Małgorzata Kapusta

Studies on floral nectary, tepals’ structure, and gynostemium morphology of Epipactis palustris (L.) Crantz (Orchidaceae)

The lip of Epipactis palustris consists of two movably joined parts: the basal part (hypochile) with central broad isthmus and epichile with callus. The analysis of flowers provides strong evidence to conclude that the whole surface of lip callus and abaxial side of isthmus are secretory. The exudation at first appears on callus, at early stages, later on isthmus. It could be a strategy to prolong the emission of volatile substances and nectar, and this means to prolong luring pollinators. The results from transmission electron microscopy (TEM) support this conclusion. The plastids noted in callus were without starch, whereas the isthmus’ cells contained partly hydrolyzed starch. Some plastids, noted in callus, had polymorphic shapes, which were often related to a starch reduction. During the depletion of starch in callus cells, the number of plastoglobuli within the plastids increased, and also lipid bodies appeared in the cytoplasm whereas, in isthmus cells, proplastids with phytoferritin were noted. The endoplasmic reticulum was in contact with plasmalemma, and the vesicles were fusing with plasmalemma in secretory cells of callus and isthmus, which is a way of granulocrine secretion. The cross-sections of sepals revealed that abaxial epidermis was tomentose, with stomata at the top of substomatal cavities. The pollen grains adhering to the rostellum-viscidium prove previous ecological observations that the rostellum-viscidium is not a barrier preventing self-pollination.

Evidence for mixed sexual and asexual reproduction in the rare European mycoheterotrophic orchid Epipogium aphyllum, Orchidaceae (ghost orchid)

BACKGROUND AND AIMS Despite their significant capacity to propagate vegetatively, most orchids reproduce via seeds. Sexual reproduction via seed is commonly reported, in contrast to apomixis, whereby seeds are clones of the mother. Although insect pollination and autonomous self-pollination exist in mycoheterotrophic plants, the reproductive embryology of these plants remains under-studied. This paper provides evidence for the co-occurrence of both sexual and apomictic reproduction in a population of mycoheterotrophic plants - Epipogium aphyllum We investigated seed formation via open pollination, induced autogamy, autogamy sensu stricto and autonomous apomixis. METHODS The study was performed on a population of E. aphyllum located in northern Poland. The research included studies of the micromorphology, histochemistry and embryology of four types of reproductive systems. Scanning, fluorescence and light microscopy accompanied by graphical and statistical analyses were employed. KEY RESULTS We observed gametophyte development, from the one-nucleate stage to maturity, in unpollinated flower buds. The lack of zygotes in flower buds indicated that fertilization did not occur at this stage. Manual self-pollination led to a zygote, followed by embryo formation. Fertilization and embryo development derived from embryogenesis via open pollination is delayed compared with hand pollination. Isolation from external pollination resulted only in structures resembling zygotes that may originate either sexually or independent of fertilization. Parthenogenetic structures that resembled zygotes were observed in flowers that were emasculated and isolated from pollination. Zygotes formed at significantly higher frequencies via open pollination and induced autogamy in comparison to the parthenogenetic structures formed in other treatments. CONCLUSIONS We showed the absence of pre-zygotic barriers for autogamy in E. aphyllum Self-pollination and self-fertilization are possible; however, natural self-pollination is unlikely or rare due to the position of the pollinia. Incidental parthenogenesis in E. aphyllum is very likely, given the biology of ovule development of this mycoheterotrophic orchid. This species therefore has the potential to produce seeds via both sexual and asexual means, although the contribution of apomixis to this process appears largely negligible.

Antennal and mouthpart sensilla of Acanthoscelides obtectus say (Coleoptera: Bruchidae)

Acanthoscelides obtectus, a serious pest with a worldwide distribution, damages stored seeds of Phaseolus sp. To acquire a better understanding of the sensory orientation of bean weevils, their antennal and palpal sensilla were investigated. The antennae and palps possess several types of sensory hairs: blunt‐tipped sensilla chaetica and trichoidea and also sharp‐tipped sensilla trichoidea, these last being the most numerous. Sensilla basiconica are present only on the flagellomeres, but Böhm bristles occur on the basal sclerite and scape of the antennae. The terminal segments of the maxillary and labial palps bear a group of sensilla styloconica with single apical pores. Sensilla coeloconica are located only on the terminal palpal segments. They were unable to detect any distinct morphological differences between males and females. The possible chemosensory, mechanosensory, and thermo‐/hygroreceptive functions of the sensilla are discussed.

Establishing the cell biology of apomictic reproduction in diploid Boechera stricta (Brassicaceae)

Abstract Background and aims In the Brassicaceae family, apomictic development is characteristic of the genus Boechera. Hybridization, polyploidy and environmental adaptation that arose during the evolution of Boechera may serve as (epi)genetic regulators of apomictic initiation in this genus. Here we focus on Boechera stricta, a predominantly diploid species that reproduces sexually. However, apomictic development in this species has been reported in several studies, indicating non-obligate sexuality. Methods A progressive investigation of flower development was conducted using three accessions to assess the reproductive system of B. stricta. We employed molecular and cyto-embryological identification using histochemistry, transmission electron microscopy and Nomarski and epifluorescence microscopy. Key Results Data from internal transcribed spacer (ITS) and chloroplast haplotype sequencing, in addition to microsatellite variation, confirmed the B. stricta genotype for all lines. Embryological data indicated irregularities in sexual reproduction manifested by heterochronic ovule development, longevity of meiocyte and dyad stages, diverse callose accumulation during meiocyte-to-gametophyte development, and the formation of triads and tetrads in several patterns. The arabinogalactan-related sugar epitope recognized by JIM13 immunolocalized to one or more megaspores. Furthermore, pollen sterility and a high frequency of seed abortion appeared to accompany reproduction of the accession ES512, along with the initiation of parthenogenesis. Data from flow cytometric screening revealed both sexual and apomictic seed formation. Conclusion These results imply that B. stricta is a species with an underlying ability to initiate apomixis, at least with respect to the lines examined here. The existence of apomixis in an otherwise diploid sexual B. stricta may provide the genomic building blocks for establishing highly penetrant apomictic diploids and hybrid relatives. Our findings demonstrate that apomixis per se is a variable trait upon which natural selection could act.

Mature Luffa Leaves (Luffa cylindrica L.) as a Tool for Gene Expression Analysis by Agroinfiltration

We exploited the potential of cucurbits for ectopic gene expression. Agroinfiltration is a simple and commonly used method to obtain transient expression of foreign genes in plants. In contrast to in vitro transformation techniques, agroinfiltration can be used for genetic modification of mature plant tissues. Although the cucurbits are commonly used as model plants for molecular biology and biotechnology studies, to date there are no literature sources on the possibility of transient gene expression in mature cucurbit tissues. Our research has shown that mature leaves of Luffa cylindrica L. (luffa), in contrast to other cucurbit species, can be successfully transiently transformed with Agrobacterium tumefaciens. We efficiently transformed luffa leaves with a reporter gene encoding β-glucuronidase (GUS). The GUS activity in transiently transformed leaf tissues was detected within 24 h after the infiltration with bacteria. Additionally, we have shown that the activity of a transiently expressed the GUS gene can be monitored directly in the EDTA-exudates collected from the cut petioles of the agroinfiltrated leaves. The results suggest that luffa leaves can be useful as a plant expression system for studies of physiological and biochemical processes in cucurbits.

Production of recombinant human deoxyribonuclease I in Luffa cylindrica L. and Nicotiana tabacum L.: evidence for protein secretion to the leaf intercellular space

The sequence encoding hDNase I with its native N-terminal signal peptide was codon-optimized for heterological expression in cucurbit plants. The obtained synthetic gene rhDN was successfully transiently expressed in L. cylindrica (luffa) leaves using agroinfiltration. The results of in silico analysis suggest that the rhDN gene can be expressed in different cucurbits and in non-cucurbit species, such as a model plant Nicotiana tabacum. For further analysis of the recombinant hDNase I (rhDNase I) produced in planta we generated stably transformed tobacco lines (NtDN) overexpressing the rhDN gene. The presence of the active rhDNase I in the total protein extracts and in the intercellular fluids isolated from the leaves of the transgenic NtDN lines was demonstrated. However, the plants producing the rhDNase I did not show any visible changes in the phenotype. Using indirect immunofluorescence, we localized the rhDNase I in the intercellular space of NtDN leaves, which suggests that the native hDNase I signal peptide can be correctly recognized in the plant cells, what results in the protein targeting to the endoplasmic reticulum and then to the secretory pathway. Our results indicate the possibility to use the human DNase I as a molecular tool for improving and optimization of the plant expression systems based on cucurbits. Additionally, the established tobacco lines overexpressing the rhDN gene can be used as a platform to produce the active human DNase I.

How Does the Sweet Violet (Viola odorata L.) Fight Pathogens and Pests – Cyclotides as a Comprehensive Plant Host Defense System

Cyclotides are cyclic plant polypeptides of 27–37 amino acid residues. They have been extensively studied in bioengineering and drug development contexts. However, less is known about the relevance of cyclotides for the plants producing them. The anti-insect larvae effects of kB1 and antibacterial activity of cyO2 suggest that cyclotides are a part of plant host defense. The sweet violet (Viola odorata L.) produces a wide array of cyclotides, including kB1 (kalata B1) and cyO2 (cycloviolacin O2), with distinct presumed biological roles. Here, we evaluate V. odorata cyclotides’ potency against plant pathogens and their mode of action using bioassays, liposome experiments and immunogold labeling for transmission electron microscopy (TEM). We explore the link between the biological activity and distribution in plant generative, vegetative tissues and seeds, depicted by immunohistochemistry and matrix assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). Cyclotides cyO2, cyO3, cyO13, and cyO19 are shown to have potent activity against model fungal plant pathogens (Fusarium oxysporum, F. graminearum, F. culmorum, Mycosphaerella fragariae, Botrytis cinerea) and fungi isolated from violets (Colletotrichum utrechtense and Alternaria alternata), with minimal inhibitory concentrations (MICs) ranging from 0.8 μM to 25 μM. Inhibition of phytopathogenic bacteria – Pseudomonas syringae pv. syringae, Dickeya dadantii and Pectobacterium atrosepticum – is also observed with MIC = 25–100 μM. A membrane-disrupting antifungal mode of action is shown. Finding cyO2 inside the fungal spore cells in TEM images may indicate that other, intracellular targets may be involved in the mechanism of toxicity. Fungi can not break down cyclotides in the course of days. varv A (kalata S) and kB1 show little potency against pathogenic fungi when compared with the tested cycloviolacins. cyO2, cyO3, cyO19 and kB1 are differentially distributed and found in tissues vulnerable to pathogen (epidermis, rizodermis, vascular bundles, protodermis, procambium, ovary walls, outer integuments) and pest (ground tissues of leaf and petiole) attacks, respectively, indicating a link between the cyclotides’ sites of accumulation and biological role. Cyclotides emerge as a comprehensive defense system in V. odorata, in which different types of peptides have specific targets that determine their distribution in plant tissues.

Atypical mechanoreceptors in larvae of biting midges Forcipomyia nigra (Diptera: Ceratopogonidae)

The dorsolateral setae of Forcipomyia nigra have been investigated. They are mechanoreceptors, sensilla trichoidea, innerved by a single neuron. The tubular body is located at the base of the hair shaft. Although the fine structure of the dorsolateral setae is similar to dorsal setae that perform a double function: secretion of a hygroscopic viscous substance through the pores and mechanoreception dorsolateral setae, they do not secrete any fluid. In both types of setae, trichogen cells (hair forming cells) produce the hair shafts and thereafter do not retract from the cavity of the setae. They contain a large polyploid nucleus and expanded bundles of microtubules. In dorsolateral mechanoreceptors, the microtubules form a network around the nucleus of the trichogen cell and are especially numerous in the cytoplasm invading the interior of the seta, which is evidenced by immunofluorescence light microscopy. No tormogen cell, responsible for the production of the setal socket, was found. Our observations indicate that the dorsolateral setae are solely mechanoreceptors but their trichogen cells reveal some glandular activity.

Metamorphic changes in abdominal spines of Forcipomyia nigra pupae (Diptera: Ceratopogonidae).

Pupae of Forcipomyia nigra biting midges bear double rows of dorsal and lateral spines. Their arrangement corresponds to the distribution of larval mechanosensory setae. They are serrated simple cuticular structures with tubercles but, in contrast to larval secretory mechanoreceptors, they are not innervated and do not exhibit any pores. The ultrastructure of abdominal spines varies among different pupal stages. They are produced by epidermal cells which fill the interior of the spine. In the youngest pupae epidermal cells are tightly packed and adhere to the cuticle. Then, the cells withdraw from the spinal cavity and the beginning of autophagy is observed. The last stage represents abdominal spines without any cellular material and then apoptosis probably proceeds in the withdrawn epidermal cells. Since the pupal spines occupied the same region of the segment as the larval setae, we consider that the same genes are responsible for their formation as for the formation of epidermal cells but that their mechanosensory and secretory function is no longer needed.