion of basal cell in vicinity of nucle-us. Cytoplasm is rich in mitochondria (M), profiles of rough endoplasmic reticulum (RER), dictyosomes (D).Occasionally, microbodies (MB), bundles of microfilaments (MF) and microtubules (MT) were observed; plastid (P).

found in the cytoplasm, both electron-dark andelectron-bright (Fig. 4b). Microfilaments and micro-tubules near the nucleus are also observed occa-sionally (Figs. 3c, 4b).

The chalazal suspensor consists of 6–9 highlyvacuolated cells (Fig. 5b). The size and structure ofthe nucleus of the chalazal suspensor cells do notdiffer from those of the nuclei of the embryo proper.Many free ribosomes are visible in the cytoplasm butthere are few RER cisternae and dictyosomes.Mitochondria are not very numerous; they arespherical or ellipsoidal in shape. The few plastidsgenerally are smaller than the basal cell plastids.The wall separating the basal cell from the chalazalsuspensor contains numerous plasmodesmata.There are a large number of plasmodesmata in thethin inner walls of the chalazal suspensor andembryo proper, but they are absent from the outerwalls of the entire embryo proper (Fig. 5b).

ULTRASTRUCTURE OF THE MICROPYLAR HAUSTORIUM

The basal cell forms an anucleate micropylar haus-torium. Together with the enlargement of the basalcell, its micropylar haustorium grows and graduallypenetrates deeper into the integument tissue (Fig. 6a). Delicate ingrowths are formed on themicropylar haustorium. There are numerous mito-chondria and rough endoplasmic reticulum profilesin close proximity to the ingrowths (Fig. 6b). Thehaustorium has very dense cytoplasm. Many dic-tyosomes are distributed throughout the cytoplasmof the micropylar haustorium. The cytoplasm isfilled with many active dictyosomes which producedictyosomal vesicles. Vacuoles, plastids and lipiddroplets of various sizes are also visible (Fig. 6b).

MATURE EMBRYO

In the next stage of senescence, the suspensor con-sists of a basal cell (~57 × 29 μm) and a few elon-gated chalazal cells. The embryo proper is ~700 μmlong (Fig. 7a). The cytoplasm is filled with a verysmall number of small vacuoles. The cytoplasm ofthe micropylar haustorium also contains vacuolesbut they are larger than in the basal cell. The chalazalcells of the suspensor are strongly vacuolized (Fig. 7b). The nucleus of the basal cell becomes lobed,loses its regular shape and becomes centrally locatedin the cell (Fig. 7b,d). As compared with the earlierstages of embryo development, the number and sizeof wall ingrowths increases slightly, especially in themicropylar part of the basal cell (Fig. 7c). Aniline BlueBlack staining suggests slightly lower protein contentin the cytoplasm of the basal cell, in chalazal cells,and in the micropylar haustorium than in earlierstages (Fig. 7d). The number of lipid droplets

decreases in both the basal cell and the micropylarhaustorium as compared with earlier stages (Fig. 7e).There is a thin layer of cuticle on the outer surfaces ofthe embryo proper (not shown).

DISCUSSION

The diversity of size, shape, longevity and cytologicalfeatures of the suspensor observed among differenttaxa is related to the mechanism of suspensor func-tioning in the nutrition of the embryo. InCrassulaceae the suspensor forms aggressive haus-toria which penetrate the surrounding ovular tissueand are thought to be involved in translocation ofnutrients from somatic cells of the ovule to thedeveloping embryo (Raghavan, 1986). The haustori-al suspensor in S. reflexum significantly differs instructure from previously investigated suspensors ofCrassulaceae taxa. One difference is in the structureof chalazal suspensor cells. The suspensor of S. reflexum is built of 5–10 single cells in a filament-like arrangement. These cells are very strongly vac-uolated. In the suspensor of S. acre, S. hispanicum,Jovibarba sobolifera or Sempervivum arach-noideum the chalazal suspensor cells are few (2–4)and arranged in two levels. Another feature distin-guishing this suspensor is the presence of very spe-cific, linearly arranged endosperm cells adjacent tothe suspensor along its entire length. These cells arehighly vacuolated and have wall ingrowths in themicropylar fragment of the wall. The endospermcells surrounding the linear portion of the suspen-sor most likely play an important role in embryodevelopment, especially when the linear suspensorcells are more vacuolated. In the suspensor of bothS. acre and S. hispanicum the cellular endospermwas very poorly developed.

In Sedum reflexum the role of the suspensor innutrient transport to the embryo is confirmed bybasal cell structure. This cell undergoes develop-mental changes during embryogenesis. In terms ofthe possible physiological functions of the suspen-sor, perhaps the most salient feature of its subcellu-lar morphology is the presence of wall ingrowths(Raghavan, 2006). The walls of the haustorium andthe micropylar part of the basal cell form prominentwall ingrowths which are covered by a plasma mem-brane, which greatly increases the ability of thesecells to absorb nutrients from the surrounding tis-sues. Numerous studies on the occurrence of trans-fer cells indicate morphological, temporal and spa-tial correlations between the degree of activity oftransfer cells and the extent of their wall ingrowthdevelopment (Gunning and Pate, 1969). In S. reflex-um the wall ingrowths of transfer cells develop dur-ing early embryogenesis and are most extensive bythe late globular and early heart-shaped stage. The



FFiigg.. 55.. Suspensor development in late globular and heart-stage embryos. Electron micrographs. (aa) Magnification ofplastid (P) with spherical body containing intertwined bundles of tubules. Rough endoplasmic reticulum (RER) profilesfrequently run parallel to surface of plastids. Arrows indicate polysomes, (bb) Portion of chalazal suspensor cells (CHS).Plasmodesmata (arrows) present in cell wall separating chalazal suspensor cells; vacuole (V), endosperm (EN).


FFiigg.. 66.. Uninucleate micropylar haustorium of basal cell in fully developed suspensor. (aa) Micropylar haustorium (MH)branching into integumentary tissues (IN); cell wall (W), (bb) Higher magnification of fragment of haustorium showingwall ingrowths (WI) and haustorium wall (W). Note the numerous mitochondria (M) near the wall ingrowths; profiles ofrough endoplasmic reticulum (RER); vacuole (V), lipid droplet (L).


wall ingrowths that develop at the micropylar end ofthe basal cell and haustorium are involved in theintensive transport of metabolites. Similar sugges-tions have been made for the wall ingrowths thatoccur in Capsella bursa-pastoris (Schulz andJensen, 1969), Phaseolus coccineus (Yeung and

Clutter, 1978), Alisma (Bohdanowicz, 1987);Arabidopsis thaliana (Mansfield and Briarty, 1991),Paphiopedilum delenatii (Lee et al., 2006), Sedumacre and S. hispanicum (Kozieradzka-Kiszkurnoand Bohdanowicz, 2006), Sempervivum arach-noideum, Jovibarba sobolifera and Graptopetalum

FFiigg.. 77.. Mature embryo proper. Results of cytochemical tests. (aa) Stage of suspensor senescence showing basal cell (BC)with micropyle haustorium and a few chalazal suspensor cells, embryo proper, (bb) Higher magnification of basal cell,micropylar haustorium and fragment of chalazal cells, (cc) Micropylar haustorium and micropylar part of basal cell wallform PAS-positive ingrowths, (dd) Section stained with Aniline Blue Black showing protein distribution. Protein distri-bution in haustorial basal cell and chalazal cells; micropylar haustorium, (ee) Section stained with Sudan Black B show-ing lipid distribution; basal cell, chalazal cells. BC – basal cell; MH – micropylar haustorium; CHS – chalazal suspen-sor cells; EP – embryo proper; WI – wall ingrowths.

bellum (Kozieradzka-Kiszkurno et al., 2011b).According to Pate and Gunning (1972), any cell thatcontains wall ingrowths and is thought to beinvolved in short-distance transport of solutes canbe termed a transfer cell. The numerous organellesthat may support active transport of nutrientsacross the plasma membrane and translocation ofthe absorbed materials to the embryo proper areclosely associated with these wall projections. Theabundance of mitochondria adjacent to theingrowths suggests high energy requirements forabsorption and movement of nutrients across thecell membrane by the cells in question. The occur-rence of such mitochondria has been found in thesuspensor cells of many species: Phaseolus coc-cineus (Yeung and Clutter, 1978), Capsella bursa-pastoris (Schulz and Jensen, 1969), Pisum sativum(Marinos, 1970a), Stellaria media (Newcomb andFowke, 1974), Tropaeolum majus (Nagl, 1976),Medicago (Sangduen et al., 1983), Alisma planta-go-aquatica and A. lanceolatum (Bohdanowicz,1987), Paphiopedilum delenatii (Lee et al., 2006),Sempervivum arachnoideum and Jovibarba sobo-lifera (Kozieradzka-Kiszkurno et al., 2011b). Wall ingrowths similar to those in S. reflexum havealso been observed in two Sedum species: S. acreand S. hispanicum (Kozieradzka-Kiszkurno andBohdanowicz, 2006). In the Sedum species studiedhere, the structural specializations along with theformation of wall ingrowths occur in the suspensoralready at the proembryo stage. Their presence,which covers most of the micropylar basal cell dur-ing the developmental stages, suggests that thisregion requires a higher level of transport because itis metabolically dynamic. Further evidence that thesuspensor functions as a channel for transport ofnutrients to the developing embryo proper from thesurrounding maternal tissues is the presence ofplasmodesmata in the chalazal-end wall of the basalcell and in the end walls of the suspensor, whileplasmodesmata are absent from the outer walls ofthe embryo proper. A distribution of simple plas-modesmata similar to that in S. reflexum wasreported in suspensors of sunflower (Newcomb,1973), Capsella bursa-pastoris (Schulz and Jensen,1969), Alisma plantago-aquatica and A. lanceola-tum (Bohdanowicz, 1987). However, the structure ofplasmodesmata in S. reflexum differs from that ofsuspensor plasmodesmata recorded in severalCrassulaceae species: Sedum acre and S. hispan-icum (Kozieradzka-Kiszkurno and Bohdanowicz,2010), Sempervivum arachnoideum and Jovibarbasobolifera (Kozieradzka-Kiszkurno et al., 2011a,b,2011c). There are branched plasmodesmata with anunusual dome of electron-dense material on the sideof the cell in those species. In S. reflexum, as in mostangiosperm species, the suspensor does not have acuticle layer to facilitate direct communication with

adjacent seed tissues, unlike the embryo proper,which is surrounded by a cuticle that covers the pro-toderm surface (Szczuka and Szczuka, 2003).

In Sedum reflexum the basal cell is the site ofintense metabolic activity. This was reflected in thecomposition and distribution of proteins (AnilineBlue Black staining), lipids (Sudan Black B stain-ing), insoluble polysaccharides (PAS-reaction) andcuticular materials at various stages of developmentof the embryo proper and suspensor. Our analysisof suspensor ultrastructure also confirmed it. Thevarious organelles found in the suspensor cells sug-gest that this embryonic organ may be the site ofmetabolic processes that do not occur within theembryo proper. The suspensor basal cell of S. reflexum undergoes developmental changes dur-ing embryogenesis. The presence of polysomes inthe haustorial suspensor of S. reflexum suggestsactive synthesis of protein, which would be neededfor rapid growth of the suspensor basal cell. Theprofiles of endoplasmic reticulum (ER), which arewell developed in the S. reflexum basal cell, oftenrun from the vicinity of the micropylar transfer wallto the chalazal end of the cell and may well play anactive part in absorption and intracellular transportof nutritive substances. In S. reflexum the endo-plasmic reticulum cisternae of the basal cell are cov-ered with ribosomes and probably participate notonly in transport but also in synthesis of varioussubstances. Well-developed RER normally is associ-ated with intensive protein synthesis (Gunning andSteer, 1975). The considerable amount of cyto-chemically detected protein in the cytoplasm of thebasal cell would appear to confirm this interpreta-tion of RER function. Transport functions have alsobeen proposed for the suspensor endoplasmic retic-ulum in Capsella (Schulz and Jensen, 1969),Helianthus (Newcomb, 1973), Stellaria (Newcomband Fowke, 1974), Phaseolus (Yeung and Clutter,1978), Alisma (Bohdanowicz, 1987), Jovibarbasobolifera and Sempervivum arachnoideum(Kozieradzka-Kiszkurno et al. 2011b). The occur-rence of lipid droplets in the cytoplasm of both thehaustorial suspensor and embryo proper of S. reflexum probably is due to the abundant tubularsmooth endoplasmic reticulum (SER) in late globu-lar and heart-stage embryos. SER participates insynthesis and secretion of lipids. The endoplasmicreticulum is also equipped with enzymes and struc-tural proteins involved in biogenesis of oil bodiesand in storage of lipids. Microbodies are not verynumerous in the suspensor and micropylar hausto-rium of S. reflexum. They are not found in theembryo proper. Most microbodies contain at least apart of either the glycolate pathway or the glyoxylatecycle. Thus it seems likely that these organelles areimportant metabolically. Important metabolicenzymes have been found in other plant microbod-


ies (Gunning and Steer, 1975; Tolbert and Essner,1981). In most angiosperm species a unique struc-tural feature of suspensor cells is the presence oflarge differentiated plastids (Yeung and Meinke,1993). According to Kozieradzka-Kiszkurno andPłachno (2013), Crassulaceae plastids are highlyvariable in shape and size. The shape of theseorganelles varies from spherical to more or lessoval, irregular, and sometimes cup-shaped. A densestroma is a typical feature of most of the plastids inangiosperms (Raghavan, 1986) but in S. reflexumthe plastids are not so intensely electron-dense as inthe other Crassulaceae examined. The plastids in S. reflexum most resemble those in the suspensorof Pisum sativum (Marinos, 1970b). They have acentrally located body consisting of intertwiningtubules surrounded by a stroma and a double mem-brane. In the Crassulaceae suspensor the plastidsare in close contact with the mitochondria, endo-plasmic reticulum profiles and the nucleus. Thisapplies to S. reflexum as well. Such close contactbetween these organelles has also been observed inthe suspensor cells of many species of, for example,Stellaria (Newcomb and Fowke, 1974), Phaseolus(Yeung and Clutter, 1978) and Medicago (Sangduenet al., 1983). The close contact between plastids andthe nuclei and plasma membranes of plant cells sug-gests that this physical interaction may enhance thefunctional interactions between the organelles. Theplastid-nucleus complex can be considered a semi-cell, that is, a structure capable of metabolism, pho-tosynthesis, protein and RNA synthesis but which isnot covered by a plasmalemma (Selga et al., 2010).

In many plant species, differentiation of sus-pensor cells is accompanied by endopolyploidiza-tion of their nuclei (for review see D'Amato, 1984).Large endopolyploid nuclei are also present in thebasal cells of Alisma (Bohdanowicz, 1987),Triglochin palustre (Kozieradzka-Kiszkurno et al.,2002), Sedum acre (Kozieradzka-Kiszkurno andBohdanowicz, 2003) and a number of otherangiosperms (Raghavan, 1986). In S. reflexum,enlargement of the basal cell nucleus is one of thefirst indications of its specialization. Multiplicationof the genome number in the nucleus of anendopolyploid cell usually leads to a proportionateincrease in its synthetic activity (D'Amato, 1984).

The diversity of suspensor morphology suggestsdiversity of suspensor function. For example, the mas-sive suspensors of Phaseolus coccineus andTropaeolum majus may be more involved in macro-molecular synthesis than smaller suspensors are, andthey may serve as storage tissue that provides nutri-tional support for embryogenesis of the developinglate-stage embryo (Raghavan, 1986), while morereduced filamentous suspensors (an example of whichis S. reflexum) may function primarily in absorbingnutrients from maternal tissues and transporting

materials to the embryo proper (Yeung and Meinke,1993). Nagl (1973) aptly compared the embryonalsuspensor in plants to the mammalian trophoblast,which acts as a supply line for nutrition of the fetus.

Rapidly progressing genomic technologies haveopened up new opportunities to study the role andevolution of the suspensor in the plant kingdom. It isnow possible to investigate gene activity in the suspen-sor of almost any plant species and uncover novelgenes and pathways that play important roles in estab-lishing suspensor form and function (Kawashima andGoldberg, 2010). In recent years, Brassica napus L.has emerged as a model system for studyingmicrospore embryogenesis. Isolated microspore cul-tures in vitro provide a window for studying the cellu-lar events that mark reprogramming and totipotencyat single-cell level (Supena et al., 2008; Soriano et al.,2013). A new microspore embryogenesis systemunder low temperature mimics zygotic embryogenesisinitials and efficiently regenerates doubled-haploidplants in B. napus (Prem, 2012).

Here we showed that suspensor structure in S. reflexum markedly differs from that found atother representatives of the Crassulaceae family. Itis a type of suspensor development not foundheretofore in Sedum. All of our observations, bothcytochemical and ultrastructural, support the sug-gestion that in Sedum reflexum the suspensor playsan important physiological role in the early stages ofembryogenesis. Our results and those of others onthe structure-function relationships of the suspen-sor make it clear that this organ is a dynamic part ofthe embryo complex, functioning in absorption,short-distance translocation and exchange ofmetabolites needed for growth of the embryo.

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