Efficacy of Silene arenosa extract on acetylcholinesterase in Bungarus sindanus (krait) venom.

OBJECTIVE: To examine the efficacy of Silene arenosa extract on acetylcholinesterase (AChE) of krait (Bungarus Sindanus) snake venom. METHODS: The present project designed to evaluate the inhibition of AChE by following standard procedures. RESULTS: Statistical analysis of the results showed that Silene arenosa exerted 73% inhibition against the krait venom acetylcholinesterase at fixed substrate acetylcholine (ACh) concentration (0.5 mM). Kinetic analysis using the Lineweaver Burk plot revealed that Silene arenosa caused a competitive type of inhibition i.e. Km values increased from 26.6 to 93.3 mM (26.6% to 93.3%) and Vmax remained constant in a concentration-dependent manner. Silene arenosa competes with the substrate to bind at the active site of the enzyme. The Kmapp of venom AChE for Silene arenosa increased from 60% to 81.6% and the Vmaxapp remains constant. Ki (inhibition constant was estimated to be 48 µg for snake venom; while the Km (Michaelis-Menten constant of AChE- substrate into AChE and product) was estimated to be 0.5 mM. The IC50 of AchE calculated for Silene arenosa was 67 µg. CONCLUSION: The present results suggest that Silene arenosa extract can be considered as an inhibitor of snake venom AChE.

Use of seed priming to improve Cd accumulation and tolerance in Silene sendtneri, novel Cd hyper-accumulator.

Changes in the environment as a result of industrialisation and urbanisation impact negatively on plant growth and crop production. Cadmium (Cd) is one of the most dangerous metals that enters the food chain, with toxic effects on plants and human health. This study evaluated the potential of Silene sendtneri as a novel hyperaccumulator and the role of seed priming in tolerance and accumulation rate of Cd. The effect of different priming agents on germination performance, root growth, seedling development, metal uptake and accumulation, antioxidant defences including enzymatic and non-enzymatic antioxidants has been assessed. Seed priming using silicic acid, proline alone or in combination with salicylic acid- enhanced germination, seedling development, and root growth under Cd stress. The same priming treatments induced an increase of water content in shoots and roots when plants were exposed to Cd. The enzymatic antioxidant response was specific for the priming agent used. An increase in ferulic acid and rutin in shoots was related to the increase of Cd concentration in the medium. The concentration of malic and oxalic acid increased significantly in shoots of plants grown on high Cd concentrations compared to low Cd concentrations. Silene sendtneri can accumulate significant levels of Cd with enhanced accumulation rate and tolerance when seeds are primed. The best results are obtained by seed priming using 1% silicic acid, proline and salicylic acid.

Effects of lead, cadmium and zinc on protein changes in Silene vulgaris shoots cultured in vitro.

In the present research, Silene vulgaris as a representative species growing on both unpolluted and heavy metal (HM) polluted terrains were used to identify ecotype-specific responses to metallic stress. Growth, cell ultrastructure and element accumulations were compared between non-metallicolous (NM), calamine (CAL) and serpentine (SER) specimens untreated with HMs and treated with Pb, Cd and Zn ions under in vitro conditions. Moreover, proteins' modifications related to their level, carbonylation and degradations via vacuolar proteases were verified and linked with potential mechanisms to cope with ions toxicity. Our experiment revealed diversified strategy of HM uptake in NM and both metallicolous ecotypes, in which antagonistic relationship of Zn and Pb/Cd ions provided survival benefits for the whole organism. Despite this similarity, growth rate and metabolic pathways induced in CAL and SER shoots varied significantly. Exposition to HMs in CAL culture led to drop in protein level by approximately 16% compared to the control. This parameter nearly correlated with the enhanced activity of proteases at pH 5.2 as well as possible glutamate changes to proline and reduced glutathione, resulting in intensified growth and first signs of cell senescence. In turn, SER shoots were characterized by growth retardation (to 53% of the control), although protein level and carbonylation were not modified, while a deeper insight into protein network showed its remodeling towards production of polyamines and 2-oxoglutarate delivered to the Krebs cycle. Contrary, an uncontrolled HM influx in NM shoots contributed to morpho-structural disorders accompanied by an increase activity of proteases involved in the degradation of oxidized proteins, what pointed to metal-induced autophagy. Taken together, S. vulgaris ecotypes respond to stress by triggering various mechanisms engaged their survival and/or death under HM treatment.

Evidence from stable-isotope labeling that catechol is an intermediate in salicylic acid catabolism in the flowers of Silene latifolia (white campion).

MAIN CONCLUSION: A stable isotope-assisted mass spectrometry-based platform was utilized to demonstrate that the plant hormone, salicylic acid, is catabolized to catechol, a widespread secondary plant compound. The phytohormone salicylic acid (SA) plays a central role in the overall plant defense program, as well as various other aspects of plant growth and development. Although the biosynthetic steps toward SA are well documented, how SA is catabolized in plants remains poorly understood. Accordingly, in this study a series of stable isotope feeding experiments were performed with Silene latifolia (white campion) to explore possible routes of SA breakdown. S. latifolia flowers that were fed a solution of [2H6]-salicylic acid emitted the volatile and potent pollinator attractant, 1,2-dimethoxybenzene (veratrole), which contained the benzene ring-bound deuterium atoms. Extracts from these S. latifolia flowers revealed labeled catechol as a possible intermediate. After feeding flowers with [2H6]-catechol, the stable isotope was recovered in veratrole as well as its precursor, guaiacol. Addition of a trapping pool of guaiacol in combination with [2H6]-salicylic acid resulted in the accumulation of the label into catechol. Finally, we provide evidence for catechol O-methyltransferase enzyme activity in a population of S. latifolia that synthesizes veratrole from guaiacol. This activity was absent in non-veratrole emitting flowers. Taken together, these results imply the conversion of salicylic acid to veratrole in the following reaction sequence: salicylic acid > catechol > guaiacol > veratrole. This catabolic pathway for SA may also be embedded in other lineages of the plant kingdom, particularly those species which are known to accumulate catechol.

Distinct co-tolerance responses to combined salinity and cadmium exposure in metallicolous and non-metallicolous ecotypes of Silene vulgaris.

This study compared co-tolerance to salinity and cadmium and investigated its mechanisms in a facultative metallophyte Silene vulgaris originating from distinct habitats. Shoots of calamine (Cal) and non-metallicolous (N-Cal) ecotypes grown in vitro were exposed to 10 and 100 mM NaCl, 5 µM CdCl2 and their combinations. Stress effects were evaluated based on growth, oxidative stress parameters, and DNA content and damage. Tolerance mechanisms were assessed by analyzing non-enzymatic antioxidants, osmolytes and ion accumulation. Irrespective of the ecotype, Cd stimulated shoot proliferation (micropropagation coefficients MC = 15.2 and 12.1 for Cal and N-Cal, respectively, growth tolerance index GTI = 148.1 and 156.7%). In Cal ecotype this was attributed to an increase in glutathione content and reorganization of cell membrane structures under Cd exposure, whereas in N-Cal to enhanced synthesis of other non-enzymatic antioxidants, mainly carotenoids and ascorbate. Low salinity stimulated growth of Cal ecotype due to optimizing Cl- content. High salinity inhibited growth, especially in Cal ecotype, where it enhanced DNA damage and disturbed ionic homeostasis. Species-specific reaction to combined salinity and Cd involved a mutual inhibition of Na+, Cl- and Cd2+ uptake. N-Cal ecotype responded to combined stresses by enhancing its antioxidant defense, presumably induced by Cd, whereas the metallicolous ecotype triggered osmotic adjustment. The study revealed that in S. vulgaris Cd application ameliorated metabolic responses to simultaneous salinity exposure. It also shed a light on distinct strategies of coping with combined abiotic stresses in two ecotypes of the species showing high plasticity in environmental conditions.

Variation in plastid genomes in the gynodioecious species Silene vulgaris.

BACKGROUND: Gynodioecious species exist in two sexes - male-sterile females and hermaphrodites. Male sterility in higher plants often results from mitonuclear interaction between the CMS (cytoplasmic male sterility) gene(s) encoded by mitochondrial genome and by nuclear-encoded restorer genes. Mitochondrial and nuclear-encoded transcriptomes in females and hermaphrodites are intensively studied, but little is known about sex-specific gene expression in plastids. We have compared plastid transcriptomes between females and hermaphrodites in two haplotypes of a gynodioecious species Silene vulgaris with known CMS candidate genes. RESULTS: We generated complete plastid genome sequences from five haplotypes S. vulgaris including the haplotypes KRA and KOV, for which complete mitochondrial genome sequences were already published. We constructed a phylogenetic tree based on plastid sequences of S. vulgaris. Whereas lowland S. vulgaris haplotypes including KRA and KOV clustered together, the accessions from high European mountains diverged early in the phylogram. S. vulgaris belongs among Silene species with slowly evolving plastid genomes, but we still detected 212 substitutions and 112 indels between two accessions of this species. We estimated elevated Ka/Ks in the ndhF gene, which may reflect the adaptation of S. vulgaris to high altitudes, or relaxed selection. We compared depth of coverage and editing rates between female and hermaphrodite plastid transcriptomes and found no significant differences between the two sexes. We identified 51 unique C to U editing sites in the plastid genomes of S. vulgaris, 38 of them in protein coding regions, 2 in introns, and 11 in intergenic regions. The editing site in the psbZ gene was edited only in one of two plastid genomes under study. CONCLUSIONS: We revealed no significant differences between the sexes in plastid transcriptomes of two haplotypes of S. vulgaris. It suggests that gene expression of plastid genes is not affected by CMS in flower buds of S. vulgaris, although both sexes may still differ in plastid gene expression in specific tissues. We revealed the difference between the plastid transcriptomes of two S. vulgaris haplotypes in editing rate and in the coverage of several antisense transcripts. Our results document the variation in plastid genomes and transcriptomes in S. vulgaris.

Stability of petal color polymorphism: the significance of anthocyanin accumulation in photosynthetic tissues.

BACKGROUND: Anthocyanins are the primary source of colour in flowers and also accumulate in vegetative tissues, where they have multiple protective roles traditionally attributed to early compounds of the metabolic pathway (flavonols, flavones, etc.). Petal-specific loss of anthocyanins in petals allows plants to escape from the negative pleiotropic effects of flavonoid and anthocyanins loss in vegetative organs, where they perform a plethora of essential functions. Herein, we investigate the degree of pleiotropy at the biochemical scale in a pink-white flower colour polymorphism in the shore campion, Silene littorea. We report the frequencies of pink and white individuals across 21 populations and underlying biochemical profiles of three flower colour variants: anthocyanins present in all tissues (pink petals), petal-specific loss of anthocyanins (white petals), and loss of anthocyanins in all tissues (white petals). RESULTS: Individuals lacking anthocyanins only in petals represent a stable polymorphism in two populations at the northern edge of the species range (mean frequency 8-21%). Whereas, individuals lacking anthocyanins in the whole plant were found across the species range, yet always at very low frequencies (< 1%). Biochemically, the flavonoids detected were anthocyanins and flavones; in pigmented individuals, concentrations of flavones were 14-56× higher than anthocyanins across tissues with differences of > 100× detected in leaves. Loss of anthocyanin pigmentation, either in petals or in the whole plant, does not influence the ability of these phenotypes to synthesize flavones, and this pattern was congruent among all sampled populations. CONCLUSIONS: We found that all colour variants showed similar flavone profiles, either in petals or in the whole plant, and only the flower colour variant with anthocyanins in photosynthetic tissues persists as a stable flower colour polymorphism. These findings suggest that anthocyanins in photosynthetic tissues, not flavonoid intermediates, are the targets of non-pollinator mediated selection.

Alternative responses to fungal attack on a metalliferous soil: Phytohormone levels and structural changes in Silene paradoxa L. growing under copper stress.

In this work, a non-metallicolous and a metallicolous population of S. paradoxa were exposed to copper excess and fungal elicitation, and investigated for phytohormone production and cytological alterations. Under the stress applied separately and in combination, S. paradoxa plants varied phytohormone concentration in a population-specific way, suggesting a different signalling in response to biotic and abiotic stimuli according to the environment of origin. Generally, the stress responses consisted in increased levels of salicylic acid, auxin, and gibberellin in the non-metallicolous population, and of jasmonic and abscisic acid in the metallicolous one. Interestingly, the metallicolous population increased the level of such phytohormones following exposure to the fungal elicitor only in the presence of copper. This alternative hormonal signalling could derive from the incompatibility between the ordinary ROS-mediated response to pathogens and the acquired mechanisms that prevent oxidative stress in the population from the metal-rich soil. Furthermore, stress-induced autophagic phenomena were more evident in the non-metallicolous plants than in the metallicolous ones, suggesting that the adaptation to the metalliferous environment has also affected autophagy intensity and signalling in response to copper excess and fungal elicitation.

Whole-plant reddening in Silene germana is due to anthocyanin accumulation in response to visible light.

The phenology of anthocyanin accumulation in leaves has been widely studied in perennial plants; several hypotheses have been proposed to explain their adaptive significance. Here, we explored the photoprotection hypothesis in Silene germana, a Mediterranean annual plant with late-spring/summer flowering. We analysed the temporal patterns of anthocyanin accumulation in photosynthetic calyces, leaves and stems and throughout the reproductive season, and their relationship with flower abortion, florivory and plant mortality due to drought. In addition, the flavonoid production and the photoinhibitory response were measured in a shading experiment. The whole plant becomes red at the end of the flowering and remains red until fruiting and senescence. Calyces were redder on the side with more sun exposition. Aborted flowers showed redder calyces than those of fruiting flowers. No effect of plant redness on florivory or plant mortality was found. The shading experiment showed a positive relationship between anthocyanin accumulation and intensity of solar radiation, but plants growing in absence of UV showed similar redness than full sunlight plants. Plants growing in natural shade lack anthocyanins but produced the same amount of non-anthocyanin flavonoids. Anthocyanic and non-anthocyanic plants showed similar photochemical efficiency (Fv /Fm ) after sun exposition, but in early morning, the former showed lower Fv /Fm values. Plants growing in full sunlight produced more fruits than those of natural shade plants. Whole-plant reddening during fruiting and senescence appears to be a property of S. germana. Our results suggest that anthocyanin accumulation depends on sunlight intensity, but non-anthocyanin flavonoids are produced constitutively.

DNA methylation and genetic degeneration of the Y chromosome in the dioecious plant Silene latifolia.

BACKGROUND: S. latifolia is a model organism for the study of sex chromosome evolution in plants. Its sex chromosomes include large regions in which recombination became gradually suppressed. The regions tend to expand over time resulting in the formation of evolutionary strata. Non-recombination and later accumulation of repetitive sequences is a putative cause of the size increase in the Y chromosome. Gene decay and accumulation of repetitive DNA are identified as key evolutionary events. Transposons in the X and Y chromosomes are distributed differently and there is a regulation of transposon insertion by DNA methylation of the target sequences, this points to an important role of DNA methylation during sex chromosome evolution in Silene latifolia. The aim of this study was to elucidate whether the reduced expression of the Y allele in S. latifolia is caused by genetic degeneration or if the cause is methylation triggered by transposons and repetitive sequences. RESULTS: Gene expression analysis in S. latifolia males has shown expression bias in both X and Y alleles. To determine whether these differences are caused by genetic degeneration or methylation spread by transposons and repetitive sequences, we selected several sex-linked genes with varying degrees of degeneration and from different evolutionary strata. Immunoprecipitation of methylated DNA (MeDIP) from promoter, exon and intron regions was used and validated through bisulfite sequencing. We found DNA methylation in males, and only in the promoter of genes of stratum I (older). The Y alleles in genes of stratum I were methylation enriched compared to X alleles. There was also abundant and high percentage methylation in the CHH context in most sequences, indicating de novo methylation through the RdDM pathway. CONCLUSIONS: We speculate that TE accumulation and not gene decay is the cause of DNA methylation in the S. latifolia Y sex chromosome with influence on the process of heterochromatinization.

Phytoavailability of Cr in Silene vulgaris: The role of soil, plant genotype and bacterial rhizobiome.

Understanding the metal behavior at the soil-root interface is of utmost significance for a successful implementation of phytoremediation. In this study, we investigated the differences in chromium (Cr) uptake, chemical changes in soil solution and the shifts in rhizosphere bacterial communities of two genotypes of Silene vulgaris (SV21, SV38) with different tolerance to Cr. A greenhouse experiment was performed in two soils that differed on pH and organic matter (OM) content. An industrial sludge with high content in Cr was used as pollution source. The soil solution in the rhizosphere was sample by Rhizon Soil Moisture Samplers. The total concentration of Cr reached the highest values in soil solution samplers from calcareous soils with poor contents in OM. Plants grown in this soil also increased the Cr uptake in roots of both genotypes, but the concentration was higher in genotype SV-38 than in SV21. The clustering analysis of denaturing gradient gel electrophoresis (DGGE) of 16S rRNA fragments revealed major differences in bacterial community structure related to Cr pollution, followed by soil type and finally, plant genotype. Diversity indices based on DGGE profiles were the highest in alkaline soil, and between genotypes, values were significantly greater in SV38. Canonical correspondence analysis (CCA) showed that changes in bacterial community structure of rhizosphere were highly correlated with total Cr concentration and soil solution pH. The isolation and identification of S. vulgaris bacterial rhizosphere revealed a different composition according to soil type and plant genotype. Results suggested the potential role of Pseudomonas fluorescens on Cr mobilization and therefore, on enhanced metal bioavailability and may provide a starting point for further studies aimed at the combined use of tolerant plants and selected metal mobilizing rhizobacteria, in the microbial-assisted phytoremediation of Cr-polluted soils.

Two Silene vulgaris copper transporters residing in different cellular compartments confer copper hypertolerance by distinct mechanisms when expressed in Arabidopsis thaliana.

Silene vulgaris is a metallophyte of calamine, cupriferous and serpentine soils all over Europe. Its metallicolous populations are hypertolerant to zinc (Zn), cadmium (Cd), copper (Cu) or nickel (Ni), compared with conspecific nonmetallicolous populations. These hypertolerances are metal-specific, but the underlying mechanisms are poorly understood. We investigated the role of HMA5 copper transporters in Cu-hypertolerance of a S. vulgaris copper mine population. Cu-hypertolerance in Silene is correlated and genetically linked with enhanced expression of two HMA5 paralogs, SvHMA5I and SvHMA5II, each of which increases Cu tolerance when expressed in Arabidopsis thaliana. Most Spermatophytes, except Brassicaceae, possess homologs of SvHMA5I and SvHMA5II, which originate from an ancient duplication predating the appearance of spermatophytes. SvHMA5II and the A. thaliana homolog AtHMA5 localize in the endoplasmic reticulum and upon Cu exposure move to the plasma membrane, from where they are internalized and degraded in the vacuole. This resembles trafficking of mammalian homologs and is apparently an extremely ancient mechanism. SvHMA5I, instead, neofunctionalized and always resides on the tonoplast, likely sequestering Cu in the vacuole. Adaption of Silene to a Cu-polluted soil is at least in part due to upregulation of two distinct HMA5 transporters, which contribute to Cu hypertolerance by distinct mechanisms.

Cell Death and Cell Cycle Arrest of Silene latifolia Stamens and Pistils After Microbotryum lychnidis-dioicae Infection.

Mechanisms of suppression of pistil primordia in male flowers and of stamen primordia in female flowers differ in diclinous plants. In this study, we investigated how cell death and cell cycle arrest are related to flower organ formation in Silene latifolia. Using in situ hybridization and a TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, we detected both cell cycle arrest and cell death in suppressed stamens of female flowers and suppressed pistils of male flowers in S. latifolia. In female flowers infected with Microbotryum lychnidis-dioicae, developmental suppression of stamens is released, and cell cycle arrest and cell death do not occur. Smut spores are formed in S. latifolia anthers infected with M. lychnidis-dioicae, followed by cell death in the endothelium, middle layer, tapetal cells and pollen mother cells. Cell death is difficult to detect using a fluorescein isothiocyanate-labeled TUNEL assay due to strong autofluorescence in the anther. We therefore combined a TUNEL assay in an infrared region with transmission electron microscopy to detect cell death in anthers. We show that following infection by M. lychnidis-dioicae, a TUNEL signal was not detected in the endothelium, middle layer or pollen mother cells, and cell death with outflow of cell contents, including the nucleoplast, was observed in tapetal cells.

Positive Selection in Rapidly Evolving Plastid-Nuclear Enzyme Complexes.

Rates of sequence evolution in plastid genomes are generally low, but numerous angiosperm lineages exhibit accelerated evolutionary rates in similar subsets of plastid genes. These genes include clpP1 and accD, which encode components of the caseinolytic protease (CLP) and acetyl-coA carboxylase (ACCase) complexes, respectively. Whether these extreme and repeated accelerations in rates of plastid genome evolution result from adaptive change in proteins (i.e., positive selection) or simply a loss of functional constraint (i.e., relaxed purifying selection) is a source of ongoing controversy. To address this, we have taken advantage of the multiple independent accelerations that have occurred within the genus Silene (Caryophyllaceae) by examining phylogenetic and population genetic variation in the nuclear genes that encode subunits of the CLP and ACCase complexes. We found that, in species with accelerated plastid genome evolution, the nuclear-encoded subunits in the CLP and ACCase complexes are also evolving rapidly, especially those involved in direct physical interactions with plastid-encoded proteins. A massive excess of nonsynonymous substitutions between species relative to levels of intraspecific polymorphism indicated a history of strong positive selection (particularly in CLP genes). Interestingly, however, some species are likely undergoing loss of the native (heteromeric) plastid ACCase and putative functional replacement by a duplicated cytosolic (homomeric) ACCase. Overall, the patterns of molecular evolution in these plastid-nuclear complexes are unusual for anciently conserved enzymes. They instead resemble cases of antagonistic coevolution between pathogens and host immune genes. We discuss a possible role of plastid-nuclear conflict as a novel cause of accelerated evolution.

Oleanane-type triterpenoid saponins from Silene armeria.

Twelve triterpenoid saponins, including seven compounds (i.e., armerosides A-G) hitherto unknown, were isolated from whole plants of Silene armeria. Their structures were established based on extensive spectroscopic analyses and chemical methods. From a biosynthetic perspective, C-23 oxidation of the sapogenin appears to be a key factor in the glycosylation pathway.

Under fungal attack on a metalliferous soil: ROS or not ROS? Insights from Silene paradoxa L. growing under copper stress.

We investigated how the adaptation to metalliferous environments can influence the plant response to biotic stress. In a metallicolous and a non-metallicolous population of Silene paradoxa the induction of oxidative stress and the production of callose and volatiles were evaluated in the presence of copper and of the PAMP fungal protein cerato-platanin, separately and in combination. Our results showed incompatibility between the ordinary ROS-mediated response to fungal attack and the acquired mechanisms of preventing oxidative stress in the tolerant population. A similar situation was also demonstrated by the sensitive population growing in the presence of copper but, in this case, with a lack of certain responses, such as callose production. In addition, in terms of the joint behaviour of emitted volatiles, multivariate statistics showed that not only did the populations respond differently to the presence of copper or biotic stress, but also that the biotic and abiotic stresses interacted in different ways in the two populations. Our results demonstrated that the same incompatibility of hyperaccumulators in ROS-mediated biotic stress signals also seemed to be exhibited by the excluder metallophyte, but without the advantage of being able to rely on the elemental defence for plant protection from natural enemies.

[Features of Clonal Micropropagation of Silene cretacea (Caryophyllaceae) in in vitro Culture].

The features of the formation of microshoots in in vitro culture of Silene cretacea­endangered species with narrow ecological amplitude, which is a promising source of medicinal raw materials­were studied. It was demonstrated that, at the micropropagation step, basic Woody Plant Medium containing vitamins according to Murashige and Skoog and supplemented with 0.2 mg/L 6-benzylaminopurine, 1.0 mg/L kinetin, 1.0 mg/L gibberellic acid, and 0.5 mg/L indole-3-acetic acid is the most effective. The combination and concentration of these growth regulators, selected using mathematical combinatorial analysis, activated axillary buds and provided a high multiplication factor (9.3 ± 1.3 microshoots per explant). Morpho-histological analysis revealed the main stages of the formation of microshoots and proved the absence of callus formation during the whole time of the cultivation of explants. The features of the dynamics of the culture during the year of continuous cultivation are presented.

The composition and depth of green roof substrates affect the growth of Silene vulgaris and Lagurus ovatus species and the C and N sequestration under two irrigation conditions.

Extensive green roofs are used to increase the surface area covered by vegetation in big cities, thereby reducing the urban heat-island effect, promoting CO2 sequestration, and increasing biodiversity and urban-wildlife habitats. In Mediterranean semi-arid regions, the deficiency of water necessitates the use in these roofs of overall native plants which are more adapted to drought than other species. However, such endemic plants have been used scarcely in green roofs. For this purpose, we tested two different substrates with two depths (5 and 10 cm), in order to study their suitability with regard to adequate plant development under Mediterranean conditions. A compost-soil-bricks (CSB) (1:1:3; v:v:v) mixture and another made up of compost and bricks (CB) (1:4; v:v) were arranged in two depths (5 and 10 cm), in cultivation tables. Silene vulgaris (Moench) Garcke and Lagurus ovatus L. seeds were sown in each substrate. These experimental units were subjected, on the one hand, to irrigation at 40% of the registered evapotranspiration values (ET0) and, on the other, to drought conditions, during a nine-month trial. Physichochemical and microbiological substrate characteristics were studied, along with the physiological and nutritional status of the plants. We obtained significantly greater plant coverage in CSB at 10 cm, especially for L. ovatus (80-90%), as well as a better physiological status, especially in S. vulgaris (SPAD values of 50-60), under irrigation, whereas neither species could grow in the absence of water. The carbon and nitrogen fixation by the substrate and the aboveground biomass were also higher in CSB at 10 cm, especially under L. ovatus - in which 1.32 kg C m(-2) and 209 g N m(-2) were fixed throughout the experiment. Besides, the enzymatic and biochemical parameters assayed showed that microbial activity and nutrient cycling, which fulfill a key role for plant development, were higher in CSB. Therefore, irrigation of 40% can maintain an adequate plant cover of both endemic species, particularly in a deeper and soil-containing substrate.

Conservative and compensatory evolution in oxidative phosphorylation complexes of angiosperms with highly divergent rates of mitochondrial genome evolution.

Interactions between nuclear and mitochondrial gene products are critical for eukaryotic cell function. Nuclear genes encoding mitochondrial-targeted proteins (N-mt genes) experience elevated rates of evolution, which has often been interpreted as evidence of nuclear compensation in response to elevated mitochondrial mutation rates. However, N-mt genes may be under relaxed functional constraints, which could also explain observed increases in their evolutionary rate. To disentangle these hypotheses, we examined patterns of sequence and structural evolution in nuclear- and mitochondrial-encoded oxidative phosphorylation proteins from species in the angiosperm genus Silene with vastly different mitochondrial mutation rates. We found correlated increases in N-mt gene evolution in species with fast-evolving mitochondrial DNA. Structural modeling revealed an overrepresentation of N-mt substitutions at positions that directly contact mutated residues in mitochondrial-encoded proteins, despite overall patterns of conservative structural evolution. These findings support the hypothesis that selection for compensatory changes in response to mitochondrial mutations contributes to the elevated rate of evolution in N-mt genes. We discuss these results in light of theories implicating mitochondrial mutation rates and mitonuclear coevolution as drivers of speciation and suggest comparative and experimental approaches that could take advantage of heterogeneity in rates of mtDNA evolution across eukaryotes to evaluate such theories.

A dynamical phyllotaxis model to determine floral organ number.

How organisms determine particular organ numbers is a fundamental key to the development of precise body structures; however, the developmental mechanisms underlying organ-number determination are unclear. In many eudicot plants, the primordia of sepals and petals (the floral organs) first arise sequentially at the edge of a circular, undifferentiated region called the floral meristem, and later transition into a concentric arrangement called a whorl, which includes four or five organs. The properties controlling the transition to whorls comprising particular numbers of organs is little explored. We propose a development-based model of floral organ-number determination, improving upon earlier models of plant phyllotaxis that assumed two developmental processes: the sequential initiation of primordia in the least crowded space around the meristem and the constant growth of the tip of the stem. By introducing mutual repulsion among primordia into the growth process, we numerically and analytically show that the whorled arrangement emerges spontaneously from the sequential initiation of primordia. Moreover, by allowing the strength of the inhibition exerted by each primordium to decrease as the primordium ages, we show that pentamerous whorls, in which the angular and radial positions of the primordia are consistent with those observed in sepal and petal primordia in Silene coeli-rosa, Caryophyllaceae, become the dominant arrangement. The organ number within the outmost whorl, corresponding to the sepals, takes a value of four or five in a much wider parameter space than that in which it takes a value of six or seven. These results suggest that mutual repulsion among primordia during growth and a temporal decrease in the strength of the inhibition during initiation are required for the development of the tetramerous and pentamerous whorls common in eudicots.