Developmental Aspects of Ultrastructure, Histochemistry and Receptivity of the Stigma of Nicotiana sylvestris

The bilobed papillate stigma of Nicotiana sylvestris Speg. andComes, is covered at maturity with a copious exudate containinglipid, protein and carbohydrate. The stigma is receptive fromthe very early stage of development and it also stains positivelyfor esterase activity. The stigma has three distinct zones:an epidermis with papillae; a subepidermal secretory zone; anda parenchymatous ground tissue. The behaviour of the cells ofthese three zones has been followed from 6 d before anthesisto one day after anthesis and pollination. The cells of theepidermis and the secretory zone stain intensively for lipids,proteins and carbohydrates in the initial stages. The secretoryzone develops large intercellular spaces containing heterogenoussecretory products which also stain positively for the aforesaidthree compounds. At maturity the secretory products are releasedto the surface through gaps formed in the epidermis by cellseparation. The main secretion of the stigma is produced bythe cells of the secretory zone. Less secretion is derived fromthe stigmatic papillae. Some amount of secretion is also releasedfrom the stylar transmitting tissue adjoining the stigma. Theglandular cells of the stigma contain numerous plastids, mitochondria,ribosomes, ER, cytoplasmic lipid droplets and some dictyosomes.The plastids and the vacuoles in the secretory cells of thestigma have a lot of electron dense (osmiophilic) inclusionsrespectively in the initial and later stages of development.The former are probably involved in the production of thesematerials. It is suggested that the proteins are directly secretedby rough ER compartments whereas smooth ER is involved in thesynthesis of lipidic materials. The carbohydrate moiety of theexudate is released by the eccrine mode (sugar mono- and dimers)with some addition of polymers by disintegration of the middlelamellae. The means by which the lipidic and osmiophilic materialis extruded remains unclear. Nicotiana sylvestris, stigma receptivity, organization, stigmatic secretory system, stigmatic exudate     

Stigma development in Nicotiana tabacum. Cell death in transgenic plants as a marker to follow cell fate at high resolution

Pistil development was studied in transgenic tobacco plants in which the stigma is ablated by expression of a stigma-specific cytotoxic gene. These plants offer a tool to investigate the process of differentiation of the secretory zone, in that cell death caused by barnase activity provides a marker to follow cell fate at high resolution. After fusion of the carpel walls in the region most distal from the ovary, the epidermal cells begin to divide in both wild-type and stigmaless plants. Divisions of the L1 layer of the pistil are immediately followed by the morphogenetic events that lead to three different cell types: rounded-angular cells showing an equal number of anti- and periclinal divisions, cells that are more oblong forming the transition zone, and the square cells of the transmitting tissue dividing mostly anticlinally with respect to the original carpel wall. In the stigmaless plants, cell death caused by the expression ofSTIG 1-barnase begins at stage –1 and proceeds gradually, but is always associated with round epidermal cells and with angular-rounded cells underneath them. Studies at the ultrastructural level show that cell death caused by barnase activity occurs first in solitary cells and gradually extends to groups of cells.In situ hybridizations using the STIG 1 RNA probe in wild-type pistils confirm these results. Most likely, the cells in whichSTIG 1 is expressed are those that have just differentiated into the secretory cell type. Our results indicate that the transition zone or neck is autonomously differentiated from the secretory zone and the transmitting tissue. Furthermore, our results indicate that in both wild-type and stigmaless pistils secretion of lipids most likely occurs through the plasmodesmata. This observation suggests that bulk transport can occur via plasmodesmata.     

Reproductive biology of Nymphaea capensis Thunb. var. zanxibariensis (Casp.) Verdc. (Nymphaeaceae)

Anthesis in Nymphaea capensis var. zanzibariensis is diurnal with flowers opening and closing for three consecutive days. On the first day of anthesis, the stigmatic papillae secrete fluid and the outermost anthers are dehiscent. On the second day of anthesis the stamens form a cone above the dry stigmatic cup. The middle stamens open and turn outward. On the third day of flowering, all the stamens open and the dry stigmatic cup is exposed. The flowers are homogamous and not protogynous as the other Nymphaea. The gynoecium of the self-compatible N. capensis var. zanzibariensis , is characterized by a wet papillate stigma, a short hollow style, and secretory cells on the ventral surface of the ovary. The pollen is released on the receptive stigma. Following initial growth in intercellular spaces in the transmitting tract of the stigma, pollen tubes travel through the stylar canal and into the ovary.     

Two novel reports of semidry stigmatic surface in Asteraceae

Research documents related to the morphology and function of style branches and stigmatic surface of Asteraceae are still rather few, and the literature reports are thus controversial. We report in the present study that the stigmatic surfaces of two non-related species of Asteraceae (Lessingianthus grandiflorus and Lucilia lycopodioides) have features of semidry stigmas. Sporodermis of both species was also analyzed so that we could understand how the stigmatic surface works during pollen deposition and rehydration. Stylar branches and pollen grains (sporodermis) were studied using scanning and transmission electron microscopy (SEM and TEM) and histochemistry techniques. The inner and marginal bands of stylar branches in these species display intermediary features between the dry and wet types of stigma: the cuticle characterizes the dry stigma and cells with secretory activity characterize the wet stigma; these showed differences from what has been described to the Asteraceae family, where stigmatic surface of species from several tribes is considered dry. Pollen grains are medium-size to large with exine ornamentation (echinate and echinolophate) and abundant secretion which latter characterizes pollenkitt. We can assume that two processes might help pollen grain hydration on stigmatic surface in Lessingianthus grandiflorus and Lucilia lycopodioides: (1) the presence of pollenkitt, as observed in the secretory content inside exine cavities and around pollen grains; and (2) the secretory activity of stigmatic surface cells, whose secretion accumulates among intercellular and subcuticular spaces and leads to cuticle disruption during the floral receptive phase. Our results suggest that ultrastructural and histochemical studies should be considered when describing stigmatic surface and that the “semidry” feature within Asteraceae should be investigated still more in detail, so that the taxonomic or adaptation value of this trait in the family can be verified.     

Pistil-Specific and Ethylene-Regulated Expression of 1-Aminocyclopropane-1-Carboxylate Oxidase Genes in Petunia Flowers

The differential expression of the petunia 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene family during flower development and senescence was investigated. ACC oxidase catalyzes the conversion of ACC to ethylene. The increase in ethylene production by petunia corollas during senescence was preceded by increased ACC oxidase mRNA and enzyme activity. Treatment of flowers with ethylene led to an increase in ethylene production, ACC oxidase mRNA, and ACC oxidase activity in corollas. In contrast, leaves did not exhibit increased ethylene production or ACC oxidase expression in response to ethylene. Gene-specific probes revealed that the ACO1 gene was expressed specifically in senescing corollas and in other floral organs following exposure to ethylene. The ACO3 and ACO4 genes were specifically expressed in developing pistil tissue. In situ hybridization experiments revealed that ACC oxidase mRNAs were specifically localized to the secretory cells of the stigma and the connective tissue of the receptacle, including the nectaries. Treatment of flower buds with ethylene led to patterns of ACC oxidase gene expression spatially distinct from the patterns observed during development. The timing and tissue specificity of ACC oxidase expression during pistil development were paralleled by physiological processes associated with reproduction, including nectar secretion, accumulation of stigmatic exudate, and development of the self-incompatible response.     

STIGMA OF NICOTIANA: ULTRASTRUCTURAL AND BIOCHEMICAL STUDIES

The aim of the present study has been to elucidate further the cytology of the glandular stigma of Nicotiana tabacum, the mechanism of the secretory process during stigma development and the biochemical composition of the exudate. The stigma consists of two distinct zones: a glandular zone formed by the papillae and 2–3 layers of cells (basal cells) immediately below them, and a non-glandular region formed by vacuolated cells which are in continuity with the transmitting tissue. The stigmatic exudate is a complex mixture of different chemical compounds such as proteins, saccharides, fatty acids and phenols. The role of stigma secretion is discussed in relation to pollen activation, recognition and pollen tube growth.     

Fungicide sprays can injure the stigmatic surface during receptivity in almond flowers

Fungicides can be detrimental to flower development, pollen function and fruit set in a number of crops. Almond is a self-incompatible nut crop that has a fruit set of only approx. 30 % of the total number of flowers. Thus, interference of pollination and fertilization by fungicide sprays is of concern, and identification of chemicals having the least detrimental effects would be desirable. The objective of this study was to evaluate the effect of fungicide sprays on stigma morphology in almond using a laboratory spray apparatus that simulated field applications. Four fungicides (azoxystrobin, myclobutanil, iprodione and cyprodinil) were applied, and fresh, unfixed stigmatic surfaces were observed using a scanning electron microscope at 4 and 24 h after spraying. Increased exudate accumulation was induced by azoxystrobin at both time periods, and localized damage and collapse of stigmatic cells were observed after 24 h. Damaged stigmatic papillae exhibited wrinkling, surface distortion or collapse. Likewise, myclobutanil caused significant damage to and collapse of papillae; these were more extensive at later observations. Iprodione had no effect on exudate accumulation but caused marked and severe collapse of stigmatic papillae which was pronounced at 24 h. Cyprodinil promoted a copious increase in exudate secretion and caused the most severe collapse of stigmatic cells of all the fungicides evaluated. Damage was somewhat localized at 4 h but more global at 24 h. This study has verified that certain fungicide sprays have direct detrimental effects on stigma morphology and enhance exudate production in almond flowers.     

Papillar integrity as an indicator of stigmatic receptivity in kiwifruit (Actinidia deliciosa)

Stigmatic receptivity is a major factor limiting fruit set inkiwifruit (Actinidia deliciosa). The aim of this work was toknow what determines the cessation of stigmatic receptivityin this species. Stigmatic receptivity has been analysed inkiwifruit through the capacity of the stigma to sustain pollengermination and has been related to stigmatic development anddegeneration.The stigma of kiwifruit flowers has a papillatesurface which from anthesis is covered with an abundant exudate.Papillae are unicellular and contain a number of phenolic deposits.During the lifetime of the flower these papillae gradually loseturgidity, while stigmatic secretion increases until 5 d afteranthesis when papillae start rupturing and the papillar contentis liberated into the germination medium. Stigmatic receptivityis high at anthesis and lasts for the next 4 d. However, itdrastically decreases 5 d after anthesis and it is nil 2 d later.The pattern of stigmatic receptivity closely fits that of papillarintegrity, indicating that stigmatic receptivity relies on thisintegrity. Since papillar integrity can easily be evaluatedin stigmatic arms, this criterion can be used as a quick methodto estimate stigmatic receptivity. Key words: Actinidia deliciosa, kiwifruit, stigma, receptivity, pollen     

Structural and Cytochemical Analysis of the Stigma and Style in Tibouchina semidecandra Cogn. (Melastomataceae)

Structural and cytochemical aspects of the pistil of Tibouchinasemidecandra Cogn. were studied. The stigma is of the wet-papillatetype and is structurally divisible into a papillar zone anda stigmatic zone. The papillar zone consists of loosely arrangedpapillae which are matchstick-shaped, unicellular, and producelipid droplets that remain entrapped below the thick cuticle.The bulk of cell volume is made up of large vacuoles rich intannin. The stigmatic zone consists of layers of secretory cellswith dense cytoplasm, actively secreting dictyosomes and numerousrough endoplasmic reticulum (RER) profiles. Free-flowing lipidexudate, produced by these cells, is initially stored in theintercellular spaces, and subsequently extruded out to coverthe surface. The style is solid with a core of transmittingtissue traversing its whole length. The transmitting tissueconsists of loosely arranged cells with numerous organellesand conspicuous intercellular substance rich in polysaccharidesand pectins. Ultrastructural details indicate that the intercellularsecretion is accompanied with fraying of the wall component.Both the transverse and longitudinal walls contain plasmodesmata.Copyright1995, 1999 Academic Press Cytochemistry, stigma and style, ultrastructure, Tibouchina semidecandra     

The Structure and Cytochemistry of the Pistil of Sternbergia lutea (Amaryllidaceae)

Studies were carried out on structural and cytochemical aspectsof the pistil of Sternbergia lutea (L.) KerGawl. The stigmais of the wet papillate type; the papillae are unicellular andare arranged densely around the rim of a funnel-shaped stigma.The stigma exudate is limited and is confined to the bases ofthe papillae and the inner lining of the stigma. The papillaeare smooth in the distal part and are covered with intact cuticle-pelliclelining. The cuticle is disrupted at places towards the baseof the papillae releasing the exudate. The exudate is rich inpectins and other polysaccharides but poor in proteins and lipids.The papillae show dense cytoplasmic profiles with extensiveendoplasmic reticulum (ER), abundant mitochondria, polyribosomesand active dictyosomes. The style is hollow. The stylar cavityis surrounded by two to four layers of glandular cells. In theyoung pistil the canal is lined with a continuous cuticle, butin the mature pistil the cuticle becomes disrupted and the canalis filled with the secretion produced by the cells of the surroundingglandular tissue. Ultrastructurally, the cells of the glandulartissue are very similar to the stigmatic papillae. The innertangential wall of the cells bordering the canal is uniformlythicker than other walls. The secretion in the stylar canal,as well as the intercellular spaces of the glandular tissue,stain intensely for pectins and polysaccharides but poorly forproteins and lipids. Pollen tubes grow through the stylar canal.Structural and cytochemical details of the pistil of Sternbergiaare compared with other hollow-styled systems. Pistil, Sternbergia lutea (L.) Ker-Gawl., stigma and style, structure and cytochemistry     

GYNOECIAL DEVELOPMENT,POLLINATION, AND THE PATH OF POLLEN TUBE GROWTH IN THE TEPARY BEAN,PHASEOLUS ACUTIFOLIUS

The gynoecium of Phaseolus acutifolius var. latifolius, a self-compatible legume, is characterized by a wet non-papillate stigma, an intermeditae hollow/solid style type, and secretory cells on the ventral surface of the ovary which direct pollen tube growth. The stigma is initially receptive 5–6 days prior to anthesis. Production of stigmatic secretions, composed primarily of carbohydrates and lipids, fragment the cuticle covering epidermal cells of the stigma early in ontogeny; the lipidic aspect of the copious secretions apparently serves to inhibit desiccation after the cuticle is ruptured. Stylar canal development occurs as a combination of elongation of a basal canal present early in development, and dissolution of part of a solid transmitting tract tissue just below the stigma. Anthers dehisce and the tricolporate pollen is released onto the receptive stigma one day before anthesis. Following initial growth in intercellular spaces in the transmitting tract of the stigma, pollen tubes adhere to epidermal secretory cells along the ventral side of the stylar canal and upper ovary; here the transmitting tract is apparently limited in the number of tubes it can accommodate, providing a possible site of selection of male gametes.     

The stigmatic orifice in Cassia,Senna, and Chamaecrista (Caesalpiniaceae): morphological variation,function during pollination,and possible adaptive significance

In species of Cassia, Senna, and Chamaecrista an orifice leading into a stigmatic cavity is present at the tip of the style. The size of the orifice, the occurrence and morphology of surrounding trichomes or papillae, and the occurrence of a drop of secretion at its outlet were recorded in 17 species belonging to the three genera of the subtribe Cassiinae. Much of the observed variation in these characters apparently reflects different modes of pollen capturing and varying degrees of secretion exposure. In some of the species studied no secretion was noticeable at the entrance to the stigmatic cavity. The unusual type of stigma in the Cassiinae does not fit into any of the recognized main groups of angiosperm stigmas. It is conjectured that in the buzz-pollinated Cassiinae species with small stigmatic orifices, the secretory material is retained within the stigmatic cavity, to be extruded by vibratory movements of the style induced by a buzzing bee. It is also hypothesized that in the wet tropics, concealment of secretory materials within a cavity prevents their washing away or dilution by rain. Furthermore, in the Cassiinae, the considerable variation in features of the style and of the stigma may function as mechanisms isolating between sympatric, visitor-sharing, concurrently blooming species.     

Structure of the style and wet non-papillate stigma of Colophospermum mopane, Caesalpinioideae: Detarieae

The capitate stigma of Colophospermum mopane (Kirk ex Benth.) Kirk ex J. Leonard is an intensely folded bilobed structure. The epidermal layer of the stigma consists of non-papillate cells. Before anthesis the epidermis is covered with a cuticle and thin proteinaceous layer. Elongated subepidermal cells constitute the secretory zone. Cell disintegration in the central region of each stigma lobe leads to cavities that become connected to the central cavity in the style. During early anthesis it appears as if the receptive surface of the stigma is confined to the depressions of the stigma surface and to the cleft between the two stigma lobes as the secretory product and pollen grains are mainly confined to these areas. The secretory products of the stigma and style are released during five different stages from prior to anthesis to late anthesis. The stigmatic exudate appears complex and consists of carbohydrates, proteins and lipids. The style has a hollow, lysigenous, fluid-filled canal that is not lined with an epidermal layer or cuticle. The stylar canal is continuous with the opening between the two stigma lobes and provides an open route for the passage of exudate. The stylar exudate is PAS-positive. The dorsal and ventral bundles that supply the style branch in such a way as to almost form a cylinder around the central transmitting tissue and stylar canal. New sieve elements proliferate before anthesis.  © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 139 , 295–304.     

The secretory stigma inLycopersicum peruvianum Mill.: Ontogenesis and glandular activity

Summary The aim of the present study has been to elucidate the cytology of the glandular stigma inLycopersicum peruvianum Mill. and the mechanism of the secretory process during the stigma development.The glandular stigma (papillae and superficial stigmatic tissue) has been studied by light and electron microscopy (S.E.M. and T.E.M.).At anthesis, the longitudinal intercellular spaces are filled with exudate in the form of heterogeneous droplets and form an intercommunicating system which allows transmitting tissue to communicate with superficial papillae.The presence of cytoplasmic droplets similar in appearance to the exudate has been noted in the developing stigmatic tissue. Cytoplasmic events which may be related to their production include transitory vacuolar accumulations, modifications of the morphology of plastids and development of smooth endoplasmic reticulum. At anthesis, observations possibly related to the origin of these droplets include their contiguity with endoplasmic reticulum cisternae and their relationship to the Golgi apparatus.Some observations suggest that cytoplasmic droplets are extruded by a process of exocytotic secretion. In addition, the cytoplasmic transport of secretory products in a non-visible form may explain the involvement of several cell compartments and the heterogeneity of the secretory products.Research partially supported by C.N.R.S. R.C.P. 429 and by C.N.R. programme Biology of reproduction.     

Structure of the stigma and style of <Emphasis Type="Italic">Callaeum psilophyllum</Emphasis> (Malpighiaceae) and its relation with potential pollinators

The family Malpighiaceae, particularly in the Neotropic, shows a similar floral morphology. Although floral attraction and rewards to pollinators are alike, stigmas and styles show more diversity. The stigmas were described covered with a thin and impermeable cuticle that needs to be ruptured by the mechanical action of the pollinators. However, this characteristic was only mentioned for a few species and the anatomy and ultrastructure of the stigmas were not explored. In this work, we analyze the morphology, anatomy, and ultrastructure of the stigma and style of Callaeum psilophyllum. Moreover, we identify the potential pollinators in order to evaluate how the disposition of the stigmas is related with their size and its role in the exposure of the receptive stigmatic surface. Our observations indicate that Centris flavifrons, C. fuscata, C. tarsata, and C. trigonoides are probably efficient pollinators of C. psilophyllum. The three stigmas are covered by a cuticle that remained intact in bagged flowers. The flowers exposed to visitors show the cuticle broken, more secretion in the intercellular spaces between sub-stigmatic cells and abundant electron-dense components inside vacuoles in stigmatic papillae. This indicates that the stigmas prepares in similar ways to receive pollen grains, but the pollinator action is required to break the cuticle, and once pollen tubes start growing, stigmatic and sub-stigmatic cells release more secretion by a granulocrine process.     

Stigma and style anatomy and ultrastructure in Italian Oxalis pes-caprae L. and their possible connection with self-incompatibility

The South-African Oxalis pes-caprae with trimorphic flowers is naturalised in many Mediterranean countries. In Italy only short-styled (S) populations are known for certain and the plant is believed to reproduce only asexually, due to self- and intramorph-incompatibility. This study aims to clarify anatomical, biochemical and ultrastructural features of the stigma and style of an Italian S population, also to define their possible role in incompatibility. Additional observations were also carried out on other Italian population and on short-, long- (L) and mid-styled (M) flowers from plants of South African origin. Morphological and biochemical features of flowers collected in different phenological stages during the whole flowering season were observed both under LM and TEM. In S flowers, three different zones could be distinguished in each stigma-style complex: zone I (stigmatic), zone II (substigmatic), zone III (stylar). The main differences concern the transmitting tissue: in zones I and III this is composed of loosely arranged cells with thick walls, with an abundant soft matrix which at anthesis is rich in mucopolysaccharides and lacks pectins. In zone II, it is more compact, with a less abundant wall matrix, at anthesis containing both mucopolysaccharides and pectins. In S flowers, subjected to illegitimate pollination, many pollen tubes penetrate the stigmatic papillae but apparently are arrested in zone II; only few—and mostly at the end of flowering period—succeed reaching zone III, where they encounter no further resistance to growth. Differently, after legitimate pollinations, pollen tubes succeed in crossing transmitting tissue of zone II, where cell walls of cells lying close to pollen tubes show a considerably reduced pectin content. In L and M flowers of South African origin, no peculiar transmitting tissue could be noticed in the substigmatic zone. In such flowers, pollen tubes seemingly grow easily from stigma to style both after legitimate and illegitimate pollinations. Results suggest that in S flowers the rigid transmitting tissue in zone II acts as a mechanical barrier for illegitimate pollen tubes, as the spaces between cells are narrower than the tube diameter and pectins maintain the rigidity of the cell walls, preventing cells from separating from one another. This obstacle can be overcome by legitimate pollen tubes, which make their way between cells, possibly releasing or activating specific pectinases. However, the blocking of illegitimate tubes is not absolute: a few of such tubes grow beyond zone II and reach the ovules, so that occasional fertilisation and embryo formation can be observed. In M and L flowers, different self-incompatibility mechanisms can be hypothesised.     

The Secretory Stigmas in Populus: Development,Cytochemistry, Ultrastructure and Their Relation to Intersectional Incompatibility

The stigmas of five species, Populus euphratica Oliv., P. alba L., P. sirnonii Carr., P. lasiocarpa Oliv. and P. nigra L. have been studied. Scanning electron microscopy reveals that exudates are present in the intercellular spaces, in the clefts between the multicellular papillae and on the receptive surface. Release and movement of exudates can be visualized when the fresh stigmas are stained with sudan Ⅲ and auramine O. Paraffin and semithin resin sections of stigmas after glutaraldehyde-osmium fixation evidence the lipidic nature of the exudates. Transmission electron microscopy reveals the glandular features of the stigmatic papillae cells, such as abundance of rough and smooth endoplasmic reticulum, polyribosomes, and well-developed dictyosomes with secretory vesicles. Peuicle and epicuticular lamellate layers which have been considered as typical features of the dry-type stigmas are also present in the species where stigmas appear extremely wet. It is concluded that stigmas in all of the five species are secretory at the receptive stage. Well-developed generative and sperm ceils were observed in the pollen tubes penetrating through the deep layers of the stigmatic tissue in the reciprocal crosses between P. euphratica and P. simonii, which indicated that there is no significant barrier in the stigma.     

Pollen-Stigma Interaction in the Leguminosae: the Organization of the Stigma in Trifolium pratense L.

The intact stigma of Trifolium pratense possesses a smooth receptivesurface fringed by a few ranks of brush hairs. This surfaceis ensheathed by a thin (75–100 nm) but highly impermeablecuticle, which encloses four to five ranks of secretory cellsimmersed in their secretory products. Experimental single-grainpollinations show that pollen cannot become hydrated or germinateon the intact surface. The cuticle is ruptured when the floweris tripped; the secretion is released, and captured pollen-selfor cross-can then germinate. As in other papilionoid Leguminosae,this mechanism provides a guard against premature selling. Thesecretory cells are elongated; they remain in communicationthrough persistent pit-fields as the intercellular spaces fillwith secretion product. The secretion forms a lipid-rich emulsion,with a mucilaginous aqueous phase which reacts cytochemicallyfor protein and carbohydrate and has esterase activity. Duringthe early development of the stigma head, the cells possessa fine-structure appropriate to their secretory function, withabundant ribosomal and smooth endoplasmic reticulum, stratifiedor in the form of ramifying and anastomosing tubules, numerousmitochondria and a well developed Golgi system. Lipid globuli,partly invested in endoplasmic reticulum, are abundant in theyoung cells, but there is as yet no indication of how the lipidis transferred to the intercellular spaces during the secretoryperiod. As the stigma matures, the secretory cells become moribund. Leguminosae, Trifolium pratenseL., pollen-stigma interaction, self-incompatibility, autofertility, stigma secretory system, lipid secretion, cuticle permeability