Elżbieta Kuta

Increased genetic diversity of Viola tricolor L. (Violaceae) in metal-polluted environments

Changes in DNA sequences affecting cryptic intraspecific variability are very important mechanisms of plant microevolutionary processes, initiating species diversification. In polluted environments, intra- and interpopulation changes at the molecular level proceed rapidly and lead to the formation of new ecotypes in a relatively short time. We used ISSR PCR fingerprinting data to analyze the genetic diversity and genetic structure of seven populations of Viola tricolor: four growing on soil contaminated with heavy metals (Zn, Pb, Cd; waste heaps) and three from control soil. The populations from the polluted sites showed higher genetic polymorphism (%(poly)=84%) and gene diversity (H(T)=0.1709) than the control populations (%(poly)=75% and H(T)=0.1448). The number of private markers we detected within metallicolous (MET) populations was more than double that found within non-metallicolous (NON) populations (15 vs. 7). The STRUCTURE and UPGMA analyses showed clear genetic differences between the NON and MET populations. Based on broad analyses of the genetic parameters, we conclude that the effect of these polluted environments on the genetic diversity of the MET populations, separating them from the NON populations, is evidence of microevolutionary processes at species level, leading to species divergence and the emergence of local ecotypes better adapted to their different environments.

The rare, endemic zinc violets of Central Europe originate from Viola lutea Huds

Two endemic zinc violets of the section Melanium Ging. occur on heavy metal soils of Central Europe. The form with yellow flowers is restricted to the area between Aachen, Germany, and Liège, Belgium, whereas the blue zinc violet exclusively thrives on a very small location at Blankenrode, Germany. Both violets are currently treated as separate species. Sequences of altogether 674 bp of the ITS1-5.8S rDNA-ITS2 regions of 61 different specimens of six taxa indicated that both violets are closely related to each other and also to Viola lutea Huds. Therefore these two zinc violets are, at best, subspecies or even only varieties of V. lutea. Thus they are termed V. lutea ssp. westfalica and V. lutea ssp. calaminaria in the present manuscript. Microsporogenesis, pollen morphology and viability of the zinc violets, particularly of the blue violet of Blankenrode, are often defective due to disturbed meiosis. The population of the blue violet might not yet be stabilized genetically but can cope with the adverse effects of the heavy metal elements.

Violets of the section Melanium, their colonization by arbuscular mycorrhizal fungi and their occurrence on heavy metal heaps

Violets of the sections Melanium were examined for their colonization by arbuscular mycorrhizal fungi (AMF). Heartsease (Viola tricolor) from several heavy metal soils was AMF-positive at many sites but not at extreme biomes. The zinc violets Viola lutea ssp. westfalica (blue zinc violet) and ssp. calaminaria (yellow zinc violet) were always AMF-positive on heavy metal soils as their natural habitats. As shown for the blue form, zinc violets germinate independently of AMF and can be grown in non-polluted garden soils. Thus the zinc violets are obligatorily neither mycotrophs nor metalophytes. The alpine V. lutea, likely ancestor of the zinc violets, was at best poorly colonized by AMF. As determined by atomic absorption spectrometry, the contents of Zn and Pb were lower in AMF colonized plants than in the heavy metal soils from where the samples had been taken. AMF might prevent the uptake of toxic levels of heavy metals into the plant organs. Dithizone staining indicated a differential deposition of heavy metals in tissues of heartsease. Leaf hairs were particularly rich in heavy metals, indicating that part of the excess of heavy metals is sequestered into these cells.

Morphological versus genetic diversity of Viola reichenbachiana and V. riviniana (sect. Viola, Violaceae) from soils differing in heavy metal content

Morphological characters, AFLP markers and flow cytometry were used to investigate the morphological and genetic variability and differentiation of Viola reichenbachiana and V. riviniana in non-metallicolous (NM) and metallicolous (M) populations. The aims were to clarify the taxonomic status of plants occurring in ore-bearing areas, to determine any relationship in V. reichenbachiana and V. riviniana from sites not polluted with heavy metals, and to examine the genetic variability and differentiation of M and NM populations of both species. Multivariate analyses based on morphological characters showed significant differences between V. reichenbachiana and V. riviniana from non-polluted sites, high levels of intra- and inter-population variability, and the occurrence of inter-specific hybrids. Plants from M populations showed hybrid characters but also fell within the range of V. riviniana or V. reichenbachiana. There were no significant differences in relative genome size between plants from polluted areas and V. riviniana from NM populations. Bayesian analysis of population genetic structure based on AFLP markers distinguished two main groups: V. reichenbachiana and V. riviniana together with the M populations. That analysis also revealed the occurrence of populations of inter-specific hybrids from non-polluted areas. Further Bayesian analysis of V. riviniana including NM and M populations separated all the studied M populations from NM populations. We conclude that plants forming the M populations are well adapted to a metal-polluted environment, and could be considered as stabilised introgressive forms resulting from unidirectional (asymmetric) introgression toward V. riviniana.

Floral structure and pollen morphology of two zinc violets (Viola lutea ssp. calaminaria and V. lutea ssp. westfalica) indicate their taxonomic affinity to Viola lutea

Two zinc violets, the yellow form of the Aachen–Liège area and the blue morph of Blankenrode in western Westphalia, have very restricted occurrence on heavy metal waste heaps. Their taxonomic affinities have not been finally resolved. The flower micromorphological analysis presented here indicates that both zinc violets are closely related to the alpine Viola lutea, in line with our earlier published molecular data, but not with the conclusions of other authors. The zinc violets are classed at the rank of subspecies as V. lutea: ssp. calaminaria for the yellow zinc violet and ssp. westfalica for its blue counterpart. Although the violets examined (V. lutea, V. lutea ssp. calaminaria, V. lutea ssp. westfalica) are closely related, there is no evidence that V. lutea ssp. westfalica is a descendent of V. tricolor. Here we provide the most detailed information on generative organ structure in the four violets studied.

Exogenous plant hormones and cyclotide expression in Viola uliginosa (Violaceae)

Plants from Violaceae produce cyclotides, peptides characterized by a circular peptide backbone and a cystine knot. This signature motif gives stability that can harness a wide spectrum of biological activities, with implications in plant defense and with applications in medicine and biotechnology. In the current work, cyclotide expressing in vitro cultures were established from Viola uliginosa. These cultures are useful models for studying biosynthesis of cyclotides and can also be used in their production. The cyclotide expression pattern is shown to be dependent on exogenous plant growth regulators, both on peptide and gene expression levels. The highest yields of cyclotides were obtained on media containing only a cytokinin and were correlated with storage material accumulation. Exposure to auxins decreased cyclotide production and caused shifting of the biosynthesis pattern to root specific cyclotides. The response to stimuli in terms of cyclotide expression pattern appears to be developmental, and related to polar auxin transportation and the auxin/cytokinin ratio regulating tissue differentiation. By the use of whole transcriptome shotgun sequencing (WTSS) and peptidomics, 20 cyclotide sequences from V. uliginosa (including 12 new) and 12 complete precursor proteins could be identified. The most abundant cyclotides were cycloviolacin O3 (CyO3), CyO8 and CyO13. A suspension culture was obtained that grew exponentially with a doubling time of approximately 3 days. After ten days of growth, the culture provided a yield of more than 4 mg CyO13 per gram dry mass.

Insight into “serpentine syndrome” of Albanian, endemic violets (Viola L., Melanium Ging. section) – Looking for unique, adaptive microstructural floral, and embryological characters

Abstract The genus Viola, particularly Melanium section, rich in metallophytes, is an excellent taxon for study of microevolutionary and adaptation processes. Pollen, ovule, and microstructural floral characters were investigated by LM, SEM, and CLSM in seven endemic Albanian violets, five serpentinophytes (Viola albanica, V. dukadjinica, V. albanica × V. dukadjinica, V. raunsiensis, and V. macedonica), two from chalk soil (V. aetolica and V. schariensis), and in their closest relatives (V. lutea ssp. sudetica, V. tricolor ssp. tricolor, and V. arvensis) for their taxonomic usefulness and adaptive value. Three among analyzed characters were common in all Albanian violets however not unique. Serpentinophytes, V. aetolica and V. schariensis possessed hairs deep inside the spur, developed pollen heteromorphism, both increase the chance of pollination in unpredictable conditions and had strongly developed tannin rich layer in the outer integument of the young ovules with a protective role. They also all exhibited high pollen viability (86.9 ± 10.2%), high frequency of normally developed, enlarged (fertilized) ovules in ovary (65.0 ± 24.0%), but also high frequency of degenerations in developing ovules (40.4 ± 9.8%). Several flower characters may be adaptive in the unfavorable, high altitude environment, including serpentine soils. High pollen viability and normally developed fertilized ovules are sufficient for Albanian species maintenance.

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.