Dorota Tchórzewska

The dynamics of the actin cytoskeleton during sporogenesis in Psilotum nudum L.

The actin cytoskeleton (microfilaments, MFs) accompanies the tubulin cytoskeleton (microtubules) during the meiotic division of the cell, but knowledge about the scope of their physiological competence and cooperation is insufficient. To cast more light on this issue, we analysed the F-actin distribution during the meiotic division of the Psilotum nudum sporocytes. Unfixed sporangia of P. nudum were stained with rhodamine-phalloidin and 4′,6-diamidino-2-phenylindole dihydrochloride, and we monitored the changes in the actin cytoskeleton and nuclear chromatin throughout sporogenesis. We observed that the actin cytoskeleton in meiotically dividing cells is not only part of the kariokinetic spindle and phragmoplast but it also forms a well-developed network in the cytoplasm present in all phases of meiosis. Moreover, in telophase I F-actin filaments formed short-lived phragmoplast, which was adjacent to the plasma membrane, exactly at the site of future cell wall formation. Additionally, the meiocytes were pre-treated with cytochalasin-B at a concentration that causes damage to the MFs. This facilitated observation of the effect of selective MFs damage on the course of meiosis and sporogenesis of P. nudum. Changes were observed that occurred in the cytochalasin-treated cells: the daughter nuclei were located abnormally close to each other, there was no formation of the equatorial plate of organelles and, consequently, meiosis did not occur normally. It seems possible that, if the actin cytoskeleton only is damaged, regular cytokinesis will not occur and, hence, no viable spores will be produced.

A new type of microtubular cytoskeleton in microsporogenesis of Lavatera thuringiaca L.

Summary.In Lavatera thuringiaca, kariokinesis and simultaneous cytokinesis during the meiotic division of microsporogenesis follow a procedure similar to that which takes place in the majority of members of the class Angiospermae. However, chondriokinesis occurs in a unique way found only in species from the family Malvaceae. Chondriokinesis in such species is well documented, but the relationship between the tubulin cytoskeleton and rearrangement of cell organelles during meiosis in L. thuringiaca has not been precisely defined so far. In this study, the microtubular cytoskeleton was investigated in dividing microsporocytes of L. thuringiaca by immunofluorescence. The meiotic stages and positions of cell organelles were identified by staining with 4′,6-diamidino-2-phenylindole. We observed that, during prophase I and II, changes in microtubular cytoskeleton configurations have unique features, which have not been described for other plant species. At the end of prophase I, organelles (mostly plastids and mitochondria) form a compact envelope around the nucleus, and the subsequent phases of kariokinesis take place within this arrangement. At this point of cell division, microtubules surround the organelle envelope and separate it from the peripheral cytoplasm, which is devoid of plastids and mitochondria. In telophase I, two newly formed nuclei are tightly surrounded by the cell organelle envelopes, and these are separated by the phragmoplast. Later, when the phragmoplast disappears, cell organelles still surround the nuclei but also move a little, starting to occupy the place of the disappearing phragmoplast. After the breakup of tetrads, the radial microtubule system is well developed, and cell organelles can still be observed as a dense envelope around the nuclei. At a very late stage of sporoderm development, the radial microtubule system disappears, and cell organelles become gradually scattered in the cytoplasm of the microspores. Using colchicines, specific inhibitors of microtubule formation, we investigated the relationship between the tubulin cytoskeleton and the distribution of cell organelles. Our analysis demonstrates that impairment of microtubule organization, which constitutes only a single component of the cytoskeleton, is enough to disturb typical chondriokinesis in L. thuringiaca. This indicates that microtubules (independent of microfilaments) are responsible for the reorganization of cell organelles during meiotic division.

Cytological and biophysical comparative analysis of cell structures at the microsporogenesis stage in sterile and fertile Allium species

AbstractMain conclusionUsing a live-cell-imaging approach and autofluorescence-spectral imaging, we showed quantitative/qualitative fluctuations of chemical compounds within the meiocyte callose wall, providing insight into the molecular basis of male sterility in plants from the genusAllium. Allium sativum (garlic) is one of the plant species exhibiting male sterility, and the molecular background of this phenomenon has never been thoroughly described. This study presents comparative analyses of meiotically dividing cells, which revealed inhibition at the different microsporogenesis stages in male-sterile A. sativum plants (cultivars Harnas and Arkus) and sterile A. ampeloprasum var. ampeloprasum (GHG-L), which is phylogenetically related to garlic. Fertile species A. ampeloprasum (leek) was used as the control material, because leek is closely related to both garlic and GHG-L. To shed more light on the molecular basis of these disturbances, autofluorescence-spectral imaging of live cells was used for the assessment of the biophysical/biochemical differences in the callose wall, pollen grain sporoderm, and the tapetum in the sterile species, in comparison with the fertile leek. The use of techniques for live-cell imaging (autofluorescence-spectral imaging) allowed the observation of quantitative/qualitative fluctuations of autofluorescent chemical compounds within the meiocyte callose wall. The biophysical characterisation of the metabolic disturbances in the callose wall provides insight into the molecular basis of male sterility in A. sativum. In addition, using this method, it was possible for the first time, to determine precisely (on the basis of fluctuations of autofluorescence compounds) the meiosis stage in which normal microsporogenesis is disturbed, which was not visible using light microscopy.

Changing of biochemical parameters and cell wall polysaccharides distribution during physiological development of tomato fruit

This study was aimed at discovering an impact of biochemical parameters (like content of cell wall polysaccharides, phenolic compounds, ascorbic acid or activity of pectinolytic enzymes) on cell wall microstructure during physiological fruit development. Cell wall microstructure as well as changes in the polysaccharides distribution were examined by confocal Raman microscopy. Also there was a need to simultaneous usage of reference method which is immunolabeling. A tomato fruit (Solanum lycopersicum cv Cerise) has been selected to observe the changes taking place in the fruit cell wall as it recently has been recognized as a model species for exploring fruit development processes such as fruit formation and ripening. Our studies showed that chemical images allows to depict changes in spatial distribution of polysaccharides in plant cell wall (including the middle lamella area), thus this technique allows to observation of cell wall degradation during tomato ripening (mainly pectic polysaccharides degradation). It seems that high level of pectinolytic enzymes activity and increasing content of ascorbate and hence decrease of pectins content have a significant impact on spatial distribution of biopolymers in fruit cell wall.

Tubulin cytoskeleton during microsporogenesis in the male-sterile genotype of Allium sativum and fertile Allium ampeloprasum L.

Key messageMicrosporogenesis in garlic.AbstractThe male-sterile Allium sativum (garlic) reproduces exclusively in the vegetative mode, and anthropogenic factors seem to be the cause of the loss of sexual reproduction capability. There are many different hypotheses concerning the causes of male sterility in A. sativum; however, the mechanisms underlying this phenomenon have not been comprehensively elucidated. Numerous attempts have been undertaken to understand the causes of male sterility, but the tubulin cytoskeleton in meiotically dividing cells during microsporogenesis has never been investigated in this species. Using sterile A. sativum genotype L13 and its fertile close relative A. ampeloprasum (leek), we have analysed the distribution of the tubulin cytoskeleton during microsporogenesis. We observed that during karyokinesis and cytokinesis, in both meiotic divisions I and II, the microtubular cytoskeleton in garlic L13 formed configurations that resembled tubulin arrangement typical of monocots. However, the tubulin cytoskeleton in garlic was distinctly poorer (composed of a few MT filaments) compared with that found in meiotically dividing cells in A. ampeloprasum. These differences did not affect the course of karyogenesis, chondriokinesis, and cytokinesis, which contributed to completion of microsporogenesis, but there was no further development of the male gametophyte. At the very beginning of the successive stage of development of fertile pollen grains, i.e. gametogenesis, there were disorders involving the absence of a normal cortical cytoskeleton and dramatically progressive degeneration of the cytoplasm in garlic. Therefore, we suggest that, due to disturbances in cortical cytoskeleton formation at the very beginning of gametogenesis, the intracellular transport governed by the cytoskeleton might be perturbed, leading to microspore decay in the male-sterile garlic genotype.

Effect of environment fluctuations on biomass and allicin level in Allium sativum (cv. Harnas, Arkus) and Allium ampeloprasum var. ampeloprasum (GHG-L)

Climate variables such as temperature and precipitation are the major abiotic environmental factors determining the yields in crop plants. Given the observed trends in climate change, it is important to carry out analyses aimed at description and selection of plant species characterised by not only the best performance traits but also the best adaptation to climate changes. This study focused on phenological-morphological-biochemical investigations, comparing Allium sativum with A. ampeloprasum var. ampeloprasum GHG-L. We present analyses of economically important traits (biomass and allicin levels) in garlic and GHG-L grown in ecological system and effect of environment fluctuations on these traits. Comparative analysis of the biomass and allicin level in the underground part of garlic and GHG-L revealed not only substantial differences between the species and cultivars, but also great impact of the climate variables on these traits. It was found that garlic and GHG-L cultivated in adverse conditions, exhibited lower yielding rates, but the content of allicin was inversely proportional to the biomass. It should be emphasised that, irrespective of the climate fluctuations, GHG-L produced higher biomass and exhibited higher allicin level than garlic grown in the same conditions, indicating that GHG-L is well adapted to adverse climate changes.