Variation in Sulfur and Selenium Accumulation Is Controlled by Naturally Occurring Isoforms of the Key Sulfur Assimilation Enzyme ADENOSINE 5′-PHOSPHOSULFATE REDUCTASE2 across the Arabidopsis Species Range

Natural variation allows the investigation of both the fundamental functions of genes and their role in local adaptation. As one of the essential macronutrients, sulfur is vital for plant growth and development and also for crop yield and quality. Selenium and sulfur are assimilated by the same process, and although plants do not require selenium, plant-based selenium is an important source of this essential element for animals. Here, we report the use of linkage mapping in synthetic F2 populations and complementation to investigate the genetic architecture of variation in total leaf sulfur and selenium concentrations in a diverse set of Arabidopsis (Arabidopsis thaliana) accessions. We identify in accessions collected from Sweden and the Czech Republic two variants of the enzyme ADENOSINE 5′-PHOSPHOSULFATE REDUCTASE2 (APR2) with strongly diminished catalytic capacity. APR2 is a key enzyme in both sulfate and selenate reduction, and its reduced activity in the loss-of-function allele apr2-1 and the two Arabidopsis accessions Hodonín and Shahdara leads to a lowering of sulfur flux from sulfate into the reduced sulfur compounds, cysteine and glutathione, and into proteins, concomitant with an increase in the accumulation of sulfate in leaves. We conclude from our observation, and the previously identified weak allele of APR2 from the Shahdara accession collected in Tadjikistan, that the catalytic capacity of APR2 varies by 4 orders of magnitude across the Arabidopsis species range, driving significant differences in sulfur and selenium metabolism. The selective benefit, if any, of this large variation remains to be explored.Sulfur is one of the essential mineral nutrients for all organisms and is required for the biosynthesis of sulfur-containing amino acids, lipids, vitamins, and secondary metabolites, the catalytic and regulatory activity of enzymes, and the stabilization of protein structures. However, animals can only utilize sulfur that has been incorporated into biomolecules primarily originating from plants. Sufficient sulfur fertilization is also important to maximize yields of various crops, including rapeseed (Brassica napus) and wheat (Triticum aestivum; Bloem et al., 2004; Dubousset et al., 2010; Steinfurth et al., 2012). Interestingly, sulfur fertilization can also be important for food quality, with well-fertilized wheat crops producing flour with improved bread-making qualities (Shahsavani and Gholami, 2008).Over the last 50 years, the levels of sulfur in soils have been impacted significantly by the release of sulfur dioxide into the atmosphere as a result of the combustion of fossil fuels (Menz and Seip, 2004). In the atmosphere, sulfur dioxide is oxidized to sulfuric acid and thereafter deposited on soils as acid rain. Regulations controlling the release of sulfur dioxide from power stations introduced in the 1970s have caused significant declines in sulfate deposition due to this acid rain (Menz and Seip, 2004). These reductions in sulfate deposition are significant enough to have increased the need for sulfur fertilization of agricultural crops such as rapeseed and wheat (Dubousset et al., 2010; Steinfurth et al., 2012). The essentiality of sulfur for plants and the relatively recent major fluctuations in soil sulfate deposition make the study of natural variation in sulfur homeostasis by plants attractive. Such studies not only have the potential to identify new molecular mechanisms involved in sulfur homeostasis but also could provide a platform for probing at the genetic level interactions between natural plant populations and a changing soil environment.The sulfur analog selenium is also widely distributed in soil and is incorporated into biomolecules in plants in place of sulfur via sulfur assimilatory processes. Although selenium is not required by plants, it is essential for animals, and plant-based selenium is the primary source of this important nutrient for humans. In animals, selenium plays a vital role in numerous different selenoproteins involved in redox reactions, selenium storage, and hormone biosynthesis (Underwood, 1981; Rayman, 2012; Roman et al., 2014). In these proteins, selenium is incorporated as seleno-Cys. Unlike plants where selenium is incorporated into biomolecules nonspecifically as a sulfur analog, seleno-Cys in animals is biosynthesized by the action of a specific seleno-Cys synthase that converts Ser-tRNA to seleno-Cys-tRNA (Roman et al., 2014). To help prevent the negative health effects of selenium deficiency in the diet, fortification of various foods with selenium is already practiced, and biofortification of crops such as wheat, through the addition of selenium to fertilizers, is being proposed (Lyons et al., 2003). Moreover, there is a growing body of studies suggesting that supraoptimal dietary levels of selenium in the form of seleno-Met and seleno-methylseleno-Cys may be helpful in preventing certain cancers, although the efficacy of these supplements is still debated (Rayman, 2012; Steinfurth et al., 2012). Therefore, the identification of genes that control variation in the uptake and metabolism of sulfur and selenium in plants is not only an essential task for understanding the molecular mechanisms of plant nutrition but also important for crop yield, food quality, and human health.Plants mainly take up sulfur from the soil in the form of sulfate. After uptake of sulfate via the sulfate transporters SULTR1;1 and SULTR1;2 in the root (Yoshimoto et al., 2007; Barberon et al., 2008), sulfate needs to be reduced to sulfide before it can be incorporated into Cys, the first sulfur-containing compound in the assimilatory process (for review, see Takahashi et al., 2011). Sulfate is first activated through adenylation by ATP SULFURYLASE (ATPS) to form adenosine 5′-phosphosulfate (APS) in both plastids and the cytosol (Rotte and Leustek, 2000). After activation, sulfate as APS is reduced to sulfite by APS REDUCTASE (APR) using reduced glutathione (GSH) as the electron donor (Gutierrez-Marcos et al., 1996; Setya et al., 1996). Alternatively, APS can be phosphorylated by APS kinase to form 3′-phosphoadenosine 5′-phosphosulfate (PAPS), which acts as the sulfate donor for sulfotransferases to incorporate sulfate directly into saccharides and secondary metabolites such as glucosinolates (Mugford et al., 2009). Sulfite produced by APR is further reduced to sulfide by sulfite reductase (Khan et al., 2010). In the final step, sulfide is combined with O-acetyl-Ser to form Cys, catalyzed by O-acetyl-Ser(thiol)lyase (Wirtz and Hell, 2006). In plants, selenium is taken up as selenate via sulfate transporters (Shibagaki et al., 2002). After uptake, it is thought that selenate is reduced to selenite via the action of ATP sulfurylase and APS reductase (Shaw and Anderson, 1972; Pilon-Smits et al., 1999; Sors et al., 2005a, 2005b). Selenite is most likely nonenzymatically reduced to selenide and combined with O-acetyl-Ser to form seleno-Cys catalyzed by O-acetyl-Ser(thiol)lyase (Ng and Anderson, 1978).Sulfate uptake and reduction represent the two control points for sulfur homeostasis (Kopriva et al., 2009). Based on sequence similarity, there are 14 genes annotated as sulfate transporters in the Arabidopsis (Arabidopsis thaliana) genome, and at least six of them have evidence supporting their functions (Kopriva et al., 2009; Takahashi, 2010). Among these, SULTR1;1 and SULTR1;2 encode high-affinity sulfate transporters in the root responsible for sulfate uptake from the soil solution (Rouached et al., 2008; Takahashi, 2010). Genes encoding both ATPS and APR also form gene families in Arabidopsis, with ATPS being encoded by four genes and APR by three (Kopriva et al., 2009). ATPS enzymes localize to both the cytosol and plastids, whereas APR enzymes localize solely to the plastids (Lunn et al., 1990; Rotte and Leustek, 2000). ATPS1 and APR2 are responsible for the majority of ATP sulfurylase and APS reductase activity of seedlings.In a quantitative trait locus (QTL) analysis of sulfate content in Arabidopsis leaves using recombinant inbred lines generated by crossing Bayreuth (Bay-0) and Shahdara (Sha), APR2 was identified as a locus controlling variation in sulfate accumulation between the Bay-0 and Sha accessions (Loudet et al., 2007). The causal quantitative trait nucleotide in the Sha allele of APR2 results in a substitution of Ala-399 to Glu-399. The substitution localizes in the thioredoxin active site, which reduces the affinity of APR2 for GSH and results in a loss of 99.8% of enzyme activity. The loss-of-function Sha APR2 allele leads to a significant decrease in sulfate reduction and a concomitant increase in leaf sulfate accumulation. In the same analysis, a second QTL for leaf sulfate accumulation was described (Loudet et al., 2007). This second QTL was recently established to be driven by an ATPS1 expression-level polymorphism between Bay-0 and Sha, with Bay-0 showing reduced expression of ATPS1 compared with Sha (Koprivova et al., 2013).Both Loudet et al. (2007) and Koprivova et al. (2013) focused their studies on the genetic architecture of natural variation for leaf sulfate using a single recombinant population created by crossing the Bay-0 and Sha accessions. This recombinant inbred population has proved a powerful tool for the identification of novel alleles controlling several different traits (Loudet et al., 2008; Jiménez-Gómez et al., 2010; Jasinski et al., 2012; Pineau et al., 2012; Anwer et al., 2014). However, because this set of recombinant inbred lines is composed of genotypes from only two accessions, its value is limited when trying to understand allelic diversity across the Arabidopsis species as a whole. To address this limitation, we performed experiments using a set of 349 Arabidopsis accessions collected from across the species range. These 349 accessions were selected from a worldwide collection of 5,810 accessions sampled to minimize redundancy and close family relatedness (Baxter et al., 2010; Platt et al., 2010). To allow us to further probe Arabidopsis species-wide allelic diversity, we also utilized the complete genome sequences of 855 Arabidopsis accessions from the 1,001 Genomes Project (http://signal.salk.edu/atg1001/index.php).Since sulfate uptake and accumulation is only one step in the complex process of sulfur assimilation in plants, uncovering the genetic architecture of sulfate accumulation, as performed by Loudet et al. (2007) and Koprivova et al. (2013), could potentially overlook genetic variation in other important aspects of sulfur metabolism. To enable us to identify natural genetic variation in sulfur homeostasis beyond just sulfate accumulation, we chose to quantify total leaf sulfur. This potentially allowed us to capture variation in the accumulation of both sulfate and other important sulfur-containing metabolites such as GSH and glucosinolates.Furthermore, to test genetically the long-held assumption that selenium in plants is metabolized as a sulfur analog, we also phenotyped the same set of plants for total leaf selenium. Through a combination of high-throughput elemental analysis, linkage mapping, genetic and transgenic complementation, reciprocal grafting, and protein haplotype analysis, we have established that the natural variation in both leaf sulfur and selenium content in Arabidopsis is controlled by several rare APR2 variants across the whole of the Arabidopsis species.     

Mycoendophytes as efficient synthesizers of bionanoparticles: nanoantimicrobials,mechanism, and cytotoxicity

Mycoendophytes are the fungi that occur inside the plant tissues without exerting any negative impact on the host plant. They are most frequently isolated endophytes from the leaf, stem, and root tissues of various plants. Among all fungi, the mycoendophytes as biosynthesizer of noble metal nanoparticles (NPs) are less known. However, some reports showing efficient synthesis of metal nanoparticles, mainly silver nanoparticles and its remarkable antimicrobial activity against bacterial and fungal pathogens of humans and plants. The nanoparticles synthesized from mycoendophytes present stability, polydispersity, and biocompatibility. These are non-toxic to humans and environment, can be gained in an easy and cost-effective manner, have wide applicability and could be explored as promising candidates for a variety of biomedical, pharmaceutical, and agricultural applications. Mycogenic silver nanoparticles have also demonstrated cytotoxic activity against cancer cell lines and may prove to be a promising anticancer agent. The present review focuses on the biological synthesis of metal nanoparticles from mycoendophytes and their application in medicine. In addition, different mechanisms of biosynthesis and activity of nanoparticles on microbial cells, as well as toxicity of these mycogenic metal nanoparticles, have also been discussed.     

Using a bistable animal opsin for switchable and scalable optogenetic inhibition of neurons

There is no consensus on the best inhibitory optogenetic tool. Since Gi/o signalling is a native mechanism of neuronal inhibition, we asked whether Lamprey Parapinopsin (“Lamplight”), a Gi/o‐coupled bistable animal opsin, could be used for optogenetic silencing. We show that short (405 nm) and long (525 nm) wavelength pulses repeatedly switch Lamplight between stable signalling active and inactive states, respectively, and that combining these wavelengths can be used to achieve intermediate levels of activity. These properties can be applied to produce switchable neuronal hyperpolarisation and suppression of spontaneous spike firing in the mouse hypothalamic suprachiasmatic nucleus. Expressing Lamplight in (predominantly) ON bipolar cells can photosensitise retinas following advanced photoreceptor degeneration, with 405 and 525 nm stimuli producing responses of opposite sign in the output neurons of the retina. We conclude that bistable animal opsins can co‐opt endogenous signalling mechanisms to allow optogenetic inhibition that is scalable, sustained and reversible.     

Programmed cell death lives

Research on cell death mechanisms gets a lot of attention. This is understandable as it underlies biology in general, as well as the insight in pathological conditions and the development of opportunities for therapeutic intervention. Over the last years a steady rise in the number of scientific reports and in the impact of this literature on the different mechanisms of programmed cell death can be observed. A number of new concepts are highlighted.

     

The influence of a synthetic growth hormone-releasing hormone analogue G11 and opioid peptide biphalin on selected fibroblasts parameters relevant to wound healing

The treatment of hard-to-heal chronic wounds is still a major medical problem and an economic and social burden. In this work, we examine the proregenerative potential of two peptides, G11 (a trypsin-resistant analogue of growth hormone-releasing hormone [GHRH]) and biphalin (opioid peptide), and their combination in vitro on human fibroblasts (BJ). G11, biphalin and their combination exhibited no toxicity against BJ cells. On the contrary, these treatments significantly stimulated proliferation and migration of fibroblasts. Under inflammatory conditions (LPS-induced BJ cells), we noticed that the tested peptides decreased the levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and interleukin 1β (IL-1β). This was correlated with diminished phosphorylation levels of p38 kinase, but not those of ERK1/2. We found also that G11, biphalin and their combination activated the ERK1/2 signalling pathway, which has been previously implicated in promigratory activity of some regeneration enhancers, including opioids or GHRH analogues. Potential application of their combination requires further work, in particular in vivo experiments, in which the organism-level relevance of the discussed cell-level effects would be proven and, additionally, analgesic action of the opioid ingredient could be quantified.     

Population genetics of the hazel hen <Emphasis Type="Italic">Bonasa bonasia</Emphasis> in Poland assessed with non-invasive samples

Despite a severe decrease in the number of hazel hens during the 20^th century, nowadays this grouse species is rather common in the forests of Northeastern and Southern Poland. We used mitochondrial control region and microsatellite markers to examine the genetic variability of Polish populations of hazel hens. We used non-invasively collected faeces to estimate genetic variability within populations, genetic differentiation among populations as well as genetic differentiation between two regions inhabited by two different subspecies of hazel hens. Our results confirm the usefulness of DNA from faeces to obtain reliable information on the population genetics of hazel hens. We found a rather high level of genetic variability in the Polish population. Genetic variability was higher in birds from continuous forests in the South of the country than in birds from fragmented habitats in the Northeast. Genetic differentiation was higher among subpopulations from Northeastern Poland. Additionally, both classes of molecular markers suggested the presence of two distinct genetic groups of birds, corresponding to previously described subspecies. We conclude that the genetic variability of the Polish hazel hen population has been influenced by habitat fragmentation and the history of the population during its post-glacial colonization of Poland from different glacial refugia.     

Nocardia aciditolerans sp. nov., isolated from a spruce forest soil

Actinomycetes growing on acidified starch-casein agar seeded with suspensions of litter and mineral soil from a spruce forest were provisionally assigned to the genus Nocardia based upon colonial properties. Representative isolates were found to grow optimally at pH 5.5, have chemotaxonomic and morphological features consistent with their assignment to the genus Nocardia and formed two closely related subclades in the Nocardia 16S rRNA gene tree. DNA:DNA relatedness assays showed that representatives of the subclades belong to a single genomic species. The isolates were distantly associated with their nearest phylogenetic neighbour, the type strain of Nocardia kruczakiae, and were distinguished readily from the latter based on phenotypic properties. On the basis of these data it is proposed that the isolates merit recognition as a new species, Nocardia aciditolerans sp. nov. The type strain is isolate CSCA68^T (=KACC 17155^T = NCIMB 14829^T = DSM 45801^T).