Range-constrained co-occurrence simulation reveals little niche partitioning among rock-dwelling Montenegrina land snails (Gastropoda: Clausiliidae).

AIM: Taxon co-occurrence analysis is commonly used in ecology, but it has not been applied to range-wide distribution data of partly allopatric taxa because existing methods cannot differentiate between distribution-related effects and taxon interactions. Our first aim was to develop a taxon co-occurrence analysis method that is also capable of taking into account the effect of species ranges and can handle faunistic records from museum databases or biodiversity inventories. Our second aim was to test the independence of taxon co-occurrences of rock-dwelling gastropods at different taxonomic levels, with a special focus on the Clausiliidae subfamily Alopiinae, and in particular the genus Montenegrina. LOCATION: Balkan Peninsula in south-eastern Europe (46N-36N, 13.5E-28E). METHODS: We introduced a taxon-specific metric that characterizes the occurrence probability at a given location. This probability was calculated as a distance-weighted mean of the taxon's presence and absence records at all sites. We applied corrections to account for the biases introduced by varying sampling intensity in our dataset. Then we used probabilistic null-models to simulate taxon distributions under the null hypothesis of no taxon interactions and calculated pairwise and cumulated co-occurrences. Independence of taxon occurrences was tested by comparing observed co-occurrences to simulated values. RESULTS: We observed significantly fewer co-occurrences among species and intra-generic lineages of Montenegrina than expected under the assumption of no taxon interaction. MAIN CONCLUSIONS: Fewer than expected co-occurrences among species and intrageneric clades indicate that species divergence preceded niche partitioning. This suggests a primary role of non-adaptive processes in the speciation of rock-dwelling gastropods. The method can account for the effects of distributional constraints in range-wide datasets, making it suitable for testing ecological, biogeographical, or evolutionary hypotheses where interactions of partly allopatric taxa are in question.

Taxonomic revision of the rock-dwelling door snail genus Montenegrina Boettger, 1877 (Mollusca, Gastropoda, Clausiliidae).

The genus Montenegrina is revised on the basis of material available at the Hungarian Natural History Museum (Budapest), Naturhistorisches Museum Wien (Vienna), and the Naturmuseum Senckenberg (Frankfurt am Main), as well as newly discovered populations. The following new taxa are described: Montenegrina haringae sp. n., Montenegrina lillae sp. n., Montenegrina prokletiana sp. n., Montenegrina sturanyana sp. n., Montenegrina grammica erosszoltani ssp. n., Montenegrina grammica improvisa ssp. n., Montenegrina hiltrudae desaretica ssp. n., Montenegrina hiltrudae selcensis ssp. n., Montenegrina laxa delii ssp. n., Montenegrina nana barinai ssp. n., Montenegrina prokletiana kovacsorum ssp. n., Montenegrina rugilabris golikutensis ssp. n., Montenegrina rugilabris gregoi ssp. n., Montenegrina skipetarica danyii ssp. n., Montenegrina skipetarica gurelurensis ssp. n., Montenegrina skipetarica pifkoi ssp. n., Montenegrina skipetarica puskasi ssp. n., Montenegrina sporadica tropojana ssp. n., Montenegrina sturanyana gropana ssp. n., Montenegrina sturanyana ostrovicensis ssp. n., and Montenegrina tomorosi hunyadii ssp. n. A neotype is designated for Montenegrina helvola (Küster, 1860), and Montenegrina cattaroensis antivaricostata nom. n. was introduced to replace the junior homonym Clausilia umbilicata costata Boettger, 1907 (non Pfeiffer, 1928). Of each taxon types or specimens from the type localities are figured, and distribution maps are provided.

Identification and functional characterisation of a novel KCNJ2 mutation, Val302del, causing Andersen-Tawil syndrome.

Loss-of-function mutations of the KCNJ2 gene encoding for the inward rectifier potassium channel subunit Kir2.1 cause Andersen-Tawil Syndrome (ATS), a rare genetic disorder characterised by periodic paralysis, ventricular arrhythmias, and dysmorphic features. Clinical manifestations of the disease appear to vary greatly with the nature of mutation, therefore, functional characterisation of ATS-causing mutations is of clinical importance. In this study, we describe the identification and functional analysis of a novel KCNJ2 mutation, Val302del, identified in a patient with ATS. Heterologously expressed wild type (WT) and Val302del mutant alleles showed similar subcellular distribution of the Kir2.1 protein with high intensity labelling from the membrane region, demonstrating normal membrane trafficking of the Val302del Kir2.1 variant. Cells transfected with the WT allele displayed a robust current with strong inward rectification, while no current above background was detected in cells expressing the Val302del Kir2.1 subunit. Co-transfection of CHO cells with the WT and the Val302del Kir2.1 revealed a dose-dependent inhibitory effect of the Val302del Kir2.1 mutant subunit on WT Kir2.1 currents. These observations indicate that the WT and the Val302del mutant subunits co-assemble in the cell membrane and that the mutation affects potassium conductivity and (or) gating of the WT/Val302del heteromeric Kir2.1 channels.

Differential expression of the brassinosteroid receptor-encoding BRI1 gene in Arabidopsis.

Brassinosteroid (BR)-regulated growth and development in Arabidopsis depends on BRASSINOSTEROID INSENSITIVE 1 (BRI1), the BR receptor that is responsible for initiating the events of BR signalling. We analysed the temporal and spatial regulation of BRI1 expression using stable transgenic lines that carried BRI1 promoter:reporter fusions. In both seedlings and mature plants the tissues undergoing elongation or differentiation showed elevated BRI1 gene activity, and it could be demonstrated that in the hypocotyl this was accompanied by accumulation of the BRI1 transcript and its receptor protein product. In seedlings the BRI1 promoter was also found to be under diurnal regulation, determined primarily by light repression and a superimposed circadian control. To determine the functional importance of transcriptional regulation we complemented the severely BR insensitive bri1-101 mutant with a BRI1-luciferase fusion construct that was driven by promoters with contrasting specificities. Whereas the BRI1 promoter-driven transgene fully restored the wild phenotype, expression from the photosynthesis-associated CAB3 and the vasculature-specific SUC2 and ATHB8 promoters resulted in plants with varying morphogenic defects. Our results reveal complex differential regulation of BRI1 expression, and suggest that by influencing the distribution and abundance of the receptor this regulation can enhance or attenuate BR signalling.

CYP90A1/CPD, a brassinosteroid biosynthetic cytochrome P450 of Arabidopsis, catalyzes C-3 oxidation.

Brassinosteroids (BRs) are steroidal phytohormones that regulate plant growth and development. Whereas in Arabidopsis the network-like routes of BR biosynthesis have been elucidated in considerable detail, the roles of some of the biosynthetic enzymes and their participation in the different subpathways remained to be clarified. We investigated the function of the cytochrome P450 monooxygenase CYP90A1/CPD, which earlier had been proposed to act as a BR C-23 hydroxylase. Our GC-MS and genetic analyses demonstrated that the cpd mutation arrests BR synthesis upstream of the DET2-mediated 5α reduction step and that overexpression of the C-23 hydroxylase CYP90C1 does not alleviate BR deficiency in the cpd mutant. In line with these results, we found that CYP90A1/CPD heterologously expressed in a baculovirus-insect cell system catalyzes C-3 oxidation of the early BR intermediates (22S)-22-hydroxycampesterol and (22R,23R)-22,23-dihydroxycampesterol, as well as of 6-deoxocathasterone and 6-deoxoteasterone. Enzyme kinetic data of CYP90A1/CPD and DET2, together with those of the earlier studied CYP90B1, CYP90C1, and CYP90D1, suggest that BR biosynthesis proceeds mainly via the campestanol-independent pathway.

Cloning the bacterial bphC gene into Nicotiana tabacum to improve the efficiency of phytoremediation of polychlorinated biphenyls.

The aim of this work was to construct transgenic plants with increased capabilities to degrade organic pollutants, such as polychlorinated biphenyls. The environmentally important gene of bacterial dioxygenase, the bphC gene, was chosen to clone into a plant of Nicotiana tabacum. The chosen bphC gene encodes 2,3-dihydroxybiphenyl-1,2-dioxygenase, which cleaves the aromatic ring of dihydroxybiphenyl, and we cloned it in fusion with the gene for ß-glucuronidase (GUS), luciferase (LUC) or with a histidine tail. Several genetic constructs were designed and prepared and the possible expression of desired proteins in tobacco plants was studied by transient expression. We used genetic constructs successfully expressing dioxygenase's genes we used for preparation of transgenic tobacco plants by agrobacterial infection. The presence of transgenic DNA , mRNA and protein was determined in parental and the first filial generation of transgenic plants with the bphC gene. Properties of prepared transgenic plants will be further studied.

C-23 hydroxylation by Arabidopsis CYP90C1 and CYP90D1 reveals a novel shortcut in brassinosteroid biosynthesis.

Brassinosteroids (BRs) are biosynthesized from campesterol via several cytochrome P450 (P450)-catalyzed oxidative reactions. We report the functional characterization of two BR-biosynthetic P450s from Arabidopsis thaliana: CYP90C1/ROTUNDIFOLIA3 and CYP90D1. The cyp90c1 cyp90d1 double mutant exhibits the characteristic BR-deficient dwarf phenotype, although the individual mutants do not display this phenotype. These data suggest redundant roles for these P450s. In vitro biochemical assays using insect cell-expressed proteins revealed that both CYP90C1 and CYP90D1 catalyze C-23 hydroxylation of various 22-hydroxylated BRs with markedly different catalytic efficiencies. Both enzymes preferentially convert 3-epi-6-deoxocathasterone, (22S,24R)-22-hydroxy-5alpha-ergostan-3-one, and (22S,24R)-22-hydroxyergost-4-en-3-one to 23-hydroxylated products, whereas they are less active on 6-deoxocathasterone. Likewise, cyp90c1 cyp90d1 plants were deficient in 23-hydroxylated BRs, and in feeding experiments using exogenously supplied intermediates, only 23-hydroxylated BRs rescued the growth deficiency of the cyp90c1 cyp90d1 mutant. Thus, CYP90C1 and CYP90D1 are redundant BR C-23 hydroxylases. Moreover, their preferential substrates are present in the endogenous Arabidopsis BR pool. Based on these results, we propose C-23 hydroxylation shortcuts that bypass campestanol, 6-deoxocathasterone, and 6-deoxoteasterone and lead directly from (22S,24R)-22-hydroxy-5alpha-ergostan-3-one and 3-epi-6-deoxocathasterone to 3-dehydro-6-deoxoteasterone and 6-deoxotyphasterol.

Diurnal regulation of the brassinosteroid-biosynthetic CPD gene in Arabidopsis.

Plant steroid hormones, brassinosteroids (BRs), are essential for normal photomorphogenesis. However, the mechanism by which light controls physiological functions via BRs is not well understood. Using transgenic plants carrying promoter-luciferase reporter gene fusions, we show that in Arabidopsis (Arabidopsis thaliana) the BR-biosynthetic CPD and CYP85A2 genes are under diurnal regulation. The complex diurnal expression profile of CPD is determined by dual, light-dependent, and circadian control. The severely decreased expression level of CPD in phytochrome-deficient background and the red light-specific induction in wild-type plants suggest that light regulation of CPD is primarily mediated by phytochrome signaling. The diurnal rhythmicity of CPD expression is maintained in brassinosteroid insensitive 1 transgenic seedlings, indicating that its transcriptional control is independent of hormonal feedback regulation. Diurnal changes in the expression of CPD and CYP85A2 are accompanied by changes of the endogenous BR content during the day, leading to brassinolide accumulation at the middle of the light phase. We also show that CPD expression is repressed in extended darkness in a BR feedback-dependent manner. In the dark the level of the bioactive hormone did not increase; therefore, our data strongly suggest that light also influences the sensitivity of plants to BRs.

Can tobacco have a potentially beneficial effect to our health?

With urgent pressure to clean up the contaminated environment, new approaches are needed. Phyto- and rhizoremediation using plants and related bacteria is a promising approach, but has its inborn limitations. To overcome the slow performance of the process, transgenic plants have been prepared specifically tailored for phytoremediation purposes. Our projects addressed a group of widespread synthetic organic xenobiotics, polychlorinated biphenyls (PCBs), and heavy metals as representatives of inorganic contaminants. Beside basic research studies in the field of phyto/rhizoremediation of the mentioned toxicants we focused on genetically modified plants as a highly promising tool for these purposes. We tried to prepare tobacco plants expressing the bacterial enzyme responsible for cleaving PCBs, coded by the gene bphC from the bacterial biphenyl operon. The expression of bphC product in fusion with the green fluorescent protein is described together with evaluation of the twice increased resistance of transgenic seeds towards PCBs. The other model is addressing improvement of cadmium accumulation by preparing plants bearing fused transgenes of metal binding protein (yeast metallothionein) with an introduced additional metal binding domain--polyhistidine anchor with high affinity to metals. The genetically modified plants exhibit 190% Cd accumulation of the control in harvestable parts, higher resistance and lower Cd content in roots. The performance of the plants in real contaminated soil is also evaluated.

Unique and overlapping expression patterns of Arabidopsis CYP85 genes involved in brassinosteroid C-6 oxidation.

Brassinosteroids (BRs) are steroid hormones that are essential for plant growth and development. To gain insight into potential sites of BR synthesis, we studied promoter activities of the two Arabidopsis BR C-6 oxidase genes (CYP85A1 and CYP85A2) in transgenic plants carrying promoter fusions with the GUS, GFP or LUC reporter genes. BR-dependent feedback regulation of the GUS reporter constructs indicated that their expression corresponded to those of the native genes. Both the CYP85A1 and CYP85A2 promoters showed maximum activity during the first week following germination, particularly in the vascular tissues. Compared to CYP85A2, CYP85A1 expression was weaker and confined to the early stages of seedling development. Stronger CYP85A2 promoter activity was evident in both juvenile and adult plants. Comparison of the 5'-UTR and TATA box sequences of CYP85A1 and CYP85A2 revealed high homology, indicating a relatively recent gene duplication. We also found that transgenic Arabidopsis plants harbouring the tomato DWARF promoter-GUS fusion had similarities in the expression pattern to the Arabidopsis genes suggesting common transcriptional regulation of CYP85 genes in the two species.

Patterns of Dwarf expression and brassinosteroid accumulation in tomato reveal the importance of brassinosteroid synthesis during fruit development.

Brassinosteroids (BRs) are essential for many physiological functions in plants, however little is known concerning where and when they are synthesized. This is especially true during flower and fruit production. To address this we have used a promoter-GUS reporter fusion and RT-PCR to determine the relative expression levels of the tomato Dwarf (D) gene that encodes a BR C-6 oxidase. In young seedlings GUS reporter activity was observed mainly in apical and root tissues undergoing expansion. In flowers GUS activity was observed in the pedicel joints and ovaries, whereas in fruits it was strongest during early seed development and was associated with the locular jelly and seeds. RT-PCR analysis showed that tissue-specific expression of Dwarf mRNA was consistent with that of the Dwarf:GUS fusion. In good correlation with the high local Dwarf activity, quantitative measurements of endogenous BRs indicated intense biosynthesis in developing tomato fruits, which were also found to contain high amounts of brassinolide. Grafting experiments showed the lack of BR transport indicating that BR action occurs at the site of synthesis.

Brassinosteroid and systemin: two hormones perceived by the same receptor.

Brassinosteroids, coordinating developmental events, and systemin, inducing systemic wound responses to attacks by insect pests, are newly recognized plant hormones that are perceived by plasma membrane-localized leucine-rich repeat receptor kinases. The recent characterization of the brassinosteroid receptor BRI1 from tomato revealed that this protein is identical to the previously isolated SR160 systemin receptor, strongly suggesting that both brassinosteroid and systemin signalling use the same surface receptor.

Regulation of transcript levels of the Arabidopsis cytochrome p450 genes involved in brassinosteroid biosynthesis.

Cytochrome P450 enzymes of the closely related CYP90 and CYP85 families catalyze essential oxidative reactions in the biosynthesis of brassinosteroid (BR) hormones. Arabidopsis CYP90B1/DWF4 and CYP90A1/CPD are responsible for respective C-22 and C-23 hydroxylation of the steroid side chain and CYP85A1 catalyzes C-6 oxidation of 6-deoxo intermediates, whereas the functions of CYP90C1/ROT3, CYP90D1, and CYP85A2 are still unknown. Semiquantitative reverse transcriptase-polymerase chain reaction analyses show that transcript levels of CYP85 and CYP90 genes are down-regulated by brassinolide, the end product of the BR biosynthesis pathway. Feedback control of the CYP90C1, CYP90D1, and CYP85A2 genes by brassinolide suggests that the corresponding enzymes might also participate in BR synthesis. CYP85 and CYP90 mRNAs show strong and transient accumulation during the 1st week of seedling development, as well as characteristic organ-specific distribution. Transcripts of CYP90A1 and CYP85A2 are preferentially represented in shoots and CYP90C1, CYP90D1, and CYP85A1 mRNAs are more abundant in roots, whereas CYP90B1 is ubiquitously expressed. Remarkably, the spatial pattern of CYP90A1 expression is maintained in the BR-insensitive cbb2 mutant, indicating the independence of organ-specific and BR-dependent regulation. Quantitative gas chromatography-mass spectrometry analysis of endogenous BRs in shoots and roots of Arabidopsis, pea (Pisum sativum), and tomato (Lycopersicon esculentum) reveal similar partitioning patterns of BR intermediates in these species. Inverse correlation between CYP90A1/CPD transcript levels and the amounts of the CYP90A1 substrate 6-deoxocathasterone in shoots and roots suggests that transcriptional regulation plays an important role in controlling BR biosynthesis.

Characterisation of BRH1, a brassinosteroid-responsive RING-H2 gene from Arabidopsis thaliana.

Although many important aspects of plant development are controlled by brassinosteroids (BRs), the early molecular events of their hormonal action are largely unknown. Using a differential-display RT-PCR screen designed to detect early response transcripts, those regulated by BR treatment in the absence of de novo protein synthesis, we identified an Arabidopsis thaliana (L.) Heynh. gene (designated BRH1) that encodes a novel RING finger protein. As deduced from a complete cDNA clone, the 170-amino-acid sequence of BRH1 forms an N-terminal hydrophobic domain and a C-terminal RING-H2 signature. In wild-type Arabidopsis, the level of the BRH1 transcript was rapidly down-regulated by brassinolide, but this effect was abolished in a BR-insensitive mutant deficient in the BRI1 receptor. BRH1 mRNA abundance was not influenced by other phytohormones, but the pathogen elicitor chitin induced a rapid and transient accumulation of the transcript. Antisense expression of BRH1 resulted in transgenic Arabidopsis plants with thicker inflorescence stems and altered leaf morphology, whereas in sense overexpression lines no phenotypic effect could be observed. Considering the potential of the RING proteins to participate in regulatory protein complexes, BR-dependent expression of BRH1 may suggest its involvement in later hormonal effects.