Locular Fluid Research Articles

Cytochemical and comparative transcriptome analyses elucidate the formation and ecological adaptation of three types of pollen coat in Zingiberaceae

BackgroundThe pollen ornate surface of flowering plants has long fascinated and puzzled evolutionary biologists for their variety. Each pollen grain is contained within a pollen wall consisting of intine and exine, over which the lipoid pollen coat lies. The cytology and molecular biology of the development of the intine and exine components of the pollen wall are relatively well characterised. However, little is known about the pollen coat, which confers species specificity. We demonstrate three types of pollen coat in Zingiberaceae, a mucilage-like pollen coat and a gum-like pollen coat, along with a pollen coat more typical of angiosperms. The morphological differences between the three types of pollen coat and the related molecular mechanisms of their formation were studied using an integrative approach of cytology, RNA-seq and positive selection analysis.ResultsContrary to the ‘typical’ pollen coat, in ginger species with a mucilage-like (Caulokaempferia coenobialis, Cco) or gum-like (Hornstedtia hainanensis, Hhn) pollen coat, anther locular fluid was still present at the bicellular pollen (BCP) stage of development. Nevertheless, there were marked differences between these species: there were much lower levels of anther locular fluid in Hhn at the BCP stage and it contained less polysaccharide, but more lipid, than the locular fluid of Cco. The set of specific highly-expressed (SHE) genes in Cco was enriched in the ‘polysaccharide metabolic process’ annotation term, while ‘fatty acid degradation’ and ‘metabolism of terpenoids and polyketides’ were significantly enriched in SHE-Hhn.ConclusionsOur cytological and comparative transcriptome analysis showed that different types of pollen coat depend on the residual amount and composition of anther locular fluid at the BCP stage. The genes involved in ‘polysaccharide metabolism’ and ‘transport’ in the development of a mucilage-like pollen coat and in ‘lipid metabolism’ and ‘transport’ in the development of a gum-like pollen coat probably evolved under positive selection in both cases. We suggest that the shift from a typical pollen coat to a gum-like or mucilage-like pollen coat in flowering plants is an adaptation to habitats with high humidity and scarcity of pollinators.

Morphology of the unusual polyad in Amazonian Parkia legume trees

An unusual polyad occurs in three Parkia species, named cavitate polyad. It has an internal central space full of lipoprotein substances, contacting all pollen grains, probably aiding pollen germination. This study details the unusual morphology of polyads of three species of Parkia (P. multijuga Benth., P. ulei (Harms) Kuhlm., and P. pendula (Willd.) Benth. ex Walp.) and suggests functions for polyad adaptive traits that are linked to the reproductive success of the species. Polyads within the anthers of the three Parkia species were analysed by surface (scanning electron microscopy) and anatomical (light microscopy) studies. Ultrastructure and development studies were carried out for P. pendula polyads. Polyads are globose and cavitated, i.e., exhibit an internal cavity that varies in size, being more conspicuous in P. ulei and P. pendula. Other differences among species are related to the polyad size, exine ornamentation and the type of substances stored in the pollen grain. The polyad internal cavity is filled with an exudate that may be related to the pollen germination through the internal pores and/or translocation of substances from the anther loculus to the inside or vice versa. This inference is supported by the following observations: the spaces between the pollen grains in a polyad are also filled with the exudate, and the exudate inside the polyad is similar to the anther locular fluid. The morphology and substances stored within the pollen grains of Parkia polyads seem to be more related to polyad functionality and physiology than to the selective pressures exerted by different pollinators on the group.

Microsporogenesis in vivo and in vitro. Autofluorescence of pollen wall of Lilium and changes in pollen wall of Gasteria in Lilium anther

Autafluarescence of the pollen wall of <em>Lilium</em> shows a correlative change with its development. When the pollen of Gasteria is injected into the anther of <em>Lilium</em>, it exhibits almost the same characteristics of wall autofluorescence as those of <em>Lilium</em>. Only the last stages of wall development are dissimilar. During this period the pollenkitt sticks to the pollen wall in <em>Lilium</em> only. The conclusion is that in the exine of <em>Gasteria</em> pollen, in contact with the locular fluid of <em>Lilium</em>, polymerization of <em>Lilium</em> sporapollenin occurs instead of a structural addition to the wall. This polymerization is a continuous process during the entire development.

Variations in Carbohydrate Content and Sucrose-Metabolizing Enzymes in Tomato (<i>Solanum lycopersicum</i> L.) Stamen Parts during Pollen Maturation

The formation of mature and fertile pollen grains, taking place inside the anther, depends on supply of assimilates, in the form of sucrose, provided mainly by the leaves. Data is limited, however, with respect to the understanding of sucrose metabolism in microspores and the supporting tissues. The aims of the present work were to 1) follow the changes in total and relative concentrations of sucrose, glucose, fructose and starch in the stamen parts and microspores up until anthesis, 2) follow the activities of sucrose-metabolism-related enzymes, in the anther walls fraction and microspores of the crop plant tomato. Sucrose was found to be partially cleaved in the filament, decreasing by more than twofold in the anther wall layers and the locular fluid, and to accumulate in the mature pollen grains, constituting 80% of total soluble sugars. Thus, sucrose was both the starting sugar, supporting microspore development, and the main carbohydrate accumulated at the end of the pollen-development program. The major invertase found to be active in both the anther wall layers and in maturing microspores was cell-wall-bound invertase. High fructokinase 2 and sucrose phosphate synthase activities during pollen maturation coincided with sucrose accumulation. The potential importance of sucrose accumulation during pollen dehydration phase and germination is discussed.

Adenoma malignum of the uterine cervix: ultrasonographic findings in 11 patients

To evaluate the ultrasonographic features of adenoma malignum, a minimal deviation adenocarcinoma of the uterine cervix. Eighteen consecutive patients with pathologically confirmed adenoma malignum were enrolled in this study at two institutions. Preoperative ultrasound examination was performed and the results were available in 11 patients. We analyzed retrospectively the gray-scale ultrasound findings for the following morphologic characteristics: cervical enlargement, as well as size, location and ultrasonographic characteristics of lesions. In five patients we also evaluated Doppler features with regard to intralesional vascularity. The cervix was enlarged in 73% (8/11) of cases. The mean greatest tumor diameter was 4.2 (range, 2.5-6.8) cm. In five (45%) cases, the cervix was completely infiltrated by the tumor. At gray-scale ultrasound examination, three (27%) tumors were multilocular lesions, four (36%) were multilocular lesions with solid components and four (36%) were solid lesions. In the multilocular lesions with or without a solid component, locules tended to be 1 cm or less in average diameter (86%, 6/7 cases) and there tended to be 11-20 in number (57%, 4/7 cases). In most (57%, 4/7) cases the locular fluid was homogeneously hypoechoic. Most (75%, 3/4) solid lesions manifested heterogeneous echogenicity. The five (100%) tumors examined with Doppler manifested moderate or abundant color content on color or power Doppler. Adenoma malignum can appear sonographically as solid, multilocular and multilocular solid cervical lesions. Awareness of its clinical and ultrasonographic features might improve diagnosis before surgery.

Relationships between Tapetum, Loculus, and Pollen during Development

Various parts of the anther (walls, tapetum, locular fluid, meiocytes, microspores) cooperate for correct pollen development. To show the different types of relationships possible between these parts, we considered the following characteristics, alone and in combination: types of tapetum, types of pollen‐dispersing unit, form of loculus, amount of locular space and fluid, number of pollen grains in transverse sections of the anther and in the loculus, and hydration status of pollen at dispersal. The different relationships between pollen, loculus, and tapetum should enable uniform nourishment of grains in the loculus and ensure a high percentage of viable grains. Two opposite modes were identified: direct and indirect nourishment of grains, the latter occurring when locular fluid is more or less abundant between the tapetum and the grains. The amount of space between developing grains determines different manners of grain nourishment and modes of pollen presentation for dispersal. Cyclosis, which is presu...

Sucrose and starch catabolism in the anther of Lilium during its development: a comparative study among the anther wall, locular fluid and microspore/pollen fractions

In order to better understand the various pathways of sucrose and starch catabolism in the anther of lily (Lilium hybrida var. "Enchantment"), invertase (EC 3.2.1.26) and amylase (EC 3.2.1.1, EC 3.2.1.2) activities were measured separately in different fractions (anther wall, locular fluid and microspore/pollen) and correlated with the sugar content during anther development. Our findings showed significant differences among the fractions analyzed, suggesting that the regulation of sucrose and starch catabolism could follow distinct pathways in each fraction. Glucose and fructose amounts progressively decreased from anther wall to fluid and from fluid to microspore/pollen. Thus, the developing pollen could act as a sink for the carbohydrates that reach the anther. In this sense, cell wall-bound invertases seem to play a major role in soluble sugar partitioning in the different fractions of the anther. Sucrose concentration was found to be substantially higher in the locular fluid than in the other fractions, indicating a probable site for storage. On the other hand, the anther wall tissues could have a buffering function, storing nutrient surplus in starch grains and thus regulating the availability of soluble sugars in the whole anther. All these results proved the advantages of the experimental model proposed here, as well as its usefulness to investigate sugar metabolism in Lilium anthers.

The occurrence of a micropylar exudate in Musa and Enset (Musaceae)

Wild bananas are diploid while most cultivated varieties are triploid. Triploidy leads to sterility that has a number of causes, but in Musa a micropylar exudate is not listed amongst them. We examined ovules of seeded diploid Musa acuminata (AA) and edible triploids (AAA) from the same cytogenetic groups (AAB, ABB) and the related genus Ensete sp. to determine whether there was any association between the presence of a micropylar exudate and sterility. Pre-anthetic, anthetic and post-anthetic specimens were embedded in methacrylate, sectioned and examined under the bright-field microscope. The micropyles of seeded diploid and seedless edible triploid cultivars of M. acuminata. contained an exudate. In contrast to the locular fluid, the exudate persisted after fixation, thus it was a viscous mucilaginous material. It was strongly PAS-positive and counter stained purple with Toluidine blue; it contained insoluble polysaccharides and proteins and appeared to arise from the nucellar cap. The exudate was not observed in ovules of pre-anthetic M. acuminata or post-anthetic Ensete, both of which are diploid. However, the exudate in post-anthetic fertilised and unfertilised diploid M. acuminata ovules was dissimilar. In the fertilised ovules the exudate was redder and less purple, indicating it contained more polysaccharides and less protein. Both pre-anthetic and post-anthetic ovules of the edible cultivars (A and B genome) exhibited a micropylar exudate. Early in ovule ontogeny the exudate was variable in presence. There appeared to be a determinate age when the triploid ovule begins to produce an exudate, between archesporia and megaspore. The production of an exudate may depend on the presence of a functioning megagametophyte or embryo sac. Embryo sac competency could be the cue to turn off pollen tube inhibition. The exudate may be linked to an ovarian self-incompatibility system.

The effect of heat stress on tomato pollen characteristics is associated with changes in carbohydrate concentration in the developing anthers.

Continuous exposure of tomato 'Trust' to high temperatures (day/night temperatures of 32/26 degrees C) markedly reduced the number of pollen grains per flower and decreased viability. The effect of heat stress on pollen viability was associated with alterations in carbohydrate metabolism in various parts of the anther during its development. Under control, favourable temperature conditions (28/22 degrees C), starch accumulated in the pollen grains, where it reached a maximum value 3 d before anthesis; it then diminished towards anthesis. During anther development, the concentration of total soluble sugars gradually increased in the anther walls and in the pollen grains (but not in the locular fluid), reaching a maximum at anthesis. Continuous exposure of the plants to high temperatures (32/26 degrees C) prevented the transient increase in starch concentration and led to decreases in the concentrations of soluble sugars in the anther walls and the pollen grains. In the locular fluid, however, a higher soluble sugar concentration was detected under the high-temperature regime throughout anther development. These results suggest that a major effect of heat stress on pollen development is a decrease in starch concentration 3 d before anthesis, which results in a decreased sugar concentration in the mature pollen grains. These events possibly contribute to the decreased pollen viability in tomato.

Pectin secretion and distribution in the anther during pollen development in Lilium.

Using the monoclonal antibodies JIM 5 and 7, pectin was immunolocalized and quantitatively assayed in three anther compartments of Lilium hybrida during pollen development. Pectin levels in both the anther wall and the loculus increased following meiosis, were maximal during the early microspore stages and declined during the remainder of pollen ontogenesis. In the microspores/pollen grains, pectin was detectable at low levels during the microspore stages but accumulated significantly during pollen maturation. During early microspore vacuolation, esterified pectin epitopes were detected both in the tapetum cytoplasm and vacuoles. In the anther loculus, the same epitopes were located simultaneously in undulations of the plasma membrane and in the locular fluid. At the end of microspore vacuolation, esterified pectin epitopes were present within the lipids of the pollenkitt, and released in the loculus at pollen mitosis. Unesterified pectin epitopes were hardly detectable in the cytoplasm of the young microspore but were as abundant in the primexine matrix as in the loculus. During pollen maturation, both unesterified and esterified pectin labelling accumulated in the cytoplasm of the vegetative cell, concurrently with starch degradation. In the mature pollen grain, unesterified pectin epitopes were located in the proximal intine whereas esterified pectin epitopes were deposited in the distal intine. These data suggest that during early microspore development, the tapetum secretes pectin, which is transferred to the primexine matrix via the locular fluid. Further, pectin is demonstrated to constitute a significant component of the pollen carbohydrate reserves in the mature grain of Lilium.

Mechanism of Septum Opening in Anthers of Two-rowed Barley (Hordeum vulgare L.)

To test the hypothesis that the rapid swelling of pollen grains driven by potassium movement opens the septum in anthers of poaceous plants, we studied (1) the behaviour of pollen grains during unfolding of the locule and (2) the distribution of potassium in the locule in two-rowed barley. In the first experiment, the unfolding of decapitated anthers was observed. The pollen grains paved the inner wall of the locule during the unfolding process, suggesting that the pollen grains bend the locule wall outward when they adhere to the wall. In the second experiment, the distribution of potassium in transverse sections of locules in dehisced and indehisced anthers was observed. In indehisced anthers, potassium was detected outside the pollen grains. In contrast, in dehisced anthers, potassium was detected inside pollen grains. This suggested potassium ions moved from the inter-pollen space (locular fluid) into the pollen grains in the locule at the time of pollen-grain swelling.

Electron-translucent angular areas in developing tapetum cell walls and pollen grains ofTilia platyphyllos

InTilia platyphyllos, the anther tapetal cell walls undergo significant modifications from the tetrad stage onwards. During the tetrad stage the inner tangential and radial parts of the tapetal walls begin to dissolve, while the distal parts swell. After the tetrad stage, the distal and outer radial tapetal cell walls become covered by a thick, irregular, highly electron-dense, polysaccharide layer. Striking features of the maturing tapetal walls (microspore stage and later) are electron-translucent, structureless, unstainable angular areas of variable dimensions. Similar electron-translucent areas occur in the exine arcades and apertures, but also isolated in the locular fluid ofT. platyphyllos. Electron-translucent areas, that are also found in the exine arcades and tapetal cells of other angiosperms, can be interpreted as the products of poorly understood metabolic processes.

Anther wall layers control pollen sugar nutrition inLilium

Anthers ofLilium were for the first time investigated at the ultrastructural level in order to appreciate the possible ways of sugar transport in the microsporangium. Our results have shown that the cells of the outer anther wall layers and the cell of the connective were interconnected by plasmodesmata, thus allowing assimilates to travel through the symplasmic pathway from the vascular bundle to the most internal middle layer (ML 1). ML 1 was devoid of cell communication throughout pollen development. Tapetal cells were also lacking plasmodesmata on their external face towards ML 1, but adjacent tapetal cells developed lateral junctions: the tapetum could represent a syncytium. Sugars destinated to pollen in the loculus have then to cross the ML 1 and the tapetal layers by the apoplasmic pathway; it is suggested that these two envelopes could be involved in the control of sugar transport from the outer anther wall layers to the locular fluid. Before microspore mitosis, the tapetum degenerated but ML 1 remained structurally unchanged. During pollen development, the guard cells of stomata were lacking cell communication, and preserved their starch content, which could be the sign of photosynthesis within the anther wall. In order to check whether these structural disconnections in anther tissues corresponded to physiological barriers, isolated pollen and stamens were cultivated during the anther maturation phase, on a medium containing increasing concentrations of sucrose (0 M, 1/6 M, 1/2 M, 1 M). After 7 days of culture, isolated pollen was engorged with starch grains and was unable to germinate, whereas in cultivated stamens, pollen did not contain any starch grain: sporophytic tissues, however, accumulated abnormal amylaceous reserves. These results strongly suggest that the anther wall layers, in particular ML 1, starve pollen with sugars during its maturation. They are acting as a physiological buffer storing nutriment surplus in starch grains.

The Transfer of Pollenkitt in Smyrnium perfoliatum (Apiaceae)

In Smyrnium perfoliatum the formation of pollenkitt within a secretory tapetum, and the subsequent breakdown of the cell organelles, is followed by the transformation of pollenkitt lumps into pollenkitt droplets. These droplets move within a locular fluid towards the pollen exine, where they enter the exine cavities after passing a fibrillar layer (remnants of the primexine-matrix) in between the tectum elements. This is followed by the fusion of pollenkitt droplets, forming a distinct layer at the bottom of the exine cavities

A novel technique for identification of sites of anion transport in intact cells and tissues using a fluorescent probe

Abstract The purported blocker of anion transport 4, 4′ di‐isothiocyano‐2‐2′ stilbene disulfonate (DIDS) has been shown to partially inhibit 36Cl− influx, 36CIO−3 influx and 35SO2−4 influx into Pisum salivum L. cv. Feltham First seedlings. This inhibitory effect could be prevented by pretreatment with the respective unlabelled medium. There was no effect of DIDS on 14C methylamine influx. The results are consistent with the hypothesis that the binding of DIDS to the site of anion‐carrier interaction is responsible for its observed inhibitory effects on anion fluxes. The fluorescent properties of DIDS upon binding to membrane proteins was exploited in an attempt to examine the major sites of anion pumping in whole roots. The results show clearly that in the presence of DIDS the epidermal layers became brightly fluorescent, while cortical layers did not fiuoresce. Lycopersicum esculentum cells taken from locular fluid were plasmolysed using sucrose solution, and the patterns of fluorescence in the presence of DIDS showed in an unambiguous way that the fluorescence is associated with cell membranes. The potential usefulness of this technique to probe sites of anion transport in whole plants and tissues is discussed.

Tomato lectin is located predominantly in the locular fluid of ripe tomatoes

We have investigated the localization of the tomato lectin within the ripe fruit of Lycopersicon esculentum Mill. All of the hemagglutinating activity was found in the fluid and placental tissues of the locules. Centrifugation of the locular material produced a clear, gel-free extract from which the tomato lectin could be purified to apparent homogeneity in one step by affinity chromatography on a column of ovomucoid-Sepharose 4B.

The tapetum: Its form, function, and possible phylogeny inEmbryophyta

It appears that the tapetum is universally present in land plants, even though it is sometimes difficult to recognize, because it serves mostly as a tissue for meiocyte/spore nutrition. In addition to this main function, the tapetum has other functions, namely the production of the locular fluid, the production and release of callase, the conveying of P.A.S. positive material towards the loculus, the formation of exine precursors, viscin threads and orbicules (= Ubisch bodies), the production of sporophytic proteins and enzymes, and of pollenkitt/tryphine. Not all these functions are present in all land plants:Embryophyta. Two main tapetal types are usually distinguished in theSpermatophyta: the secretory or parietal type and the amoeboid or periplasmodial type; in lower groups, however, other types may be recognized, with greater or lesser differences. A hypothetical phylogenesis of the tapetum is proposed on the basis of its morphological appearance and of the nutritional relations with meiocytes/spores. The evolutionary trends of the tapeta tend towards a more and more intimate and increasingly greater contact with the spores/pollen grains. Three evolutionary trends can be recognized: 1) an intrusion of the tapetal cells between the spores, 2) a loss of tapetal cell walls, and 3) increasing nutrition through direct contact in narrow anthers.

A route for the passage of substances through the developing pteridophyte exine

The developing exine ofLycopodium gnidioides is traversed from the outer to the inner surface by a series of anastomosing channels filled or lined with fibrillar glycoprotein. When living sporangia are incubated in colloidal iron, particles of ferric iron can be detected in the exine channels, the intine and the spore cytoplasm, and some iron is retained by the surface coatings on the spore. Although there is some diffuse iron staining of the exine between the channels, the main concentration of particles is associated with these structures. This, together with the fact that the proximal lasurae of the spore are closed during development, is taken as evidence that the iron has passed from the locular fluid to the surface of the protoplast principally by way of the exine channels. Results obtained from fixation in a glutaraldehyde-lanthanum nitrate mixture support this interpretation. While the exine channels are in existence, therefore, the spore protoplast is in open communication with the locular environment. The study provides no evidence to suggest that the iron which entered the spore cytoplasm did so by endocytosis. It is possible that iron altered the permeability of the plasma membrane by damaging its structure; entry of iron to the cytoplasm being effected through the damaged membrane.