Differentiation and Propagation Potential of Arnica montana L. Achenes as a Consequence of the Morphological Diversity of Flowers and the Position of Flower Heads on the Plant.

Arnica montana L. is a very important medicinal plant and simultaneously a European endemic endangered plant species. The morphological features and details of seed development and achene variability are poorly recognized. The aim of this study was to determine the impact of the achene position in the infructescence and the location of the inflorescence on the plant on the (i) morphological characteristics and germination ability of achenes, and (ii) recruitment of seedlings and their biometric features. Infructescences containing fully ripe achenes were randomly collected from A. montana individuals for the measurements and the germination experiment. Scanning electron microscopy, fluorescence microscopy, and light microscopy were used for characterization of flowers and achenes. The morphological traits of achenes and reproductive characteristics of A. montana were determined by the position of the achenes in the infructescence and the location of the inflorescence on the plant. The surface of arnica achenes is equipped with non-glandular and glandular trichomes, which is very rarely presented in species of the family Asteraceae. It is possible that the fluid-containing glandular trichomes are a source of essential oils. The peripherally located achenes were longer, thinner, and lighter. They were characterized by lower embryo weight, lower embryo/achene weight ratio, and lower germination capacity in comparison to the centrally located ones. The results presented in this article fill the gap in the knowledge of the morphology of achenes and the biology of the species, and provide information that can help in breeding programs, active protection, and field cultivation.

Essential Oil from Arnica Montana L. Achenes: Chemical Characteristics and Anticancer Activity.

Mountain arnica Arnica montana L. is a source of several metabolite classes with diverse biological activities. The chemical composition of essential oil and its major volatile components in arnica may vary depending on the geographical region, environmental factors, and plant organ. The objective of this study was to characterize the chemical composition of essential oil derived from A. montana achenes and to investigate its effect on induction of apoptosis and autophagy in human anaplastic astrocytoma MOGGCCM and glioblastoma multiforme T98G cell lines. The chemical composition of essential oil extracted from the achenes was examined with the use of Gas Chromatography-Mass Spectrometry GC-MS. Only 16 components of the essential oil obtained from the achenes of 3-year-old plants and 18 components in the essential oil obtained from the achenes of 4-year-old plants constituted ca. 94.14% and 96.38% of the total EO content, respectively. The main components in the EO from the arnica achenes were 2,5-dimethoxy-p-cymene (39.54 and 44.65%), cumene (13.24 and 10.71%), thymol methyl ether (8.66 and 8.63%), 2,6-diisopropylanisole (8.55 and 8.41%), decanal (7.31 and 6.28%), and 1,2,2,3-tetramethylcyclopent-3-enol (4.33 and 2.94%) in the 3- and 4-year-old plants, respectively. The essential oils were found to exert an anticancer effect by induction of cell death in anaplastic astrocytoma and glioblastoma multiforme cells. The induction of apoptosis at a level of 25.7-32.7% facilitates the use of this secondary metabolite in further studies focused on the development of glioma therapy in the future. Probably, this component plays a key role in the anticancer activity against the MOGGCCM and T98G cell lines. The present study is the first report on the composition and anticancer activities of essential oil from A. montana achenes, and further studies are required to explore its potential for future medicinal purposes.

Distribution of plastids and mitochondria during male gametophyte formation in Tinantia erecta (Jacq.) Fenzl.

During meiosis in microsporogenesis, autonomous cellular organelles, i.e., plastids and mitochondria, move and separate into daughter cells according to a specific pattern. This process called chondriokinesis is characteristic for a given plant species. The key criterion for classification of the chondriokinesis types was the arrangement of cell organelles during two meiosis phases: metaphase I and telophase I. The autonomous organelles participate in cytoplasmic inheritance; therefore, their precise distribution to daughter cells determines formation of identical viable microspores. In this study, the course of chondriokinesis during the development of the male gametophyte in Tinantia erecta was analyzed. The study was conducted using optical and transmission electron microscopes. During microsporogenesis in T. erecta, autonomous cell organelles moved in a manner defined as a neutral-equatorial type of chondriokinesis. Therefore, metaphase I plastids and mitochondria were evenly dispersed around the metaphase plate and formed an equatorial plate between the daughter nuclei in early telophase I. Changes in the ultrastructure of plastids and mitochondria during pollen microsporogenesis were also observed.

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

MAIN CONCLUSION: Using 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 genus Allium. 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.

A study on calcium oxalate crystals in Tinantia anomala (Commelinaceae) with special reference to ultrastructural changes during anther development.

Calcium oxalate (CaOx) crystals in higher plants occur in five forms: raphides, styloids, prisms, druses, and crystal sand. CaOx crystals are formed in almost all tissues in intravacuolar crystal chambers. However, the mechanism of crystallization and the role of CaOx crystals have not been clearly explained. The aim of this study was to explore the occurrence and location of CaOx crystals in organs of Tinantia anomala (Torr.) C.B. Clarke (Commelinaceae) with special attention to ultrastructural changes in the quantity of tapetal raphides during microsporogenesis. We observed various parts of the plant, that is, stems, leaves, sepals, petals, anthers, staminal trichomes and stigmatic papillae and identified CaOx crystals in all parts except staminal trichomes and stigmatic papillae in Tinantia anomala. Three morphological forms: styloids, raphides and prisms were found in different amounts in different parts of the plant. Furthermore, in this species, we identified tapetal raphides in anthers. The number of tapetal raphides changed during microsporogenesis. At the beginning of meiosis, the biosynthesis of raphides proceeded intensively in the provacuoles. These organelles were formed from the endoplasmic reticulum system. In the tetrad stage, we observed vacuoles with needle-shaped raphides (type I) always localised in the centre of the organelle. When the amoeboid tapetum was degenerating, vacuoles also began to fade. We observed a small number of raphides in the stage of mature pollen grains.

Activity of selected hydrolytic enzymes in Allium sativum L. anthers.

The aim of the study was to determine enzymatic activity in sterile Allium sativum anthers in the final stages of male gametophyte development (the stages of tetrads and free microspores). The analysed enzymes were shown to occur in the form of numerous isoforms. In the tetrad stage, esterase activity was predominant, which was manifested by the greater number of isoforms of the enzyme. In turn, in the microspore stage, higher numbers of isoforms of acid phosphatases and proteases were detected. The development of sterile pollen grains in garlic is associated with a high level of protease and acid phosphatase activity and lower level of esterase activities in the anther locule. Probably this is the first description of the enzymes activity (ACPH, EST, PRO) in the consecutives stages of cell wall formation which is considered to be one of the causes of male sterility in flowering plant.

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

KEY MESSAGE: Microsporogenesis in garlic. The 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.

Male gametogenesis and sterility in garlic (Allium sativum L.): barriers on the way to fertilization and seed production.

Commercial cultivars of garlic (Allium sativum) do not produce flowers and seed; hence, information on microgametogenesis and genetic knowledge of this important crop is unavailable. Recently, physiological studies enabled flowering and fertility restoration in garlic bolting genotypes by environmental manipulations, thus broadening of the genetic variation and facilitating genetic studies. The present report provides first detailed description of the development of male gametophytes in 11 garlic genotypes varying in their fertility traits. Morphological and anatomical studies revealed completely fertile genotypes, as well as variation in anther and pollen development and disruption of the male organs and gametes at different developmental stages. Three types of plant sterility were observed, including complete sterility, male sterility and environmentally induced male sterility. The ITS1 and ITS2 regions of rRNA of the studied genotypes proved to be strongly conservative and thus did not correspond with the phenotypic expression of fertility or sterility in garlic. On the other hand, two-dimensional protein separation maps revealed significant differences between fertile and sterile genotypes, as well as between developmental stages of microsporogenesis. Further research is needed to investigate the internal mechanisms and environmental component of garlic sterility, as well as the possible molecular markers of these traits.

Characterization of callase (ß-1,3-D-glucanase) activity during microsporogenesis in the sterile anthers of Allium sativum L. and the fertile anthers of A. atropurpureum.

We examined callase activity in anthers of sterile Allium sativum (garlic) and fertile Allium atropurpureum. In A. sativum, a species that produces sterile pollen and propagates only vegetatively, callase was extracted from the thick walls of A. sativum microspore tetrads exhibited maximum activity at pH 4.8, and the corresponding in vivo values ranged from 4.5 to 5.0. Once microspores were released, in vitro callase activity peaked at three distinct pH values, reflecting the presence of three callase isoforms. One isoform, which was previously identified in the tetrad stage, displayed maximum activity at pH 4.8, and the remaining two isoforms, which were novel, were most active at pH 6.0 and 7.3. The corresponding in vivo values ranged from pH 4.75 to 6.0. In contrast, in A. atropurpureum, a sexually propagating species, three callase isoforms, active at pH 4.8-5.2, 6.1, and 7.3, were identified in samples of microsporangia that had released their microspores. The corresponding in vivo value for this plant was 5.9. The callose wall persists around A. sativum meiotic cells, whereas only one callase isoform, with an optimum activity of pH 4.8, is active in the acidic environment of the microsporangium. However, this isoform is degraded when the pH rises to 6.0 and two other callase isoforms, maximally active at pH 6.0 and 7.3, appear. Thus, factors that alter the pH of the microsporangium may indirectly affect the male gametophyte development by modulating the activity of callase and thereby regulating the degradation of the callose wall.

Differential influence of bacitracin on plant proteolytic enzyme activities.

The effect of bacitracin on the activity of proteases extracted from pollen and sprouts of various plant species and compared to five commercially available proteases was studied. Bacitracin stimulates some pollen proteolytic enzyme activities, contrary to its inhibitory influence on proteases from the other sources. Proteases from maize pollen, inhibited by pepstatin and phenylmethylsulfonyl fluoride, immediately accelerate their activities after addition of bacitracin to the reaction mixture. The stimulating influence of peptide antibiotic on pollen proteases of some plants is unexpected and molecular mechanism of this phenomenon requires a further elucidation. The augmentation of allergenic response caused by pollen enzymes and drugs containing bacitracin is discussed.