The impact of mechanical stress on anatomy, morphology, and gene expression in Urtica dioica L.

MAIN CONCLUSION: Mechanical stress induces distinct anatomical, molecular, and morphological changes in Urtica dioica, affecting trichome development, gene expression, and leaf morphology under controlled conditions The experiments were performed on common nettle, a widely known plant characterized by high variability of leaf morphology and responsiveness to mechanical touch. A specially constructed experimental device was used to study the impact of mechanical stress on Urtica dioica plants under strictly controlled parameters of the mechanical stimulus (touching) and environment in the growth chamber. The general anatomical structure of the plants that were touched was similar to that of control plants, but the shape of the internodes' cross section was different. Stress-treated plants showed a distinct four-ribbed structure. However, as the internodes progressed, the shape gradually approached a rectangular form. The epidermis of control plants included stinging, glandular and simple setulose trichomes, but plants that were touched had no stinging trichomes, and setulose trichomes accumulated more callose. Cell wall lignification occurred in the older internodes of the control plants compared to stress-treated ones. Gene analysis revealed upregulation of the expression of the UdTCH1 gene in touched plants compared to control plants. Conversely, the expression of UdERF4 and UdTCH4 was downregulated in stressed plants. These data indicate that the nettle's response to mechanical stress reaches the level of regulatory networks of gene expression. Image analysis revealed reduced leaf area, increased asymmetry and altered contours in touched leaves, especially in advanced growth stages, compared to control plants. Our results indicate that mechanical stress triggers various anatomical, molecular, and morphological changes in nettle; however, further interdisciplinary research is needed to better understand the underlying physiological mechanisms.

Transcriptomic Changes in Internode Explants of Stinging Nettle during Callogenesis.

Callogenesis, the process during which explants derived from differentiated plant tissues are subjected to a trans-differentiation step characterized by the proliferation of a mass of cells, is fundamental to indirect organogenesis and the establishment of cell suspension cultures. Therefore, understanding how callogenesis takes place is helpful to plant tissue culture, as well as to plant biotechnology and bioprocess engineering. The common herbaceous plant stinging nettle (Urtica dioica L.) is a species producing cellulosic fibres (the bast fibres) and a whole array of phytochemicals for pharmacological, nutraceutical and cosmeceutical use. Thus, it is of interest as a potential multi-purpose plant. In this study, callogenesis in internode explants of a nettle fibre clone (clone 13) was studied using RNA-Seq to understand which gene ontologies predominate at different time points. Callogenesis was induced with the plant growth regulators α-napthaleneacetic acid (NAA) and 6-benzyl aminopurine (BAP) after having determined their optimal concentrations. The process was studied over a period of 34 days, a time point at which a well-visible callus mass developed on the explants. The bioinformatic analysis of the transcriptomic dataset revealed specific gene ontologies characterizing each of the four time points investigated (0, 1, 10 and 34 days). The results show that, while the advanced stage of callogenesis is characterized by the iron deficiency response triggered by the high levels of reactive oxygen species accumulated by the proliferating cell mass, the intermediate and early phases are dominated by ontologies related to the immune response and cell wall loosening, respectively.

Polyploid evolution: The ultimate way to grasp the nettle.

Polyploidy is one of the major forces of plant evolution and widespread mixed-ploidy species offer an opportunity to evaluate its significance. We therefore selected the cosmopolitan species Urtica dioica (stinging nettle), examined its cytogeography and pattern of absolute genome size, and assessed correlations with bioclimatic and ecogeographic data (latitude, longitude, elevation). We evaluated variation in ploidy level using an extensive dataset of 7012 samples from 1317 populations covering most of the species' distribution area. The widespread tetraploid cytotype (87%) was strongly prevalent over diploids (13%). A subsequent analysis of absolute genome size proved a uniform Cx-value of core U. dioica (except for U. d. subsp. cypria) whereas other closely related species, namely U. bianorii, U. kioviensis and U. simensis, differed significantly. We detected a positive correlation between relative genome size and longitude and latitude in the complete dataset of European populations and a positive correlation between relative genome size and longitude in a reduced dataset of diploid accessions (the complete dataset of diploids excluding U. d. subsp. kurdistanica). In addition, our data indicate an affinity of most diploids to natural and near-natural habitats and that the tetraploid cytotype and a small part of diploids (population from the Po river basin in northern Italy) tend to inhabit synanthropic sites. To sum up, the pattern of ploidy variation revealed by our study is in many aspects unique to the stinging nettle, being most likely first of all driven by the greater ecological plasticity and invasiveness of the tetraploid cytotype.

Native Phytoremediation Potential of Urtica dioica for Removal of PCBs and Heavy Metals Can Be Improved by Genetic Manipulations Using Constitutive CaMV 35S Promoter.

Although stinging nettle (Urtica dioica) has been shown to reduce HM (heavy metal) content in soil, its wider phytoremediation potential has been neglected. Urtica dioica was cultivated in soils contaminated with HMs or polychlorinated biphenyls (PCBs). After four months, up to 33% of the less chlorinated biphenyls and 8% of HMs (Zn, Pb, Cd) had been removed. Bacteria were isolated from the plant tissue, with the endophytic bacteria Bacillus shackletonii and Streptomyces badius shown to have the most significant effect. These bacteria demonstrated not only benefits for plant growth, but also extreme tolerance to As, Zn and Pb. Despite these results, the native phytoremediation potential of nettles could be improved by biotechnologies. Transient expression was used to investigate the functionality of the most common constitutive promoter, CaMV 35S in Urtica dioica. This showed the expression of the CUP and bphC transgenes. Collectively, our findings suggest that remediation by stinging nettle could have a much wider range of applications than previously thought.

RAPD-PCR and real-time PCR HRM based genetic variation evaluations of Urtica dioica parts, ecotypes and evaluations of morphotypes in Turkey.

Urtica dioica is an ethnobotanically and medicinally important Complementary and Alternative Medicine (CAM) plant worldwide and in Turkey; 90 % of herbal CAM applications depend on it in Turkey. It has a wide range of habitats in nearly all continents. It is found in all three phytogeographical regions in Turkey (Euro-Siberian, Irano-Turanian, Mediterranean) with high adaptivity to heterogeneous geographies such as climate, soil types and altitudes. This fact in relation to the assessment of chemical constituents of the plant and combining with further genetic and morphological variation data can assist and enhance the works for the utility and reliability of CAM applications in effect and activity of this plant species. In this work we have made some preliminary experiments with novel approaches to reveal the ecotypes and genetic variation of mighty ecotypes of Urtica dioica from different phytogeographical regions of Turkey (Euro-Siberian and Mediterranean). The ecotypes have heterogeneity in both its parts (leaf, stem, root) as revealed by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) using random primers and High-resolution Melt (HRM) analysis using Urtica dioica specific primers and universal chloroplast DNA (cpDNA) primers and morphological traits such as phenolic contents and antioxidant capacities of plants' leaf infusions as used in medicinal applications in Turkey. This work will contribute a lot for the development of molecular markers to detect the genetic variation and heterogeneity of Urtica dioica to further relate with expected phenotypes that are most useful and relevant in CAM applications.

Inheritance of progeny sex ratio in Urtica dioica.

Seed samples collected from female Urtica dioica plants in the field showed considerable inter-family variation in the sex ratio (faction of males). To investigate the inheritance pattern of the sex ratio trait, crosses were performed between individual male and female plants from different sex ratio families. Our results suggest, at least for the families studied here, that maternal parents strongly contribute to the variation in the primary sex ratio. Furthermore, progeny sex ratios from reciprocal crosses were significantly different and resembled the sex ratios produced by their maternal parents. We discuss the possible mechanisms underlying maternal control.

Molecular cloning and tissue-specific transcriptional regulation of the first peroxidase family member, Udp1, in stinging nettle (Urtica dioica).

A full-length cDNA clone, designated Udp1, was isolated from Urtica dioica (stinging nettle), using a polymerase chain reaction based strategy. The putative Udp1 protein is characterized by a cleavable N-terminal signal sequence, likely responsible for the rough endoplasmic reticulum entry and a 310 amino acids mature protein, containing all the important residues, which are evolutionary conserved among different members of the plant peroxidase family. A unique structural feature of the Udp1 peroxidase is defined into the short carboxyl-terminal extension, which could be associated with the vacuolar targeting process. Udp1 peroxidase is differentially regulated at the transcriptional level and is specifically expressed in the roots. Interestingly, wounding and ultraviolet radiation stress cause an ectopic induction of the Udp1 gene expression in the aerial parts of the plant. A genomic DNA fragment encoding the Udp1 peroxidase was also cloned and fully sequenced, revealing a structural organization of three exons and two introns. The phylogenetic relationships of the Udp1 protein to the Arabidopsis thaliana peroxidase family members were also examined and, in combination with the homology modelling approach, dictated the presence of distinct structural elements, which could be specifically involved in the determination of substrate recognition and subcellular localization of the Udp1 peroxidase.

Heritable variation in seed sex ratio of the stinging nettle (Urtica dioica).

Male and female flowering plants of the dioecious Urtica dioica occur in approximately equal numbers in our study area on the coastal sand dunes of Meijendel. The seed sex ratio (SSR, fraction of males) collected from female plants in the field varied between 0.05 and 0.76, and differed significantly between maternal parents. After one generation of selection for either high or low SSR, female plants produced seed batches with sex ratios as extreme as 0.08 and 0.73. Natural populations of U. dioica harbour considerable genetic variation in SSR.

Direct and ecological costs of resistance and tolerance in the stinging nettle.

Plant resistance and tolerance to herbivores, parasites, pathogens, and abiotic factors may involve two types of costs. First, resistance and tolerance may be costly in terms of plant fitness. Second, resistance and tolerance to multiple enemies may involve ecological trade-offs. Our study species, the stinging nettle ( Urtica dioicaL.) has significant variation among seed families in resistance and tolerance as well as costs of resistance and tolerance to the holoparasitic plant Cuscuta europaea L. Here we report on variation among seed families (i.e. genetic) in tolerance to nutrient limitation and in resistance to both mammalian herbivores (i.e. number of stinging trichomes) and an invertebrate herbivore (i.e. inverse of the performance of a generalist snail, Arianta arbustorum). Our results indicate direct fitness costs of snail resistance in terms of host reproduction whereas we did not detect fitness costs of mammalian resistance or tolerance to nutrient limitation. We further tested for ecological trade-offs among tolerance or resistance to the parasitic plant, herbivore resistance, and tolerance to nutrient limitation in the stinging nettle. Tolerance of nettles to nutrient limitation and resistance to mammalian herbivores tended to correlate negatively. However, there were no significant correlations among resistance and tolerance to the different natural enemies (i.e. parasitic plants, snails, and mammals). The results of this greenhouse study thus suggest that resistance and tolerance of nettles to diverse enemies are free to evolve independently of each other but not completely without direct costs in terms of plant fitness.

Population structure of a parasitic plant and its perennial host.

Characterization of host and parasite population genetic structure and estimation of gene flow among populations are essential for the understanding of parasite local adaptation and coevolutionary interactions between hosts and parasites. We examined two aspects of population structure in a parasitic plant, the greater dodder (Cuscuta europaea) and its host plant, the stinging nettle (Urtica dioica), using allozyme data from 12 host and eight parasite populations. First, we examined whether hosts exposed to parasitism in the past contain higher levels of genetic variation. Second, we examined whether host and parasite populations differ in terms of population structure and if their population structures are correlated. There was no evidence that host populations differed in terms of gene diversity or heterozygosity according to their history of parasitism. Host populations were genetically more differentiated (F(ST) = 0.032) than parasite populations (F(ST) = 0.009). Based on these F(ST) values, gene flow was high for both host and parasite. Such high levels of gene flow could counteract selection for local adaptation of the parasite. We found no significant correlation between geographic and genetic distance (estimated as pairwise F(ST)), either for the host or for the parasite. Furthermore, host and parasite genetic distance matrices were uncorrelated, suggesting that sites with genetically similar host populations are unlikely to have genetically similar parasite populations.

Resistance and tolerance in a host plant-holoparasitic plant interaction: genetic variation and costs.

Host organisms are believed to evolve defense mechanisms (i.e., resistance and/or tolerance) under selective pressures exerted by natural enemies. A prerequisite for the evolution of resistance and tolerance is the existence of genetic variation in these traits for natural selection to act. However, selection for resistance and/or tolerance may be constrained by negative genetic correlations with other traits that affect host fitness. We studied genetic variation in resistance and tolerance against parasitic infection and the potential fitness costs associated with these traits using a novel study system, namely the interaction between a flowering plant and a parasitic plant. In this system, parasitic infection has significant negative effects on host growth and reproduction and may thus act as a selective agent. We conducted a greenhouse experiment in which we grew host plants, Urtica dioica, that originated from a single natural population and represented 20 maternal families either uninfected or infected with the holoparasitic dodder, Cuscuta europaea. that originated from the same site. We calculated correlations among resistance, tolerance, and host performance to test for costs of resistance and tolerance. We measured resistance as parasite performance (quantitative resistance) and tolerance as the slopes of regressions relating the vegetative and reproductive biomass of host plants to damage level (measured as parasite biomass). We observed significant differences among host families in parasite resistance and in parasite tolerance in terms of reproductive biomass, a result that suggests genetic variation in these traits. Furthermore, we found differences in resistance and tolerance between female and male host plants. In addition, the correlations indicate costs of resistance in terms of host growth and reproduction and costs of tolerance in terms of host reproduction. Our results thus indicate that host tolerance and resistance can evolve as a response to infection by a parasitic plant and that costs of resistance and tolerance may be one factor maintaining genetic variation in these traits.