A Promising Ash Supplementation Strategy in the Cultivation of Spirodela polyrrhiza Plants.

An innovative approach to the management of waste in the form of ash obtained during biomass combustion is justified due to its specific properties, including the presence of macro- and microelements. The aim of the current study was to determine the concentration of ash obtained from Sorghum combustion regarding its fertilizer value and its effect on the cytological structures, physiological parameters, growth and development of Lemnaceae plants, thereby demonstrating the possibility of using this waste to supplement culture media. The analyses showed that the use of ash in the in vitro cultivation of Lemnaceae aquatic plants had a dose-dependent effect. The addition of 2% ash favorably affected the condition of plant roots, i.e., meristem elongation and an increase in nucleoli sizes as well as improving the chlorophyll content index, gas exchange parameters, chemical oxygen demand (COD) and plant vigor via PSII, which was confirmed by a chlorophyll fluorescence measurement. On the other hand, too high of a concentration, i.e., 10% ash, adversely affected the plant development and parameters studied. Concluding, the use of ash at a low concentration favorably affected the yielding of Spirodela polyrrhiza, whose biomass can be used for energy purposes in the production of bioethanol, plant biogas or the phytoremediation of industrial waters and leachate.

The Role of Cutinsomes in Plant Cuticle Formation.

The cuticle commonly appears as a continuous lipophilic layer located at the outer epidermal cell walls of land plants. Cutin and waxes are its main components. Two methods for cutin synthesis are considered in plants. One that is based on enzymatic biosynthesis, in which cutin synthase (CUS) is involved, is well-known and commonly accepted. The other assumes the participation of specific nanostructures, cutinsomes, which are formed in physicochemical self-assembly processes from cutin precursors without enzyme involvement. Cutinsomes are formed in ground cytoplasm or, in some species, in specific cytoplasmic domains, lipotubuloid metabolons (LMs), and are most probably translocated via microtubules toward the cuticle-covered cell wall. Cutinsomes may additionally serve as platforms transporting cuticular enzymes. Presumably, cutinsomes enrich the cuticle in branched and cross-linked esterified polyhydroxy fatty acid oligomers, while CUS1 can provide both linear chains and branching cutin oligomers. These two systems of cuticle formation seem to co-operate on the surface of aboveground organs, as well as in the embryo and seed coat epidermis. This review focuses on the role that cutinsomes play in cuticle biosynthesis in S. lycopersicum, O. umbellatum and A. thaliana, which have been studied so far; however, these nanoparticles may be commonly involved in this process in different plants.

Cutinsomes as building-blocks of Arabidopsis thaliana embryo cuticle.

Cutinsomes, spherical nanoparticles containing cutin mono- and oligomers, are engaged in cuticle formation. Earlier they were revealed to participate in cuticle biosynthesis in Solanum lycopersicum fruit and Ornithogalum umbellatum ovary epidermis. Here, transmission electron microscopy (TEM) and immunogold labeling with antibody against the cutinsomes were applied to aerial cotyledon epidermal cells of Arabidopsis thaliana mature embryos. TEM as well as gold particles conjugated with the cutinsome antibody revealed these structures in the cytoplasm, near the plasmalemma, in the cell wall and incorporated into the cuticle. Thus, the cutinsomes most probably are involved in the formation of A. thaliana embryo cuticle and this model plant is another species in which these specific structures participate in the building of cuticle in spite of the lack of the lipotubuloid metabolon. In addition, a mechanism of plant cuticle lipid biosynthesis based on current knowledge is proposed.

Cutinsomes and cuticle enzymes GPAT6 and DGAT2 seem to travel together from a lipotubuloid metabolon (LM) to extracellular matrix of O. umbellatum ovary epidermis.

In the ovary epidermis of O. umbellatum there are lipotubuloid metabolons (LMs), in which synthesis of lipids takes place. This process partly provides nourishment, and partly cuticle building blocks, transformed, among others, with the participation of cutinsomes. The cutinsomes are cutin-building structures, 40-200nm in size, which are formed as a result of self-assembly and self-esterification of hydroxy fatty acids. The cutinsomes, by binding to the cuticle, introduce into it nonlinear, amorphous and cross-linked polymers. Double-immunogold EM observations revealed that enzymes producing elements of cutin (GPAT6) and waxes (WS/DGAT) were found not only as free cytoplasmic molecules but also in many cases they were bound to carboxylate-carboxylic shell of cuntinsomes. Hence, we suppose that these enzymes can move alone or together with the cutinsomes through cytoplasm (pH 6.8-7.0), plasmalemma and the polysaccharide layer of a cell wall to the site of their functioning i.e. to the cuticle (pH 5.0).

Lipotubuloids in ovary epidermis of Ornithogalum umbellatum act as metabolons: suggestion of the name 'lipotubuloid metabolon'.

A metabolon is a temporary, structural-functional complex formed between sequential metabolic enzymes and cellular elements. Cytoplasmic domains called lipotubuloids are present in Ornithogalum umbellatum ovary epidermis. They consist of numerous lipid bodies entwined with microtubules, polysomes, rough endoplasmic reticulum (RER), and actin filaments connected to microtubules through myosin and kinesin. A few mitochondria, Golgi structures, and microbodies are also observed and also, at later development stages, autolytic vacuoles. Each lipotubuloid is surrounded by a tonoplast as it invaginates into a vacuole. These structures appear in young cells, which grow intensively reaching 30-fold enlargement but do not divide. They also become larger due to an increasing number of lipid bodies formed in the RER by the accumulation of lipids between leaflets of the phospholipid bilayer. When a cell ceases to grow, the lipotubuloids disintegrate into individual structures. Light and electron microscope studies using filming techniques, autoradiography with [(3)H]palmitic acid, immunogold labelling with antibodies against DGAT2, phospholipase D1 and lipase, and double immunogold labelling with antibodies against myosin and kinesin, as well as experiments with propyzamide, a microtubule activity inhibitor, have shown that lipotubuloids are functionally and structurally integrated metabolons [here termed lipotubuloid metabolons (LMs)] occurring temporarily in growing cells. They synthesize lipids in lipid bodies in cooperation with microtubules. Some of these lipids are metabolized and used by the cell as nutrients, and others are transformed into cuticle whose formation is mediated by cutinsomes. The latter were discovered in planta using specific anti-cutinsome antibodies visualized by gold labelling. Moreover, LMs are able to rotate autonomously due to the interaction of microtubules, actin filaments, and motor proteins, which influence microtubules by changing their diameter.

Cutinsomes and lipotubuloids appear to participate in cuticle formation in Ornithogalum umbellatum ovary epidermis: EM-immunogold research.

The outer wall of Ornithogalum umbellatum ovary and the fruit epidermis are covered with a thick cuticle and contain lipotubuloids incorporating (3)H-palmitic acid. This was earlier evidenced by selective autoradiographic labelling of lipotubuloids. After post-incubation in a non-radioactive medium, some marked particles insoluble in organic solvents (similar to cutin matrix) moved to the cuticular layer. Hence, it was hypothesised that lipotubuloids participated in cuticle synthesis. It was previously suggested that cutinsomes, nanoparticles containing polyhydroxy fatty acids, formed the cuticle. Thus, identification of the cutinsomes in O. umbellatum ovary epidermal cells, including lipotubuloids, was undertaken in order to verify the idea of lipotubuloid participation in cuticle synthesis in this species. Electron microscopy and immunogold method with the antibodies recognizing cutinsomes were used to identify these structures. They were mostly found in the outer cell wall, the cuticular layer and the cuticle proper. A lower but still significant degree of labelling was also observed in lipotubuloids, cytoplasm and near plasmalemma of epidermal cells. It seems that cutinsomes are formed in lipotubuloids and then they leave them and move towards the cuticle in epidermal cells of O. umbellatum ovary. Thus, we suggest that (1) cutinsomes could take part in the synthesis of cuticle components also in plant species other than tomato, (2) the lipotubuloids are the cytoplasmic domains connected with cuticle formation and (3) this process proceeds via cutinsomes.

Occurrence of calreticulin during the exchange of nucleohistones into protamine-type proteins in Chara vulgaris spermiogenesis.

During spermiogenesis of an alga Chara vulgaris, which resembles that of animals, nucleohistones are replaced by protamine-type proteins. This exchange takes place in a spermatid nucleus during the key V spermiogenesis stage, in which rough endoplasmic reticulum is the site of protamine-type protein synthesis and is also the pathway guiding the proteins to their destination, nucleus. In the present work, it was shown that a chaperon protein, calreticulin (CRT), abundantly present at this significant V stage of spermiogenesis in a few cellular compartments, i.e., a nucleus, lumen of cisternae, and vesicles of significantly swollen ER as well as outside these structures, e.g., in Golgi apparatus, could have taken part in the process of exchange of nuclear proteins. Colocalization of two proteins, protamine-type proteins, crucial for reproduction, and CRT, was especially visible in a nucleus, mainly on its peripheries where condensed chromatin was present. Localization of protamine-type proteins and CRT in nucleus is in agreement with our previous results showing that protamine-type proteins were twofold more labelled in the peripheral area in comparison to the nucleus center occupied by noncondensed chromatin. The role of CRT in the reproduction of both plants and animals is also discussed.

DGAT2 revealed by the immunogold technique in Arabidopsis thaliana lipid bodies associated with microtubules.

The immunogold technique with anti-diacylglycerol acyltransferase 2 (DGAT2) antibody revealed in A. thaliana embryo and root meristematic cells gold particles manifesting the presence of DGAT2 in ER as well as in lipid bodies. This being so, lipid synthesis could take place both in ER and in the lipid bodies. The presence of microtubules around the lipid bodies was evidenced under transmission EM. Detection of tubulin around the lipid bodies using the immunogold technique with anti-a-tubulin is in agreement with the above observations. Connection of lipid bodies with microtubules was also detected by us in other plants where they probably participated in lipid synthesis. A similar phenomenon may take place in A. thaliana.

Lipid body biogenesis and the role of microtubules in lipid synthesis in Ornithogalum umbellatum lipotubuloids.

Lipid bodies present in lipotubuloids of Ornithogalum umbellatum ovary epidermis take the form of a lens between leaflets of ER (endoplasmic reticulum) membrane filled with a highly osmiophilic substance. The two enzymes, DGAT1 [DAG (diacylglycerol) acyltransferase 1] and DGAT2 (DAG acyltransferase 2), involved in this process are synthesized on rough ER and localized in the ER near a monolayer surrounding entities like lipid bodies. After reaching the appropriate size, newly formed lipid bodies transform into mature spherical lipid bodies filled with less osmiophilic content. They appear to be surrounded by a half-unit membrane, with numerous microtubules running adjacently in different directions. The ER, no longer continuous with lipid bodies, makes contact with them through microtubules. At this stage, lipid synthesis takes place at the periphery of lipid bodies. This presumption, and a hypothesis that microtubules are involved in lipid synthesis delivering necessary components to lipid bodies, is based on strong arguments: (i) silver grains first appear over microtubules after a short [3H]palmitic acid incubation and before they are observed over lipid bodies; (ii) blockade of [3H]palmitic acid incorporation into lipotubuloids by propyzamide, an inhibitor of microtubule function; and (iii) the presence of gold grains above the microtubules after DGAT1 and DGAT2 reactions, as also near microtubules after an immunogold method that identifies phospholipase D1.

Microtubule heterogeneity of Ornithogalum umbellatum ovary epidermal cells: non-stable cortical microtubules and stable lipotubuloid microtubules.

Lipotubuloids, structures containing lipid bodies and microtubules, are described in ovary epidermal cells of Ornithogalum umbellatum. Microtubules of lipotubuloids can be fixed in electron microscope fixative containing only buffered OsO(4) or in glutaraldehyde with OsO(4) post-fixation, or in a mixture of OsO(4) and glutaraldehyde. None of these substances fixes cortical microtubules of ovary epidermis of this plant which is characterized by dynamic longitudinal growth. However, cortical microtubules can be fixed with cold methanol according immunocytological methods with the use of ß-tubulin antibodies and fluorescein. The existence of cortical microtubules has also been evidenced by EM observations solely after the use of taxol, microtubule stabilizer, and fixation in a glutaraldehyde/OsO(4) mixture. These microtubules mostly lie transversely, sometimes obliquely, and rarely parallel to the cell axis. Staining, using Ruthenium Red and silver hexamine, has revealed that lipotubuloid microtubules surface is covered with polysaccharides. The presumption has been made that the presence of a polysaccharide layer enhances the stability of lipotubuloid microtubules.

Diameters of microtubules change during rotation of the lipotubuloids of Ornithogalum umbellatum stipule epidermis as a result of varying protofilament monomers sizes and distance between them.

Microtubules in lipotubuloids of the Ornithogalum umbellatum stipule epidermis cells change their diameters depending on the motion of the cytoplasmic domains rich in microtubules and lipid bodies. Microtubules fixed during rotary and progressive motion of the lipotubuloids composed of the same number of protofilaments fall into two populations - wide (43-58 nm) and narrow (24-39 nm) in size. Following blockage of the motion with 2,4-dinitrophenol (DNP), the range of this diversity is smaller, microtubules become a medium-sized population (34-48 nm). When DNP is removed and the motion reactivated, 2 populations of microtubules reappear. Analysis of the structure of the microtubule wall revealed that changes in the microtubule diameters resulted from varying distances between the adjacent protofilaments, and stretching and compression of tubulin subunits in the protofilaments. A supposition has been put forward that the changes in the sizes of O. umbellatum microtubule diameters: 1) are connected with the interactions between microtubules and actin microfilaments lying along these microtubules; 2) can be the driving force of the rotary motion of lipotubuloids.

Immunogold evidence suggests that endoplasmic reticulum is the site of protamine-type protein synthesis and participates in translocation of these proteins into the nucleus during Chara vulgaris spermiogenesis.

During spermiogenesis of an alga Chara vulgaris, which in many aspects resembles that of animals, histones are replaced by protamine-type proteins. Our earlier immunocytochemical studies showed that this replacement started during the short stage V of spermiogenesis, when electronograms revealed an extensive system of cisternae and vesicles of endoplasmic reticulum (ER). The present studies revealed at stage V intensive incorporation of labeled (3)H-arginine and (3)H-lysine quickly translocating into a nucleus visualized with pulse-chase autoradiography of semithin sections. The immunogold technique with the use of the antibodies to protamine-type proteins isolated from Chara tomentosa show that both ER cisternae and vesicles are labeled with gold grains, which are absent from the spermatids not treated with the antibodies; thus, the ER is probably the site of the protamine-type protein synthesis. These proteins then are translocated to a nucleus through ER channels connected with the nuclear envelope, as suggested by gold labeling of an inner membrane of the nuclear envelope adjacent to condensed chromatin. The above results correspond with those of other authors showing that in animals, protamines bind with lamin B receptors localized in the inner membrane of the nuclear envelope. A hypothesis has been put forward that during Chara spermiogenesis the inner membrane of the nuclear envelope invaginates into a nucleus together with protamine-type proteins, which become separated from the membrane and penetrate into chromatin.

Role of DNA endoreduplication, lipotubuloids, and gibberellic acid in epidermal cell growth during fruit development of Ornithogalum umbellatum.

Cytophotometry of individual nuclei was used to examine the level of endoreduplication in epidermal cells from the upper and lower parts of the ovary during Ornithogalum umbellatum flower and fruit development. An increase in DNA content from 2-4C to 2-8C in both parts of the ovary was observed, while the epidermal cell surface area grew about 6-fold and 15-fold in the lower and upper parts of the ovary, respectively. However, the correlation between mean epidermal cell size and ploidy was distinct during epidermis growth. Lipotubuloids became bigger in the upper than in the lower part during ovary and fruit development. In addition, more dynamic growth of the epidermal cells of the upper than of the lower part of the ovary was connected to the higher content of gibberellic acid. A hypothesis has been put forward that the role of DNA endoreduplication in epidermal cell growth was modulated by the function of lipotubuloids and the gradient of gibberellin.

Cytochemical and immunocytochemical studies of the localization of histones and protamine-type proteins in spermatids of Chara vulgaris and Chara tomentosa.

Spermiogenesis in Chara algae, which has been divided into 10 phases (sp I-X), is similar to spermiogenesis in animals. The most important process during spermiogenesis in animals is remodeling of chromatin leading to "sleeping genome", being the result the exchange of histone proteins into protamine-like proteins. Cytochemical studies showed in both Chara species (C. vulgaris, C. tomentosa) that at spI-IV phases only histones were present, at spV-VIII phases--the amount of nuclear protamine-type proteins progressively increased and that of histones decreased while at spIX-X only pro-tamine-type proteins were present. This was also confirmed with capillar electrophoresis. In order to localize more precisely both histones and protamines the immunocytochemical studies with the use of anti-protamine antibodies (protamine-type proteins were obtained from C. tomentosa antheridia) and anti-histone H3 antibodies, have been carried out. More specific immunocytochemical studies confirmed cytochemical results including the exchange of histones into protamine-type during spermiogenesis (spV-VIII) in both Chara species. At phase V spermiogenesis these strong strand-like anti-protamine signals were observed in cytoplasm which might suggest that protamine synthesis took place in ER.

Microtubules with different diameter, protofilament number and protofilament spacing in Ornithogalum umbellatum ovary epidermis cells.

Microtubules present in the epidermis of Ornithogalum umbellatum ovary in the area of lipotubuloids (i.e. aggregates of lipid bodies surrounded by microtubules) are 25-51 nm in diameter. They consist mainly of 10 and 11, sometimes 9 and 12 protofilaments. An average diameter of microtubule consisting of 9 subunits is about 32 nm, of 10-35 nm, of 11-38 nm and of 12-43 nm, however, individual microtubules in each category significantly vary in size. These differences result from varying distance between protofilaments in microtubule walls and diameters of protofilaments: in thin microtubules they are densely packed and smaller while in thicker ones they are loosely arranged and bigger. A hypothesis has been put forward that changes in microtubule diameter depend on structural changes associated with their functional status and are executed by modifications of protofilament arrangement density and their diameters in microtubule wall. The above hypothesis seems to be in agreement with the opinion formed on the basis of in vitro image of microtubules, that lateral contact between tubulin subunits in neighboring protofilaments indicates some flexibility and changeability during microtubule function.

The influence of epoxomicin, inhibitor of proteasomal proteolytic activity, on spermiogenesis in Chara vulgaris.

The influence of 48-h treatment with epoxomicin, an inhibitor of proteolytic activity of proteasomes, at the concentration 10 microM, on spermiogenesis in algae Chara vulgaris was examined. In the presence of the inhibitor, the frequency of early spermiogenesis phases significantly increased, the number of spermatids in mid-phases decreased and disappearance of late phases was observed. A hypothesis has been put forward that epoxomicin stops spermiogenesis during the period of preparation to further deep reorganisation of spermatids by blocking proteolysis of short-lived regulatory proteins which are responsible among others for triggering the exchange of nucleohistones into nucleoprotamines.

Further ultrastructural research of Chara vulgaris spermiogenesis: endoplasmic reticulum, structure of chromatin, 3H-lysine and 3H-arginine incorporation.

On the basis of morphological features, 10 consecutive structural phases of spermatids were identified in Chara vulgaris spermiogenesis. They were schematically presented. In early and middle spermiogenesis, i.e. during the period preceding formation of fibrillar structure of mature spermatozoid nucleus, a slight remodelling of chromatin, accompanied by proplastid transformation into an amyloplast as well as by development of 2 flagella and a microtubular manchette, is observed. First, condensed chromatin concentrates around the nuclear envelope (phases III-V) and then it transforms into a network-like structure (phase VI). This change in chromatin structure is preceded by nucleolar extrusion to the cytoplasm where nucleoli become degraded (phase IV) and by a dynamic development of rough endoplasmic reticulum (RER) (phase V) which is continuous with the nuclear envelope and with RER of the adjacent spermatids via plasmodesmata. The inner membrane of the nuclear envelope invaginates into the nucleoplasm in which "nuclear reticulum" appears. It all happens during increased 3H-arginine and 3H-lysine incorporation into proteins which are rapidly translocated into the nucleus. In medium-late spermiogenesis (phases VI-VIII), network-like condensed chromatin disappears. Next, the structure of the nucleus changes dramatically. Short, randomly positioned fibrils (phase VII) appear and gradually become longer (phase VIII), thicker (phase IX) and more distinct, lying parallel to the surface of elongating and curling nucleus. Membranes of the nuclear envelope become closer to each other and a distinct dark layer--probably lamin--appears adhering to the inner membrane of the nuclear envelope. Towards the end of spermiogenesis (phase X), very densely packed parallel helices, ca 2 nm in diameter, are visible. The surfaces of flagella and the spermatozoid are covered with diamond-shaped larger and smaller scales, respectively. Helically coiled spermatozoids are liberated from antheridial filament cells through earlier created (phase VIII) "liberation pores" with pads of unknown nature.