A field portable method for the semi-quantitative estimation of dehydration tolerance of photosynthetic tissues across distantly related land plants.

Desiccation tolerant (DT) plants withstand complete cellular dehydration, reaching relative water contents (RWC) below 30% in their photosynthetic tissues. Desiccation sensitive (DS) plants exhibit different degrees of dehydration tolerance (DHT), never surviving water loss >70%. To date, no procedure for the quantitative evaluation of DHT extent exists that is able to discriminate DS species with differing degrees of DHT from truly DT plants. We developed a simple, feasible and portable protocol to differentiate between DT and different degrees of DHT in the photosynthetic tissues of seed plants and between fast desiccation (< 24 h) tolerant (FDT) and sensitive (FDS) bryophytes. The protocol is based on (1) controlled desiccation inside Falcon tubes equilibrated at three different relative humidities that, consequently, induce three different speeds and extents of dehydration and (2) an evaluation of the average percentage of maximal photochemical efficiency of PSII (Fv /fm) recovery after rehydration. Applying the method to 10 bryophytes and 28 tracheophytes from various locations, we found that (1) imbibition of absorbent material with concentrated salt-solutions inside the tubes provides stable relative humidity and avoids direct contact with samples; (2) for 50 ml capacity tubes, the optimal plant amount is 50-200 mg fresh weight; (3) the method is useful in remote locations due to minimal instrumental requirements; and (4) a threshold of 30% recovery of the initial Fv /fm upon reaching RWC ≤ 30% correctly categorises DT species, with three exceptions: two poikilochlorophyllous species and one gymnosperm. The protocol provides a semi-quantitative expression of DHT that facilitates comparisons of species with different morpho-physiological traits and/or ecological attributes.

Metabolomic Profiling of the Desiccation-Tolerant Medicinal Shrub Myrothamnus flabellifolia Indicates Phenolic Variability Across Its Natural Habitat: Implications for Tea and Cosmetics Production.

The leaves and twigs of the desiccation-tolerant medicinal shrub Myrothamnus flabellifolia are harvested for use in traditional and commercial teas and cosmetics due to their phenolic properties. The antioxidant and pharmacological value of this plant has been widely confirmed; however, previous studies typically based their findings on material collected from a single region. The existence of phenolic variability between plants from different geographical regions experiencing different rainfall regimes has thus not been sufficiently evaluated. Furthermore, the anthocyanins present in this plant have not been assessed. The present study thus used an untargeted liquid chromatography-tandem-mass spectrometry approach to profile phenolics in M. flabellifolia material collected from three climatically distinct (high, moderate, and low rainfall) regions representing the western, southern, and eastern extent of the species range in southern Africa. Forty-one putative phenolic compounds, primarily flavonoids, were detected, nine of which are anthocyanins. Several of these compounds are previously unknown from M. flabellifolia. Using multivariate statistics, samples from different regions could be distinguished by their phenolic profiles, supporting the existence of regional phenolic variability. This study indicates that significant phenolic variability exists across the range of M. flabellifolia, which should inform both commercial and traditional cultivation and harvesting strategies.

A molecular physiological review of vegetative desiccation tolerance in the resurrection plant Xerophyta viscosa (Baker).

MAIN CONCLUSION: Provides a first comprehensive review of integrated physiological and molecular aspects of desiccation tolerance Xerophyta viscosa. A synopsis of biotechnological studies being undertaken to improve drought tolerance in maize is given. Xerophyta viscosa (Baker) is a monocotyledonous resurrection plant from the family Vellociacea that occurs in summer-rainfall areas of South Africa, Lesotho and Swaziland. It inhabits rocky terrain in exposed grasslands and frequently experiences periods of water deficit. Being a resurrection plant it tolerates the loss of 95% of total cellular water, regaining full metabolic competency within 3 days of rehydration. In this paper, we review some of the molecular and physiological adaptations that occur during various stages of dehydration of X. viscosa, these being functionally grouped into early and late responses, which might be relevant to the attainment of desiccation tolerance. During early drying (to 55% RWC) photosynthesis is shut down, there is increased presence and activity of housekeeping antioxidants and a redirection of metabolism to the increased formation of sucrose and raffinose family oligosaccharides. Other metabolic shifts suggest water replacement in vacuoles proposed to facilitate mechanical stabilization. Some regulatory processes observed include increased presence of a linker histone H1 variant, a Type 2C protein phosphatase, a calmodulin- and an ERD15-like protein. During the late stages of drying (to 10% RWC) there was increased expression of several proteins involved in signal transduction, and retroelements speculated to be instrumental in gene silencing. There was induction of antioxidants not typically found in desiccation-sensitive systems, classical stress-associated proteins (HSP and LEAs), proteins involved in structural stabilization and those associated with changes in various metabolite pools during drying. Metabolites accumulated in this stage are proposed, inter alia, to facilitate subcellular stabilization by vitrification process which can include glass- and ionic liquid formation.

Erosive processes after tectonic uplift stimulate vicariant and adaptive speciation: evolution in an Afrotemperate-endemic paper daisy genus.

BACKGROUND: The role of tectonic uplift in stimulating speciation in South Africa's only alpine zone, the Drakensberg, has not been explicitly examined. Tectonic processes may influence speciation both through the creation of novel habitats and by physically isolating plant populations. We use the Afrotemperate endemic daisy genus Macowania to explore the timing and mode (geographic versus adaptive) of speciation in this region. Between sister species pairs we expect high morphological divergence where speciation has happened in sympatry (adaptive) while with geographic (vicariant) speciation we may expect to find less morphological divergence and a greater degree of allopatry. A dated molecular phylogenetic hypothesis for Macowania elucidates species' relationships and is used to address the potential impact of uplift on diversification. Morphological divergence of a small sample of reproductive and vegetative characters, used as a proxy for adaptive divergence, is measured against species' range distributions to estimate mode of speciation across two subclades in the genus. RESULTS: The Macowania crown age is consistent with the hypothesis of post-uplift diversification, and we find evidence for both vicariant and adaptive speciation between the two subclades within Macowania. Both subclades exhibit strong signals of range allopatry, suggesting that geographic isolation was important in speciation. One subclade, associated with dry, rocky environments at high altitudes, shows very little morphological and ecological differentiation but high range allopatry. The other subclade occupies a greater variety of habitats and exhibits far greater morphological differentiation, but contains species with overlapping distribution ranges. CONCLUSIONS: Species in Macowania are likely to have diversified in response to tectonic uplift, and we invoke uplift and uplift-mediated erosion as the main drivers of speciation. The greater relative morphological divergence in sympatric species of Macowania indicates that speciation in the non-sympatric taxa may not have required obvious adaptive differences, implying that simple geographic isolation was the driving force for speciation ('neutral speciation').

Risks to biodiversity from temperature overshoot pathways.

Temperature overshoot pathways entail exceeding a specified global warming level (e.g. 1.5°C or 2°C) followed by a decline in warming, achieved through anthropogenically enhanced CO2 removal from the atmosphere. However, risks to biodiversity from temperature overshoot pathways are poorly described. Here, we explore biodiversity risks from overshoot by synthesizing existing knowledge and quantifying the dynamics of exposure and de-exposure to potentially dangerous temperatures for more than 30 000 species for a 2°C overshoot scenario. Our results suggest that climate risk to biodiversity from temperature overshoot pathways will arrive suddenly, but decrease only gradually. Peak exposure for biodiversity occurs around the same time as peak global warming, but the rate of de-exposure lags behind the temperature decline. While the global overshoot period lasts around 60 years, the duration of elevated exposure of marine and terrestrial biodiversity is substantially longer (around 100 and 130 years, respectively), with some ecological communities never returning to pre-overshoot exposure levels. Key biodiversity impacts may be irreversible and reliance on widespread CO2 removal to reduce warming poses additional risks to biodiversity through altered land use. Avoiding any temperature overshoot must be a priority for reducing biodiversity risks from climate change, followed by limiting the magnitude and duration of any overshoot. More integrated models that include direct and indirect impacts from overshoot are needed to inform policy. This article is part of the theme issue 'Ecological complexity and the biosphere: the next 30 years'.

The phenolic profile extracted from the desiccation-tolerant medicinal shrub Myrothamnus flabellifolia using Natural Deep Eutectic Solvents varies according to the solvation conditions.

Natural Deep Eutectic Solvents (NaDES) have been proposed as designer solvents for the green extraction of bioactive products from plants. Myrothamnus flabellifolia is a desiccation-tolerant medicinal shrub that has been widely studied for its phenolic properties; however, a NaDES-based approach for the extraction of phenolics has not been tested in this species. Our aim was thus to evaluate the extraction of phenolics from M. flabellifolia using four different NaDES with differing acidities using a non-targeted liquid chromatography-quantitative time-of-flight-tandem mass spectrometry (LC-QTOF-MS/MS) metabolomics approach. Anthocyanin pigments were quantified using targeted high-performance LC. Leaf material from M. flabellifolia was extracted in four different NaDES solutions (sucrose-fructose-glucose; proline-malic acid; sucrose-citric acid; and glucose-choline chloride), and the results were subjected to multivariate statistical analysis to evaluate the phenolic profiles of the different NaDES extracts. The NaDES were effective at extracting phenolic compounds from M. flabellifolia and also exhibited specificity in the suites of phenolics that they extracted, as indicated by principal component analysis. Using partial least squares-discriminant analysis, we were able to identify the phenolics that were most differentially abundant between the extracts, and a heatmap provided an indication of the types of phenolics that were extracted by the different NaDES. Furthermore, the NaDES also extracted several compounds not previously detected in M. flabellifolia using conventional organic solvents, demonstrating their use in compound discovery. The NaDES also differentially targeted anthocyanins, with the more acidic NaDES extracting higher quantities of anthocyanins and polymeric pigments. A green chemistry-based extraction technique using NaDES can thus effectively target phenolics in M. flabellifolia and offers a promising solution for future phytochemical investigations in medicinal plants using a highly efficient non-toxic solvent system that can be tailored to target particular compounds.

Metabolomics as a complement to phylogenetics for assessing intraspecific boundaries in the desiccation-tolerant medicinal shrub Myrothamnus flabellifolia (Myrothamnaceae).

The desiccation-tolerant shrub Myrothamnus flabellifolia has colonised a unique and harsh niche that provides little protection from the elements. It has a wide distribution range in southern Africa, occurring across an environmental gradient that is exceptionally arid in the southwest and highly mesic in the northeast. It is also harvested for use in medicinal preparations, both traditionally and commercially. However, the phytochemical variability of plants from different rainfall regions has not been assessed, nor have the intraspecific relationships been evaluated by means of a rigorously tested phylogeny. The aims of the present study were thus (1) to test a phylogenetic hypothesis for intraspecific relationships in M. flabellifolia; (2) to assess, based on the global metabolomic profiles, whether accessions collected from the three different geographic locations in southern Africa across a rainfall gradient can be differentiated, and if this corroborates the phylogenetic signature; and (3) with the aid of multivariate statistical analysis, identify and evaluate the most significant discriminatory metabolites between the three sampled regions that could act as potential barcodes. The results show that the phylogenetic and metabolomic signatures were congruent, and the metabolomic data were better able to discriminate the different populations collected from the three regions. Several potential barcodes for discriminating the material from the three regions are proposed. Quercetin-rhamnoside and 3-O-methylquercetin, both significant antioxidants, were present at significantly higher quantities in the material from the driest region in the west than from the more mesic regions in the south and east, whereas quercetin-3-O-glucuronide was significantly higher in the latter. A naringenin-like compound or arbutin derivative could discriminate the southern samples from the eastern samples, whereas digalloylglucose differentiated the eastern samples from the southern samples. In summary, the findings of this study imply that the origin of the material should be considered when used in medicinal and cosmetic preparations.

The rediscovery of the Great Winterberg endemic Lotononis harveyi B.-E.van Wyk after 147 years, and notes on the poorly known Amathole endemic Macowania revoluta Oliv. (southern Great Escarpment, South Africa).

South Africa's 800 km-long southern Great Escarpment hosts numerous endemic plant species only known from their type specimens or from very few records. This is a legacy of a 100-150 year lag between the pioneer work of 19(th) century botanists and repeat fieldwork in the 21(st) century. As a result, population and ecological data are lacking for many local endemic species. Here we report on the rediscovery of Lotononis harveyi B.-E.van Wyk 147 years after its original description, and provide the first detailed ecological notes on the poorly known shrub Macowania revoluta Oliv. Both species are locally endemic to the Great Winterberg-Amatholes (Eastern Cape Province). With only six known individuals, Lotononis harveyi is recommended the conservation status of Critically Endangered, with fire (and potentially grazing) being the main population constraints. Macowania revoluta is locally abundant, and it is surprising that it has been so poorly collected in recent decades. It occupies an important local niche as a keystone montane wetland species, and its narrow distribution range - combined with pressure from woody alien invasive species - suggests that its conservation status should be Rare. The research further highlights the need for continued biodiversity field research along South Africa's poorly explored Great Escarpment.

Thiazolides as novel antiviral agents. 1. Inhibition of hepatitis B virus replication.

We report the syntheses and activities of a wide range of thiazolides [viz., 2-hydroxyaroyl-N-(thiazol-2-yl)amides] against hepatitis B virus replication, with QSAR analysis of our results. The prototypical thiazolide, nitazoxanide [2-hydroxybenzoyl-N-(5-nitrothiazol-2-yl)amide, NTZ] 1 is a broad spectrum antiinfective agent effective against anaerobic bacteria, viruses, and parasites. By contrast, 2-hydroxybenzoyl-N-(5-chlorothiazol-2-yl)amide 3 is a novel, potent, and selective inhibitor of hepatitis B replication (EC(50) = 0.33 µm) but is inactive against anaerobes. Several 4'- and 5'-substituted thiazolides show good activity against HBV; by contrast, some related salicyloylanilides show a narrower spectrum of activity. The ADME properties of 3 are similar to 1; viz., the O-acetate is an effective prodrug, and the O-aryl glucuronide is a major metabolite. The QSAR study shows a good correlation of observed EC(90) for intracellular virions with thiazolide structural parameters. Finally we discuss the mechanism of action of thiazolides in relation to the present results.

Thiazolides as novel antiviral agents. 2. Inhibition of hepatitis C virus replication.

We report the activities of a number of thiazolides [2-hydroxyaroyl-N-(thiazol-2-yl)amides] against hepatitis C virus (HCV) genotypes IA and IB, using replicon assays. The structure-activity relationships (SARs) of thiazolides against HCV are less predictable than against hepatitis B virus (HBV), though an electron-withdrawing group at C(5') generally correlates with potency. Among the related salicyloylanilides, the m-fluorophenyl analogue was most promising; niclosamide and close analogues suffered from very low solubility and bioavailability. Nitazoxanide (NTZ) 1 has performed well in clinical trials against HCV. We show here that the 5'-Cl analogue 4 has closely comparable in vitro activity and a good cell safety index. By use of support vector analysis, a quantitative structure-activity relationship (QSAR) model was obtained, showing good predictive models for cell safety. We conclude by updating the mode of action of the thiazolides and explain the candidate selection that has led to compound 4 entering preclinical development.

Synthesis of the 3'-thio-nucleosides and subsequent automated synthesis of oligodeoxynucleotides containing a 3'-S-phosphorothiolate linkage.

Oligodeoxynucleotides containing 3'-S-phosphorothiolate (3'-PS) linkages have become useful tools for probing enzyme-catalyzed cleavage processes in DNA. This protocol describes the synthesis of the phosphorothioamidite monomers derived from thymidine and 2'-deoxycytidine, and their application to a fully automated procedure for synthesising oligodeoxynucleotides containing 3'-PS linkages. The synthesis of the 5'-protected-3'-amidites is achievable in 2 weeks with the DNA synthesis and purification taking another 1 week.

Duplex stability of DNA.DNA and DNA.RNA duplexes containing 3'-S-phosphorothiolate linkages.

3'-S-Phosphorothiolate (3'-SP) linkages have been incorporated into the DNA strand of both a DNA.RNA duplex and a DNA.DNA duplex. Thermal melting (T(m)) studies established that this modification significantly stabilises the DNA.RNA duplex with an average increase in T(m) of about 1.4 degrees C per modification. For two or three modifications, the increase in T(m) was larger for an alternating, as compared to the contiguous, arrangement. For more than three modifications their arrangement had no effect on T(m). In contrast to the DNA.RNA duplex, the 3'-S-phosphorothiolate linkage destabilised the DNA.DNA duplex, irrespective of the arrangement of the 3'-SP linkages. The effect of ionic strength on duplex stability was similar for both the phosphorothiolate-substituted and the unmodified RNA.DNA duplexes. The results are discussed in terms of the influence that the sulfur atom has on the conformation of the furanose ring and comparisons are also drawn between the current study and those previously conducted with other modifications that have a similar conformational effect.