Role of Sulfate Transporters in Chromium Tolerance in Scenedesmus acutus M. (Sphaeropleales).

Sulfur (S) is essential for the synthesis of important defense compounds and in the scavenging potential of oxidative stress, conferring increased capacity to cope with biotic and abiotic stresses. Chromate can induce a sort of S-starvation by competing for uptake with SO42- and causing a depletion of cellular reduced compounds, thus emphasizing the role of S-transporters in heavy-metal tolerance. In this work we analyzed the sulfate transporter system in the freshwater green algae Scenedesmus acutus, that proved to possess both H+/SO42- (SULTRs) and Na+/SO42- (SLTs) plasma membrane sulfate transporters and a chloroplast-envelope localized ABC-type holocomplex. We discuss the sulfate uptake system of S. acutus in comparison with other taxa, enlightening differences among the clade Sphaeropleales and Volvocales/Chlamydomonadales. To define the role of S transporters in chromium tolerance, we analyzed the expression of SULTRs and SULPs components of the chloroplast ABC transporter in two strains of S. acutus with different Cr(VI) sensitivity. Their differential expression in response to Cr(VI) exposure and S availability seems directly linked to Cr(VI) tolerance, confirming the role of sulfate uptake/assimilation pathways in the metal stress response. The SULTRs up-regulation, observed in both strains after S-starvation, may directly contribute to enhancing Cr-tolerance by limiting Cr(VI) uptake and increasing sulfur availability for the synthesis of sulfur-containing defense molecules.

Role of DNA methylation in the chromium tolerance of Scenedesmus acutus (Chlorophyceae) and its impact on the sulfate pathway regulation.

DNA methylation is a very important epigenetic modification that participates in many biological functions. Although many researches on DNA methylation have been reported in various plant species, few studies have assessed the global DNA methylation pattern in algae. Even more the complex mechanisms by which DNA methylation modulates stress in algae are yet largely unresolved, mainly with respect to heavy metal stress, for which in plants, metal- and species- specific responses were instead evidenced. In this work, we performed a comparative Whole-Genome Bisulfite Sequencing (WGBS) on two strains of the green alga Scenedesmus acutus with different Cr(VI) sensitivity. The pattern of distribution of 5-mC showed significant differences between the two strains concerning both differentially methylated local contexts (CG, CHG and CHH) and Differentially Methylated Regions (DMRs) as well. We also demonstrated that DNA methylation plays an important role in modulating some genes for sulfate uptake/assimilation confirming the involvement of the sulfate pathway in the Cr-tolerance. Our results suggest that DNA methylation may be of particular importance in defining signal specificity associated with Cr-tolerance and in establishing new epigenetic marks which contribute to the adaptation to metal stress and also to transmit the epigenomic traits to the progeny.

The relationship between sulfur metabolism and tolerance of hexavalent chromium in Scenedesmus acutus (Spheropleales): Role of ATP sulfurylase.

Sulfur availability and the end products of its metabolism, cysteine, glutathione and phytochelatins, play an important role in heavy metal tolerance, chromium included. Sulfate and chromate not only compete for the transporters but also for assimilation enzymes and chromium tolerance in various organisms has been associated to differences in this pathway. We investigated the mechanisms of Cr(VI)-tolerance increase induced by S-starvation focusing on the role of ATP sulfurylase (ATS) in two strains of Scenedesmus acutus with different chromium sensitivity. S-starvation enhances the defence potential by increasing sulfate uptake/assimilation and decreasing chromium uptake, thus suggesting a change in the transport system. We isolated two isoforms of the enzyme, SaATS1 and SaATS2, with different sensitivity to sulfur availability, and analysed them in S-sufficient and S-replete condition both in standard and in chromium supplemented medium. SaATS2 expression is different in the two strains and presumably marks a different sulfur perception/exploitation in the Cr-tolerant. Its induction and silencing are compatible with a role in the transient tolerance increase induced by S-starvation. This enzyme can however hardly be responsible for the large cysteine production of the Cr-tolerant strain after starvation, suggesting that cytosolic rather than chloroplastic cysteine production is differently regulated in the two strains.

Authentication of Punica granatum L.: Development of SCAR markers for the detection of 10 fruits potentially used in economically motivated adulteration.

The large commercial success of pomegranate increase the likelihood of economically motivated adulteration (EMA), which has been gradually spotted with the undeclared addition of anthocyanin-rich plants or cheaper fruit juices used as bulking and diluting agents. A method based on Sequence-Characterized Amplified Regions (SCARs) was developed to detect the presence of Aristotelia chilensis, Aronia melanocarpa, Dioscorea alata, Euterpe oleracea, Malus×domestica, Morus nigra, Sambucus nigra, Vaccinium macrocarpon, Vaccinium myrtillus, Vitis vinifera as bulking agents in Punica granatum. The method enabled the unequivocal detection of up to 1% of each adulterant, allowing the preemptive rejection of suspect samples. The recourse to such method may reduce the number of samples to be subjected to further phytochemical analyses when multiple batches have to be evaluated in a short time. Vice versa, it allows the cross-check of suspect batches previously tested only for their anthocyanin profile. The dimension of the amplicons is suitable for the analysis of degraded DNA obtained from stored and processed commercial material. Proper SCAR markers may represent a fast, sensitive, reliable and low-cost screening method for the authentication of processed commercial pomegranate material.

Phytotoxic Effects and Phytochemical Fingerprinting of Hydrodistilled Oil, Enriched Fractions, and Isolated Compounds Obtained from Cryptocarya massoy (Oken) Kosterm. Bark.

The hydrodistilled oil of Cryptocarya massoy bark was characterized by GC-FID and GC/MS analyses, allowing the identification of unusual C10 massoia lactone (3, 56.2%), C12 massoia lactone (4, 16.5%), benzyl benzoate (1, 12.7%), C8 massoia lactone (3.4%), δ-decalactone (5, 1.5%), and benzyl salicylate (2, 1.8%) as main constituents. The phytotoxic activities of the oil, three enriched fractions (lactone-rich, ester-rich, and sesquiterpene-rich), and four constituents (compounds 1, 2, 5, and δ-dodecalactone (6)) against Lycopersicon esculentum and Cucumis sativus seeds and seedlings were screened. At a concentration of 1000 µl/l, the essential oil and the massoia lactone-rich fraction caused a complete inhibition of the germination of both seeds, and, when applied on tomato plantlets, they induced an 85 and 100% dieback, respectively. These performances exceeded those of the well-known phytotoxic essential oils of Syzygium aromaticum and Cymbopogon citratus, already used in commercial products for the weed and pest management. The same substances were also evaluated against four phytopathogenic bacteria and ten phytopathogenic fungi, providing EC50 values against the most susceptible strains in the 100-500 µl/l range for the essential oil and in the 10-50 µl/l range for compound 6 and the lactone-rich fraction. The phytotoxic behavior was related mainly to massoia lactones and benzyl esters, while a greater amount of 6 may infer a good activity against some phytopathogenic fungi. Further investigations of these secondary metabolites are warranted, to evaluate their use as natural herbicides.

Phytochemical profile and bioactivity of traditional ayurvedic decoctions and hydro-alcoholic macerations of Boerhaavia diffusa L. and Curculigo orchioides Gaertn.

Decoctions (DECs) and hydro-alcoholic extracts (HEs) prepared from roots of Boerhaavia diffusa L. (Nyctaginaceae) and Curculigo orchioides Gaertn. (Hypoxidaceae) were phytochemically characterised by HPLC-DAD and profiled for their antioxidant, antigenotoxic and cytotoxic activities. B. diffusa DEC was rich in ferulic acid and vanillin, while the HE also contained boeravinone B and eupalitin. Both C. orchioides HE and DEC displayed the main occurrence of orcinol-ß-d-glucoside and curculigoside A. Antioxidant activity was assayed through spectrophotometric DPPH, ABTS and ß-carotene bleaching test, and using (HP)TLC bioautographic strategies. For both crude drugs, HE was the best performing preparation. Properly modified SOS-Chromotest evidenced a 10% inhibition by phytocomplexes against 4-nitroquinoline-N-oxide, and a higher bioactivity for vanillin (36.60 ± 1.68%) and ferulic acid (35.09 ± 1.53%). C. orchioides HE was the preparation which showed higher cytotoxicity against drug-sensitive human T-lymphoblastoid cell line (CCRF-CEM) and multidrug-resistant leukaemia cell line (CEM/ADR5000), and eupalitin was the only pure compound to exhibit an IC50 value.

Quality control of saffron (Crocus sativus L.): development of SCAR markers for the detection of plant adulterants used as bulking agents.

A method based on sequence-characterized amplified regions (SCARs) was developed from random amplified polymorphic DNA markers (RAPDs) specific for Arnica montana L., Bixa orellana L., Calendula officinalis L., Carthamus tinctorius L., Crocus vernus L. (Hill), Curcuma longa L., and Hemerocallis sp. to detect these common bulking agents in commercial saffron (Crocus sativus). The method enabled the unequivocal detection of low amounts (up to 1%) of each adulterant, allowing the preemptive rejection of suspect samples. Its enforcement limits the number of samples to be subjected to further evaluation with pharmacognostic or phytochemical analyses, especially when multiple batches have to be evaluated in a short time. The dimension of the amplicons is suitable for the analysis of degraded DNA obtained from dried, stored, processed, and finely ground commercial material. Proper SCAR markers may represent a fast, sensitive, reliable, and low-cost screening method for the authentication of dried commercial saffron material.

RAPD-based method for the quality control of Mediterranean oregano and its contribution to pharmacognostic techniques.

A pharmacognostic survey of 84 commercial samples of Mediterranean oregano, obtained from wholesale traders between 2001 and 2007, pinpointed the presence of extraneous plant material in 90.5% of the samples. In 59% of them extraneous material of plant origin was above 20%. Two major groups of botanical foreign matter were identified: oregano-like flavored plants ( Satureja montana L., Origanum majorana L.) and plants lacking a clearly detectable essential oil profile ( Rubus sp., Cistus incanus L., Rhus coriaria L.), added as bulk extraneous material. A random amplified polymorphic DNA (RAPD) method was developed to make the detection of the second group of adulterants easier and speed pharmacognostic analysis of large batches of samples. Thirteen primers discriminating between Origanum spp. and Rubus caesius , R.coriaria, and C. incanus were individuated, allowing their detection in oregano samples with a limit of detection of 1%. The utilization of RAPD as a reliable test to probe the authenticity of Mediterranean oregano or previously screen the presence of specific contaminants is proposed as a complementary approach to pharmacognostic and phytochemical screening.

Identification of S2-T A63: a cDNA fragment corresponding to a gene differentially expressed in a Cr-tolerant strain of the unicellular green alga Scenedesmus acutus.

The gene expression of the wild type (S2-N) and a Cr-tolerant strain (S2-T) of the unicellular green alga Scenedesmus acutus has been compared in order to get more insight on their different chromium sensitivity. The RNA of the two strains was extracted after 4 days of culture in standard medium without chromium and analyzed by means of RNA differential display. The two strains showed differential gene transcription even in the absence of the heavy metal and six putatively differential amplicons were evidenced in the Cr-tolerant strain. Among the isolated amplicons, S2-T A63 was much more pronouncedly transcribed in the tolerant than in the wild type strain and was further characterized. S2-T A63 corresponding gene is present with the same copy number in the wild type and tolerant genomes and corresponds to an mRNA of about 2000 nt. The corresponding transcript is overexpressed in the Cr-tolerant strain after a 4-day culture and is not up-regulated by chromium exposure. The S2-T A63 sequence, obtained up to now, does not show significant homologies with any known gene. However, the analysis of the putative translation product reveals the presence of an interrupted fasciclin domain. This extracellular domain has been found in proteins from mammals, insects, echinoderms, plants, yeast and bacteria and is usually involved in cell adhesion. This finding suggests that the product for the S2-T A63 translation has an extracellular collocation, maybe as surface or secreted protein involved in external chromium detoxification.