Complex sex determination in the stinging nettle Urtica dioica

The dioecious species Urtica dioica harbours wide variation in sex ratio of seeds. We conducted a series of crosses to analyse the genetic basis of sex determination in this species. Dutch populations of U. dioica contain low proportions of monoecious individuals beside male and female plants. Self-pollination of monoecious plants always yielded female, male and monoecious plants, generally in a ratio of one female to three male/monoecious individuals. This motivated us to write down a simple model in which gender is determined by one major sex-determination locus with four alleles. In the model males and monoecious plants have distinct genotypes but are both heterozygous at the sex-determination locus. We first made crosses among progeny obtained after self-pollination of monoecious plants. These crosses showed that the monoecious trait generally showed Mendelian inheritance and was passed on to the next generation via both pollen and seeds. Further crosses between monoecious plants and plants from dioecious system indicated that alleles from the dioecious system are often dominant. However, many exceptions to our genetic model are observed which suggest that dominance is incomplete and/or that more genes are involved in sex determination. We discuss to what extent sex determination genes explain the strongly biased seed sex ratios and argue that additional genes, for instance genes for female choice, must also be involved.     

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.     

Carbohydrate binding properties of the stinging nettle (Urtica dioica) rhizome lectin

The interaction of the stinging nettle rhizome lectin (UDA) with carbohydrates was studied by using the techniques of quantitative precipitation, hapten inhibition, equilibrium dialysis, and uv difference spectroscopy. The Carbohydrate binding site of UDA was determined to be complementary to an N,N',N"-triacetylchitotriose unit and proposed to consist of three subsites, each of which has a slightly different binding specificity. UDA also has a hydrophobic interacting region adjacent to the carbohydrate binding site. Equilibrium dialysis and uv difference spectroscopy revealed that UDA has two carbohydrate binding sites per molecule consisting of a single polypeptide chain. These binding sites either have intrinsically different affinities for ligand molecules, or they may display negative cooperativity toward ligand binding.     

The role of the perennial stinging nettle, Urtica dioica, as a reservoir of beneficial natural enemies

A wide range of natural enemies, including predators, parasites and entomophagous fungi were observed to feed on the stinging nettle aphid, Microlophium carnosum, populations of which increased rapidly in late April and early May. Patches of stinging nettles thus served as an important alternate feeding site for some beneficial natural enemies before pest aphids appeared on cultivated plants. Anthocoridae, Miridae and Coccinellidae were the most abundant specific predators sampled on nettles; only the Coccinellidae appeared to disperse over a defined period to other habitats. The hymenopterous parasites Aphidius ervi and Ephedrus lacertosus parasitized up to 10% of M. carnosum populations in June: two species of the fungus Entomophthora occurred spasmodically. Cutting patches of nettles in May or June had the most striking effect on the species and numbers of Coccinellidae. Cutting in mid-June might increase the numbers and impact of natural enemies on nearby pest infestations.     

Purification and characterization of polyphenol oxidase from nettle (Urtica dioica L.) and inhibitory effects of some chemicals on enzyme activity

Polyphenol oxidase (PPO) of nettle (Urtica dioica L.) was extracted and purified through (NH₄)₂SO₄ precipitation, dialysis, and CM-Sephadex ion-exchange chromatography and was used for its characterization. The PPO showed activity to catechol, 4-methylcatechol, L-3,4-dihydroxyphenylalanine (L-DOPA), L-tyrosine, p-cresol, pyrogallol, catechin and trans-cinnamic acid. For each of these eight substrates, optimum conditions such as pH and temperature were determined and L-tyrosine was found to be one of the most suitable substrates. Optimum pH and temperature were found at pH 4.5 and 30°C respectively and K_m and V_max values were 7.90?×?10^?4?M, and 11290?EU/mL for with L-tyrosine as substrate. The inhibitory effect of several inhibitors, L-cysteine chloride, sodium azide, sodium cyanide, benzoic acid, salicylic acid, L-ascorbic acid, glutathione, thiourea, sodium diethyl dithiocarbamate, β-mercaptoethanol and sodium metabisulfite were tested. The most effective was found to be sodium diethyl dithiocarbamate which acted as a competitive inhibitor with a K_i value of 1.79?×?10^?9?M. In addition one isoenzyme of PPO was detected by native polacrylamide slab gel electrophoresis.     

Purification and characterization of polyphenol oxidase from nettle (Urtica dioica L.) and inhibitory effects of some chemicals on enzyme activity

Polyphenol oxidase (PPO) of nettle (Urtica dioica L.) was extracted and purified through (NH4)2SO4 precipitation, dialysis, and CM-Sephadex ion-exchange chromatography and was used for its characterization. The PPO showed activity to catechol, 4-methylcatechol, L-3,4-dihydroxyphenylalanine (L-DOPA), L-tyrosine, p-cresol, pyrogallol, catechin and trans-cinnamic acid. For each of these eight substrates, optimum conditions such as pH and temperature were determined and L-tyrosine was found to be one of the most suitable substrates. Optimum pH and temperature were found at pH 4.5 and 30 degrees C respectively and Km and Vmax values were 7.90 x 10(-4) M, and 11290 EU/mL for with L-tyrosine as substrate. The inhibitory effect of several inhibitors, L-cysteine chloride, sodium azide, sodium cyanide, benzoic acid, salicylic acid, L-ascorbic acid, glutathione, thiourea, sodium diethyl dithiocarbamate, beta-mercaptoethanol and sodium metabisulfite were tested. The most effective was found to be sodium diethyl dithiocarbamate which acted as a competitive inhibitor with a Ki value of 1.79 x 10(-9)M. In addition one isoenzyme of PPO was detected by native polacrylamide slab gel electrophoresis.     

Choline acetyltransferase

No multiple forms of choline acetyltransferase were found in extracts of human, mouse, rabbit, guinea pig, cat, and rat brain. A single form of this enzyme only was also demonstrated in bovine nervous tissue, including brain, dorsal and ventral roots, spinal cord, and femoral nerve. The difference from other published findings is believed to be due to ammonium sulfate fractionation, which was not used in the present study. In addition, multiple forms of the enzyme were obtained by others using isoelectric focusing, whereas this study employed gel filtration. Choline acetyltransferase was highly purified from mouse brain using a procedure similar to that used for the enzyme from bovine brain. The steps involved: (1) making an acetone-chloroform powder from whole mouse brains, (2) extracting the powder and chromatographing the soluble fraction with organomercurial Sepharose, (3) passing the enzyme solution through a column of DEAE-cellulose, (4) eluting from hydroxyapatite, and (5) removing contaminants by subunit exchange chromatography. The final preparation was essentially homogeneous as revealed by polyacrylamide gel electrophoresis.     

The primary structure of stinging nettle (Urtica dioica) agglutinin. A two-domain member of the hevein family.

The primary structure of stinging nettle (Urtica dioica) agglutinin has been determined by sequence analysis of peptides obtained from three overlapping proteolytic digests. The sequence of 80 residues consists of two hevein-like domains with the same spacing of half-cystine residues and several other conserved residues as observed earlier in other proteins with hevein-like domains. The hinge region between the two domains is four residues longer than those between the four domains in cereal lectins like wheat germ agglutinin.     

Biochemical and hemato-immunological parameters in juvenile beluga (Huso huso) following the diet supplemented with nettle (Urtica dioica)

The present study investigated the effects of different dietary nettle (Urtica dioica) levels on biochemical, hematological and immunological parameters in beluga (Huso huso). Fish were divided into 4 groups before being fed for 8 weeks with 0%, 3%, 6% and 12% of nettle. The blood samples were collected on week 4 and 8. The use of nettle did not significantly change the mean cell volume, mean cell haemoglobin, lymphocytes, eosinophils, albumin, glucose, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and lysozyme activity on week 4 and 8. After 4 weeks, the total red blood cell (RBC) and hematocrit (Ht) showed a significant increase in 12% nettle group compared to the 3% nettle and control groups but haemoglobin (Hb) had a significant change in 12% nettle compared to the control. At the same time was not found a significant change in the mean cell haemoglobin concentration (MCHC), total white blood cell (WBC), neutrophils, respiratory burst activity (RB), total immunoglobulin (Ig) and total protein (TP), triglyceride (Tri) and cholesterol (Chol). After 8 weeks, the fish treated with nettle exhibited significantly increase in neutrophil and Hb levels compared to the control and between treatment groups, 12% nettle group shown the highest Hb while RBC and Hct values significantly rose in fish fed by 12% compared to the control. Supplementing 6% and 12% nettle increased the WBC and MCHC compared to the other groups. The group fed 12% showed a highly significant difference in RB, TP and Ig after 8 weeks. However, Tri and Chol were significantly decreased in the juvenile beluga fed by the 6% and 12% nettle diet compared to the other groups. The results suggest that by using this herb there will be an improvement in hemato-biochemical parameters and immune function of juvenile beluga.     

Growth responses of Urtica dioica L. to different water table depth

The study has examined the effect of water table depth (WTD) on production, biomass allocation, allometric relationships and transpiration rate in Urtica dioica. The essential importance of WTD for occurence and spread of Urtica has been documented. Water table depths were: 60, 50, 40, 30, 20, and 10 cm below the soil surface. Thirty individuals of Urtica for each WTD, established from apical parts of young rhizomes, has been planted in containers and placed at WTD from the 8th May to the 24th July. The height of all individuals and the length and width of the largest leaf blade of each individual were measured after 42, 55 and 83 days. Transpiration rate was measured after 53 and 82 days. Above-ground biomass of all individuals was harvested on 24 of July and was separated into individual organs. Various biometric parameters were measured. Results showed that biomass, plant height, branching of stems and rhizomes and rhizomes length decreased in containers with a more shallow WTD. Particularly, allometric relationships between plant height and other characteristics such as basal diameter, length of longest branch and rhizome were affected by the water level. The biometric parameters were highly dependent on plant height. Transpiration strongly decreased with decreasing WTD. Therefore, the high water level in the soil suppresses growth of particular organs and water regime of Urtica plants/. Generally, long-term high water content in the soils of floodplains, particularly during floods, limits rapidly, particularly vegetative spread of Urtica in the wetland habitats.     

Aphidophagous guilds on nettle （Urtica dioica） strips close to fields of green pea, rape and wheat

The common nettle （Urtica dioica L.） is a perennial and cosmopolitan plant species and is known to be the source of food for a great diversity of insects. To understand the importance of the nettle in agro-ecosystems, a field experiment was carried out in an experimental farm at Gembloux （Belgium） to study the effect of nettle margin strips on aphid and aphidophagous populations in close field crops, namely wheat （Triticum aestivum L.）, green pea （Pisum sativum L.） and rape （Brassicae napus L.）. The aphids and related beneficial populations were weekly assessed, from March to August 2005, by visual observations in two plots per field crop. A higher abundance of aphidophagous beneficials was collected in nettle strips when compared to the field crops. Particularly, the presence of predatory anthocorids, mirids and green lacewings was observed on nettle only. Nevertheless, the most abundant aphid predatory family, the Coccinellidae, was distributed in both environments, in nettle strips and in crop fields. The field margin supported a significantly higher density ofHarmonia axyridis than the field crops. In contrast, the field crops, green pea particularly, supported a higher density of Coccinella septempunctata. The distribution of the aphidophagous species, mainly the ladybirds, was discussed in relation to the host plant and related aphid species and their potential effect on integrated pest management.     

The gene for stinging nettle lectin (Urtica dioica agglutinin) encodes both a lectin and a chitinase.

Chitin-binding proteins are present in a wide range of plant species, including both monocots and dicots, even though these plants contain no chitin. To investigate the relationship between in vitro antifungal and insecticidal activities of chitin-binding proteins and their unknown endogenous functions, the stinging nettle lectin (Urtica dioica agglutinin, UDA) cDNA was cloned using a synthetic gene as the probe. The nettle lectin cDNA clone contained an open reading frame encoding 374 amino acids. Analysis of the deduced amino acid sequence revealed a 21-amino acid putative signal sequence and the 86 amino acids encoding the two chitin-binding domains of nettle lectin. These domains were fused to a 19-amino acid "spacer" domain and a 244-amino acid carboxyl extension with partial identity to a chitinase catalytic domain. The authenticity of the cDNA clone was confirmed by deduced amino acid sequence identity with sequence data obtained from tryptic digests, RNA gel blot, and polymerase chain reaction analyses. RNA gel blot analysis also showed the nettle lectin message was present primarily in rhizomes and inflorescence (with immature seeds) but not in leaves or stems. Chitinase enzymatic activity was found when the chitinase-like domain alone or the chitinase-like domain with the chitin-binding domains were expressed in Escherichia coli. This is the first example of a chitin-binding protein with both a duplication of the 43-amino acid chitin-binding domain and a fusion of the chitin-binding domains to a structurally unrelated domain, the chitinase domain.     

Choline acetyltransferase.

Acetylcholine is essential to neural function. It synthesis is catalyzed by choline acetyltransferase, the enzyme responsible for the acetylation of choline by acetyl coenzye A, a reaction favored slightly thermodymodynamically and not at all kinetically. An analytically pure enzyme still has not been obtained; however, method of purification have been greatly improved recently. Numerous inhibitors of the enzyme have been synthesized and their structure-action relationships examained. Evidence has been accumulated showing the essential involvement of an imidazole group in the active site of choline acetyltransferase. The literature regarding the controversial role to thiol groups in choline acetyltransferase is reviewed. Recently, derivatives of coenzyme A have been introduced as inhibitors of this enzyme and the specificity of coenzyme A binding has been examined. Possible mechanisms responsible for the control fo acetylcholine synthesis are discussed.     

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.     

Cytokinins in the perennial herb Urtica dioica L. as influenced by its nitrogen status

The effect of nitrogen on the cytokinin relations of Urtica dioica, the stinging nettle, has been investigated. The plants were grown in quartz sand and nutrient solutions providing levels of nitrate ranging from 1 to 22 mM. Nitrogen supply did not affect biomass production within the range of 3–15 mM NO ₃ ^- . However, the shoot: root ratio of biomass was significantly higher at 15 mM (standard plants) than at 3 mM (low-nitrogen plants) nitrate supply. The cytokinin patterns of the roots, stems and adult, as well as meristematic leaves of plants grown at these two levels of nitrate supply, were determined by means of high-performance liquid chromatography (HPLC) and immunoassays. Enzyme-linked immunosorbent assays (ELISAs) for zeatin riboside, dihydrozeatin riboside, isopentenyladenosine, benzyladenosine and o-hydroxybenzyladenosine enabled the quantification of 17 cytokinins, 13 of which were found in the various tissues of Urtica. trans-Zeatin and its conjugates were the predominant cytokinins in all examined samples. While the free base trans-zeatin and its O-glucoside were the major cytokinins in adult leaves, trans-zeatin riboside was prominent in the other tissues of at least the standard plants. Glucosides of the trans-zeatin type cytokinins were present only in lower amounts. However, considerable amounts of a compound, tentatively identified as cis-zeatin riboside-O-glucoside, were found, particularly in roots and meristematic leaves. Comparatively high amounts of trans-zeatin nucleotide as well as isopentenyladenosine phosphate were also demonstrated in these tissues. Analysis of the root-pressure exudates similarly showed trans-zeatin riboside and, at a lower concentration, trans-zeatin to be the only substantial components. In the low-nitrogen plants, shortage of nitrogen was manifest only in the roots; the nitrogen contents of the shoots did not respond to the nitrogen supply. Likewise, the total content of cytokinins in the shoots of the low-nitrogen plants equaled that of the standard-plant shoots, while it was lower by about 25% in the roots of the low-nitrogen plants. In the latter, the amounts of cytokinins exuded via the root-pressure fluid were also approximately 25% lower. Since the levels of only the trans-zeatin cytokinins in the roots showed a linear correlation with the shoot-to-root ratios, these cytokinins may play an important role in biomass partitioning in Urtica dioica.Abbreviations DHZ dihydrozeatin - ELISA enzyme-linked immunosorbent assay - -G glucoside - HPLC high-performance liquid chromatography - 2iP isopentenyladenine - 2iPA isopentenyladenosine - -N nucleotide (ribotide) - -OG O-glucoside - -R riboside - S/R shoot-to-root (ratio) - Z zeatin This work was supported by the Deutsche Forschungsgemeinschaft within the scope of the SFB 137. The authors wish to thank Mrs. A. Fischbach for skilful technical assistence and Dr. Paul Ziegler (Lehrstuhl für Pflanzenphysiologie, University of Bayreuth, FRG) for linguistic suggestions.