Stigmatic Exudate Research Articles

Reproductive biology in the primitive relic AngiospermDrimys brasiliensis (Winteraceae)

From field observations onDrimys brasiliensis, principally in the Botucatu region of Sao Paulo State, Brazil, new data on the reproductive biology, the rhythm of growth, and the development of lateral cymose inflorescences, flowers and fruits are presented. Pollination accelerates the rate of flower-development for about 4–6 days. Pollination experiments show thatD. brasiliensis is not self-sterile; because of mechanical devices the sticky pollen grains do not normally come into contact with the stigmata unless an animal pollen vector is involved. The pollinators are diurnalColeoptera, Diptera andThysanoptera which eat from the pollen, lick from the stigmatic exudates and (in case of the flies) probably also from the staminal glands. Fruit- and seedeaters are birds which seem to be the main dispersal agents. Establishment of new individuals normally is through seedlings, but also by vegetative propagation through plagiotropous branches which may root and separate from the mother plant. The morphological, developmental and reproductive aspects inD. brasiliensis are discussed in a wider context, compared with data from otherMagnoliidae, and related to aspects of early Angiosperm evolution.

ハッサクの自家不和合性に関する研究 (第1報)

In the present experiment, some morphological and histological observations were made by means of both scanning electron and fluorescent microscopy concerning self-incompatibility in Hassaku, Citrus hassaku hort. ex Tanaka. Meanwhile, various treatments were tried in search of an effective method to overcome the self-incompatibility.1. The pollen behavior on stigmas of self-pollinated mature flower buds was compared with that of buds cross-pollinated with Hyuganatsu, Citrus tamurana hort. ex Tanaka. Soon after cross-pollination, the stigma exudate began to co ver the pollen grains; 9 hours after pollination, the pollen grains were completely covered by the exudate. In contrast, self-pollinated pollen grains were not covered by the exudate so quickly as cross-pollinated grains, and individual grains were observed rather clearly on some parts of the stigma even 9 hours after pollination. Furthermore, the germination vigour in the case of self-pollination seemed to be a little weaker than that in the case of cross-pollination.2. The amount of stigma exudate observed on immature flower buds, half the length of mature ones, was clearly less than that on mature flower buds which were to open the next day. Many papilla cells were clearly observed on the stigmas of immattre buds. When immatre flower buds were self-pollinated, and their stigmas were observed 24 hours after pollination, almost all the pollen grains were still cleary visible, because of the lesser secretion of stigma exudate. Germination vigour was also somewhat weaker than in the case of self-pollination on mature flower buds.3. The pollen tube growth was observed after self-and cross-pollination on both mature flower buds and immature ones. In the case of mature flower buds, the pollen tubes of Hyuganatsu penetrated into the ovaries by the 5th day after pollination. However, almost all the pollen tubes of Hassaku were still in the stigma even 9 days after pollination. In the case of immature flower buds, the pollen tubes of Hyuganatsu grew rapidly into the styles; the number of the pollen tubes which had penetrated into ovaries by the 9th day after pollination was five times larger than that observed in the case of cross-pollination onto mature buds. On the other hand, all the pollen tubes of Hassaku had not reached the lower portion of thepeated e styles even 9 days after pollination.4. Repated pollination of immature flower buds for 4 continuous days was found to be most effective for obtaining the self-fertilized seeds among all the treatments used.

The Stigmatic Exudate of Phaseolus vulgaris L.

Stigmatic exudate of Phaseolus vulgaris L. includes lipids, amino acids, protein, reducing acids, phenols, and sugar. Alkaloids are not present. In addition to its primary role in retention and germination of its pollen, the exudate may function as a nutritive source for floral visitors, including thrips and bees, and as a protectant against desiccation of the stigma and against invasion by undesirable insects or spores.

Development and Histochemistry of the Pistil of the Grape, Vitis vinifera

The development of the grape pistil is followed for a period of 9 weeks from flower initiation to anthesis. Three phases of pericarp differentiation are revealed: ring meristem formation; cell proliferation by anticlinal cell divisions; and a maturation phase characterized by periclinal cell division and differentiation. Both the stigma papillae and the transmitting tissue of the style originate by periclinal cell divisions. The receptive stigma is of the wet type and comprises many filamentous papillae, each composed of about 20 cells and covered by a loose cuticle. The stigma exudate shows similar cytochemical properties to the material in the intercellular spaces of the transmitting tissue and is physically continuous with it. After pollination and coincident with withering of the stigma, a single layer of stylar cells becomes suberized, forming a protective layer of cicatrix.

Post-x-irradiation effects on petunia pollen germinating in vitro and in vivo

The germination of Petunia hybrida L. pollen grains in germination medium, containing 10% sucrose and 0.01% H 3BO 3, was linearly related to relative humidity (RH): being minimal at 0% RH and maximal at 100% RH. The low germination at 0% RH was completely restored after transfer to 100% RH. Germination in medium decreased with increasing X-ray exposures between 0 and 400 kR. This decrease was caused by pollen rupture. No in vitro germination occurred at exposures of 400 kR and more. The radiosensitivity of pollen in vitro was minimal at 80% RH. Transfer of pollen to the stigma post-X-irradiation resulted in resistance to much higher exposures of irradiation (750 kR). The differences in radiosensitivity of the pollen germinated in vitro and in vivo are due possibly to the differences in composition of the germination medium and the stigmatic exudate. Pollen tube growth of irradiated pollen after compatible or incompatible pollination at first showed retarded then normal tube growth. A conclusion is that X-irradiation of pollen cannot influence the characteristics of pollen tube growth after compatible or incompatible pollination.

Polysaccharide component in the stigmatic exudate from Lilium longiflorum

The polysaccharide component of the stigmatic exudate from Lilium longiflorum has the composition, arabinose (26%), rhamnose (6%), galactose (57%) and glucuronic acid (11%). The highly branched polysaccharide bears a striking resemblance to the acidic polysaccharide exudate from Araucaria bidwillii in belonging to the galactan group and in carrying outer chains terminated by arabinofuranose, rhamnopyranose, galactopyranose and glucuronic acid residues. Both polysaccharides contain the sequence O-rhamnopyranosyl-(1→4)-glucopyranosyluronic acid-(1→6)-galactopyranose in some of the outer chains.

Lipochemistry of the progamic stage of a self-incompatible species: Neutral lipids and fatty acids of the secretory stigma during its glandular activity, and of the solid style, the ovary and the anther in Forsythia intermedia Zab. (Heterostylic species)

Chromatographic (thin-layer, gas column, column chromatography) analyses of neutral lipids and fatty acids of reproductive tissues of Forsythia intermedia Zab., a self-incompatible species, were performed with two objectives in mind: 1. To determine whether there is a qualitative evolution of the different classes of lipids and fatty acids that could be correlated with the three functional stages observed during previous histochemical and ultrastructural studies. The stigmatic exudate and intracellular accumulations consist mainly of neutral lipids. 2. To compare the lipid composition of the stigma (both "thrum" and "pin" forms) with that of the style, the ovary, and the anther, and to investigate the possible existence of a stigma-specific lipid compound. Stigmatic neutral lipids are found mostly in a glyceridic mixture probably containing hydrocarbons and terpenes. The fatty acids identified are between C:7 and C: 12, with the maximum unsaturated form being a C: 18. During the secretory process there is no great qualitative diference between the neutral lipids and fatty acids found in the stigmas of "thrum" and "pin" forms. Sterols are present in styles, ovaries, and anthers, but not in stigmas. They represent the only difference in the lipid composition of these various floral structures.

The effect of prepollination injection with stigmatic exudate on interspecific pollen tube growth in Lilium longiflorum thunb. styles

Prepollination injection of Lilium longiflorum styles with exudate from the stigmas of L. longiflorum significantly increased the length of pollen tubes normally inhibited by short-growth unilateral interspecific incompatibility (UI). However, stigmatic exudate injection had no effect on nor significantly retarded pollen tube growth of lily species expressing reciprocal interspecific incompatibility with L. longiflorum. These results suggest that the inhibition of short-growth pollen tubes in unilateral incompatible combinations is achieved through a different process than the inhibition-limiting pollen tube growth in reciprocal interspecific incompatibility.

Incorporation of radioactive label into nucleic acids of compatible and incompatible pollen tubes of Lilium longiflorum Thunb.

Stylar canal cells of Lilium longiflorum were labeled before pollination with 5-(3)H-uridine or 5-(3)H-orotic acid dissolved in water, or the stylar canal was filled 6 hr after pollination with stigmatic exudate supplemented with 5-(3)H-uridine, 2-(14)C-uridine or H3 (32)PO4. Nucleic acids extracted from compatible and incompatible pollen tubes grown in these styles were separated by methylated albumin kieselguhr column chromatography. Label occurred in all portions of the RNA profile, but no label appeared in the DNA peak. Using 5-(3)H-uridine, pollen tubes of different genotype had different labeling patterns.

The Nutritional Role of Pistil Exudate in Pollen Tube Wall Formation in Lilium longiflorum

Detached pistils of Lilium longiflorum were labeled with d-glucose-U-(14)C 24 hours after anthesis and then sampled for the next 6 days to determine the appearance of label into exudate from the stylar canal and the stigmatic surface of the pistil. Results were obtained with unpollinated cv. Ace pistils and pollinated cv. Ace pistils, selfed or crossed (cv. Croft pollen). Limited data were also obtained on cv. Croft pistils, selfed or crossed (cv. Ace pollen).Exudate appeared in the canal and on the stigmatic surface soon after anthesis. In unpollinated pistils it continued to accumulate for about 5 days in the canal and for the full term of the experiment, 7 days, on the stigmatic surface. Canal exudate eventually mixed with stigmatic exudate in unpollinated pistils. Pollination interrupted the flow of exudate, and a portion of the pistil secretion product was diverted toward the synthetic requirements of the developing pollen tubes. Two days after pollination, the specific radioactivity of pollen tube cytoplasm had reached a level comparable to canal exudate. Twenty-four hours later, the specific radioactivity of pollen tube wall substance was about 80% of the value found in canal exudate. Similar patterns of (14)C incorporation and similar carbohydrate contents were obtained from self- and cross-pollinated pistils, although the former contained pollen tubes of less than one-half the length of the latter.

Contribution à l’Étude cyto-physiologique du stigmate: I. Les étapes observées durant les processus glandulaires chez une Oleaceae: Forsythia intermedia Zabel

The first demonstration of the stigmatic exudate is related to the endoplasmic reticulum. This exudate is present inside the cytoplasm in the form of droplets around the vacuome and related to the tonoplast. These droplets are transported to the plasmalemma which acts in the exudate droplets migration to the cellulose wall. This exudate accumulates under the epidermal cuticle and in the intercellular system of the stigmatic tissue. Eventually, the cuticle breaks and the exudate flows out. Les premières manifestations visibles des phénomènes glandulaires stigmatiques ont pour siège le réticulum endoplasmique. L’exsudat est présent dans le cytoplasme sous forme de gouttelettes situées sur le pourtour de la vacuole et en relation avec le tonoplaste. Ces produits migrent ensuite vers le plasmalemme puis à travers la paroi cellulosique. L’exsudat s'accumule sous la cuticule et dans les espaces inter-cellulaires du tissu stigmatique. La cuticule se rompt et les produits d’exsudation sont rejetés.

The Nutritional Role of Pistil Exudate in Pollen Tube Wall Formation in Lilium longiflorum

A quantity of labeled stigmatic exudate, collected from detached Lilium longiflorum (cv. Ace) pistils labeled with d-glucose-1-(14)C, was fractionated on Sephadex G-100 and the polysaccharide component, G-100-I, was injected into the hollow styles of unlabeled detached pistils (cv. Ace) which had been removed on the day after anthesis from the plant. Injected pistils were immediately cross-pollinated with L. longiflorum (cv. No. 44) pollen. Eighty-four hours later, pistils were dissected to recover the pollen tubes, expended exudate, and labeled tissues of the stigma and style. Distribution of label revealed that at least 25% of the carbohydrate substance in excised pollen tubes was derived from G-100-I. The composition of expended exudate adhering to pollen tubes, of pollen tube cytoplasm, and of pollen tube walls suggests that utilization of exudate by growing pollen tubes involves uptake and incorporation into pollen tube cytoplasm of exudate polysaccharide fragments followed by extensive metabolism of at least a portion of the incorporated carbohydrate prior to its utilization for pollen tube wall biosynthesis. Results suggest the presence of at least two polysaccharide components in G-100-I, one which resists major degradation following injection into the style and another which undergoes measurable degradation both before and after entry into the pollen tube.

Secretory Cells of Lily Pistils. II. Electron Microscope Cytochemistry of Canal Cells

The secretory cells which line the canal of Lilium longiflorum pistils possess, on the side facing the canal, an elaborate wall which, with associated structures, Rosen and Thomas (1970) termed the “secretion zone.” We examined the secretion zone in the electron microscope following treatment of excised pistil slices with extraction procedures which remove pectin, hemicellulose, cellulose, lipid, or protein. The outer, fibrillar wall (layer 1) of the secretion zone contains protein, pectin, and cellulose. Internal to layer 1 is a granular-fibrillar wall (layer 2) several microns thick. It consists of outer and inner regions which can be distinguished from each other cytochemically. The granular component is composed of pectin which is not esterified with methyl groups and which may be complexed with protein. The short, randomly dispersed microfibrils of layer 2 were sensitive to procedures which dissolve cellulose. The extraction procedures did not reveal the chemical nature of the “osmiophilic islands” of layer 2. Paramural body membranes appear to be composed of glycoprotein and may function in secretion by serving as sites of pinocytic interchange at the plasmalemma. The origin of stigmatic exudate and the release of canal cell secretion product are discussed.

SECRETORY CELLS OF LILY PISTILS. II. ELECTRON MICROSCOPE CYTOCHEMISTRY OF CANAL CELLS

The secretory cells which line the canal of Lilium longiflorum pistils possess, on the side facing the canal, an elaborate wall which, with associated structures, Rosen and Thomas (1970) termed the “secretion zone.” We examined the secretion zone in the electron microscope following treatment of excised pistil slices with extraction procedures which remove pectin, hemicellulose, cellulose, lipid, or protein. The outer, fibrillar wall (layer 1) of the secretion zone contains protein, pectin, and cellulose. Internal to layer 1 is a granular‐fibrillar wall (layer 2) several microns thick. It consists of outer and inner regions which can be distinguished from each other cytochemically. The granular component is composed of pectin which is not esterified with methyl groups and which may be complexed with protein. The short, randomly dispersed microfibrils of layer 2 were sensitive to procedures which dissolve cellulose. The extraction procedures did not reveal the chemical nature of the “osmiophilic islands” of layer 2. Paramural body membranes appear to be composed of glycoprotein and may function in secretion by serving as sites of pinocytic interchange at the plasmalemma. The origin of stigmatic exudate and the release of canal cell secretion product are discussed.

THE STIGMATIC SECRETION OF THE SWEETPOTATO

The stigmatic exudate of sweetpotato, when removed with organic solvents, consisted chiefly of lipid and phenolic compounds. Only traces of sugar were obtained. The esterified lipids were similar in chain length to capric and lauric acids. The two prinicpal phenolic compounds have UV absorption peaks, and bathochromic shifts on ionization with NaOH similar to those of esters of caffeic acid. On acid hydrolysis, caffeic acid was obtained from these and from three minor phenolic compounds. From two of the minor phenolic compounds, glucose was released by hydrolysis. The phenolic content of stigmas increased up to 20–24 hr before anthesis, and then gradually decreased. Neither compatible nor incompatible pollination affects the amount or composition of the stigmatic exudate.

The influence of RNA-synthesis inhibitors on in vivo pollen tube growth and the self-incompatibility reaction in Lilium longiflorum thunb

Injection of 3 X 10(-4) g/ml acridine orange, 2 X 10(-5) g/ml actinomycin-D, or 1.34 X 10(-3) g/ml 6-methyl-purine in stigmatic exudate into the hollow styles of Lilium longiflorum immediately before, or 6 or 12 hr after pollination restricted compatible pollen tube growth to lengths typical of incompatible tubes. Treatment 24 hr after pollination resulted either in no effect on compatible tube growth or in a significant retardation of compatible tube growth but not to the length of tubes in incompatibly pollinated styles. Aqueous solutions of these inhibitors at the same concentrations injected into lily styles 6 or 12hr before pollination significantly stimulated incompatible pollen tube growth. These results suggest that RNA synthesis in the style is necessary for the self-incompatibility reaction and RNA synthesis in the pollen tube is required for compatible tube growth. The RNA synthesis in the pollen tube may be completed by 24 hr after pollination.

Stigmatic Exudate: A Potential Carrier of Exogenous Substances into the Style of Lilium longiflorum Thunb.1

Abstract The exudate produced on the stigma of mature flowers of L. longiflorum Thunb. did not adversely affect pollen tube growth or the self-compatibility reaction when injected into lily styles before, 6, or 12 hr after pollination. Stigmatic exudate diluted with an equal volume of distilled water before addition to the style had no effect on pollen tube growth when injection occurred before or 6 hr after pollination. Although injection of exudate alone 24 hr after pollination or injection of diluted exudate 12 or 24 hr after pollination retarded pollen tube growth, the addition of exudate or diluted exudate did not obscure the self-incompatibility reaction. Stigmatic exudate appears to be an excellent milieu in which to carry exogenous materials into the environment of pollen tubes of L. longiflorum growing in vivo.

The Composition of Stigmatic Exudate from Lilium longiflorum

Stigmatic exudate, a secretion product recovered from the upper surface of Lilium longiflorum pistils, has been examined. Over 99% of the exudate is accounted for as water, carbohydrate, and protein. Exclusive of water, 95% is a high molecular weight, protein-containing polysaccharide composed of galactose, arabinose, rhamnose, glucuronic acid, and galacturonic acid.Detached pistils supplied with myo-inositol-U-(14)C, myo-inositol-2-(3)H, d-glucose-1-(14)C, or l-proline-U-(14)C produce labeled stigmatic exudate. When myo-inositol is supplied, the exudate is rich in labeled arabinose and uronic acids, but some label also recycles through the hexose phosphate pool of secreting cells, causing label to appear in galactose and rhamnose residues. When glucose is provided, galactose is the major constituent labeled but all of the other carbohydrate constituents are also labeled. Proline produces a pattern very similar to that obtained with glucose.Stigmatic exudate also contains a small amount of low molecular weight carbohydrate. If myo-inositol is used to label exudate, free labeled myo-inositol cannot be detected in the low molecular weight fraction until it has been subjected to acid hydrolysis. Similarly, if d-glucose is the source of label, free labeled glucose is found in the low molecular weight fraction only after acid hydrolysis.

The effect of cycloheximide and 6-methylpurine on in vivo compatible and incompatible pollen tube growth in Lilium longiflorum

Cycloheximide, a potent inhibitor of protein synthesis, placed in styles of Lilium longiflorum at 10(-4) M in stigmatic exudate before, 6, or 12 hr after compatible or incompatible pollination retarded all pollen tube growth. An inhibitor of RNA synthesis, 6-methylpurine, placed in the style at 10(-4) M in stigmatic exudate before, 6, or 12 hr after pollination restricted compatible pollen tube growth to lengths not significantly different thanincompatiblepollen tubes in treated or nontreated styles. While pollen tube growth in the style of L. longiflorum appears to require protein synthesis, only compatible pollen tube growth requires RNA synthesis. Stigmatic exudate proved to be an excellent carrier of exogenous substances into the style of L. longiflorum.

INFLUENCE OF FATTY ACIDS ON PETUNIA POLLEN GRAINS

Fatty acids seem to be strong inhibitors of pollen germination in vitro, especially those with a carbon chain of 10 to 12 atoms. With at least one of these acids, namely 10-hydroxy-2-decenoic acid, this property is of practical importance. It prevents the germination of pollen stored by honeybees in their combs. This particular acid is added by the collecting workerbees from the mandibular glands. 10-OH-2 decenoic acid inhibits the respiration of pollen in culture solution, which could be the result of the blockage of one or more enzymes active in pollen-metabolism. In situ on the stigma, however, no inhibition of the germination occurs, probably on account of esterification with glycerides present in the stigma exudate.