The Salt-Stress Response of the Transgenic Plum Line J8-1 and Its Interaction with the Salicylic Acid Biosynthetic Pathway from Mandelonitrile.

Salinity is considered as one of the most important abiotic challenges that affect crop productivity. Plant hormones, including salicylic acid (SA), are key factors in the defence signalling output triggered during plant responses against environmental stresses. We have previously reported in peach a new SA biosynthetic pathway from mandelonitrile (MD), the molecule at the hub of the cyanogenic glucoside turnover in Prunus sp. In this work, we have studied whether this new SA biosynthetic pathway is also present in plum and the possible role this pathway plays in plant plasticity under salinity, focusing on the transgenic plum line J8-1, which displays stress tolerance via an enhanced antioxidant capacity. The SA biosynthesis from MD in non-transgenic and J8-1 micropropagated plum shoots was studied by metabolomics. Then the response of J8-1 to salt stress in presence of MD or Phe (MD precursor) was assayed by measuring: chlorophyll content and fluorescence parameters, stress related hormones, levels of non-enzymatic antioxidants, the expression of two genes coding redox-related proteins, and the content of soluble nutrients. The results from in vitro assays suggest that the SA synthesis from the MD pathway demonstrated in peach is not clearly present in plum, at least under the tested conditions. Nevertheless, in J8-1 NaCl-stressed seedlings, an increase in SA was recorded as a result of the MD treatment, suggesting that MD could be involved in the SA biosynthesis under NaCl stress conditions in plum plants. We have also shown that the plum line J8-1 was tolerant to NaCl under greenhouse conditions, and this response was quite similar in MD-treated plants. Nevertheless, the MD treatment produced an increase in SA, jasmonic acid (JA) and reduced ascorbate (ASC) contents, as well as in the coefficient of non-photochemical quenching (qN) and the gene expression of Non-Expressor of Pathogenesis-Related 1 (NPR1) and thioredoxin H (TrxH) under salinity conditions. This response suggested a crosstalk between different signalling pathways (NPR1/Trx and SA/JA) leading to salinity tolerance in the transgenic plum line J8-1.

Discovery and molecular and biocatalytic properties of hydroxynitrile lyase from an invasive millipede, Chamberlinius hualienensis.

Hydroxynitrile lyase (HNL) catalyzes the degradation of cyanohydrins and causes the release of hydrogen cyanide (cyanogenesis). HNL can enantioselectively produce cyanohydrins, which are valuable building blocks for the synthesis of fine chemicals and pharmaceuticals, and is used as an important biocatalyst in industrial biotechnology. Currently, HNLs are isolated from plants and bacteria. Because industrial biotechnology requires more efficient and stable enzymes for sustainable development, we must continuously explore other potential enzyme sources for the desired HNLs. Despite the abundance of cyanogenic millipedes in the world, there has been no precise study of the HNLs from these arthropods. Here we report the isolation of HNL from the cyanide-emitting invasive millipede Chamberlinius hualienensis, along with its molecular properties and application in biocatalysis. The purified enzyme displays a very high specific activity in the synthesis of mandelonitrile. It is a glycosylated homodimer protein and shows no apparent sequence identity or homology with proteins in the known databases. It shows biocatalytic activity for the condensation of various aromatic aldehydes with potassium cyanide to produce cyanohydrins and has high stability over a wide range of temperatures and pH values. It catalyzes the synthesis of (R)-mandelonitrile from benzaldehyde with a 99% enantiomeric excess, without using any organic solvents. Arthropod fauna comprise 80% of terrestrial animals. We propose that these animals can be valuable resources for exploring not only HNLs but also diverse, efficient, and stable biocatalysts in industrial biotechnology.

Sponge derived bromotyrosines: structural diversity through natural combinatorial chemistry.

Sponge derived bromotyrosines are a multifaceted class of marine bioactive compounds that are important for the chemical defense of sponges but also for drug discovery programs as well as for technical applications in the field of antifouling constituents. These compounds, which are mainly accumulated by Verongid sponges, exhibit a diverse range of bioactivities including antibiotic, cytotoxic and antifouling effects. In spite of the simple biogenetic building blocks, which consist only of brominated tyrosine and tyramine units, an impressive diversity of different compounds is obtained through different linkages between these precursors and through structural modifications of the side chains and/or aromatic rings resembling strategies that are known from combinatorial chemistry. As examples for bioactive, structurally divergent bromotyrosines psammaplin A, Aplysina alkaloids featuring aerothionin, aeroplysinin-1 and the dienone, and the bastadins, including the synthetically derived hemibastadin congeners, have been selected for this review. Whereas all of these natural products are believed to be involved in the chemical defense of sponges, some of them may also be of particular relevance to drug discovery due to their interaction with specific molecular targets in eukaryotic cells. These targets involve important enzymes and receptors, such as histone deacetylases (HDAC) and DNA methyltransferases (DNMT), which are inhibited by psammaplin A, as well as ryanodine receptors that are targeted by bastadine type compounds. The hemibastadins such as the synthetically derived dibromohemibastadin are of particular interest due to their antifouling activity. For the latter, a phenoloxidase which catalyzes the bioglue formation needed for firm attachment of fouling organisms to a given substrate was identified as a molecular target. The Aplysina alkaloids finally provide a vivid example for dynamic wound induced bioconversions of natural products that generate highly efficient chemical weapons precisely when and where needed.

Simulated climate change impact on summer dissolved organic carbon release from peat and surface vegetation: implications for drinking water treatment.

Uncertainty regarding changes in dissolved organic carbon (DOC) quantity and quality has created interest in managing peatlands for their ecosystem services such as drinking water provision. The evidence base for such interventions is, however, sometimes contradictory. We performed a laboratory climate manipulation using a factorial design on two dominant peatland vegetation types (Calluna vulgaris and Sphagnum Spp.) and a peat soil collected from a drinking water catchment in Exmoor National Park, UK. Temperature and rainfall were set to represent baseline and future conditions under the UKCP09 2080s high emissions scenario for July and August. DOC leachate then underwent standard water treatment of coagulation/flocculation before chlorination. C. vulgaris leached more DOC than Sphagnum Spp. (7.17 versus 3.00 mg g(-1)) with higher specific ultraviolet (SUVA) values and a greater sensitivity to climate, leaching more DOC under simulated future conditions. The peat soil leached less DOC (0.37 mg g(-1)) than the vegetation and was less sensitive to climate. Differences in coagulation removal efficiency between the DOC sources appears to be driven by relative solubilisation of protein-like DOC, observed through the fluorescence peak C/T. Post-coagulation only differences between vegetation types were detected for the regulated disinfection by-products (DBPs), suggesting climate change influence at this scale can be removed via coagulation. Our results suggest current biodiversity restoration programmes to encourage Sphagnum Spp. will result in lower DOC concentrations and SUVA values, particularly with warmer and drier summers.

Metabolites of amygdalin under simulated human digestive fluids.

In the present study, degradation of amygdalin in the human digestive fluids and absorption of its metabolites by the human small intestine were evaluated by simulating a gastrointestinal digestion model combined with a human intestinal cell culture. Orally administered amygdalin was degraded into prunasin by digestive enzymes after passing through the salivary and gastrointestinal phases. Prunasin, the major metabolite of amygdalin in the digestive fluids, was incubated in a caco-2 cell culture system. Prunasin was degraded into the mandelonitrile by ß-glucosidase and then hydroxylated across the small intestinal wall, producing hydroxymandelonitrile (149 Da). Results from this study suggest that risk assessment of amygdalin from food consumption can be done in a more accurate way by determining a pathway of amygdalin metabolism in the simulating human upper gastrointestinal tract.

AnhE, a metallochaperone involved in the maturation of a cobalt-dependent nitrile hydratase.

Acetonitrile hydratase (ANHase) of Rhodococcus jostii RHA1 is a cobalt-containing enzyme with no significant sequence identity with characterized nitrile hydratases. The ANHase structural genes anhA and anhB are separated by anhE, predicted to encode an 11.1-kDa polypeptide. An anhE deletion mutant did not grow on acetonitrile but grew on acetamide, the ANHase reaction product. Growth on acetonitrile was restored by providing anhE in trans. AnhA could be used to assemble ANHase in vitro, provided the growth medium was supplemented with 50 microM CoCl(2). Ten- to 100-fold less CoCl(2) sufficed when anhE was co-expressed with anhA. Moreover, AnhA contained more cobalt when produced in cells containing AnhE. Chromatographic analyses revealed that AnhE existed as a monomer-dimer equilibrium (100 mm phosphate, pH 7.0, 25 degrees C). Divalent metal ions including Co(2+), Cu(2+), Zn(2+), and Ni(2+) stabilized the dimer. Isothermal titration calorimetry studies demonstrated that AnhE binds two half-equivalents of Co(2+) with K(d) of 0.12 +/- 0.06 nM and 110 +/- 35 nM, respectively. By contrast, AnhE bound only one half-equivalent of Zn(2+) (K(d) = 11 +/- 2 nM) and Ni(2+) (K(d) = 49 +/- 17 nM) and did not detectably bind Cu(2+). Substitution of the sole histidine residue did not affect Co(2+) binding. Holo-AnhE had a weak absorption band at 490 nM (epsilon = 9.7 +/- 0.1 m(-1) cm(-1)), consistent with hexacoordinate cobalt. The data support a model in which AnhE acts as a dimeric metallochaperone to deliver cobalt to ANHase. This study provides insight into the maturation of NHases and metallochaperone function.

Simultaneous determination of 11 characteristic components in three species of Curcuma rhizomes using pressurized liquid extraction and high-performance liquid chromatography.

A high-performance liquid chromatography coupled with diode array detection (HPLC-DAD) and pressurized liquid extraction was developed for simultaneous quantitative determination of 11 characteristic compounds, including curcumenone, curcumenol, neocurdione, curdione, isocurcumenol, furanodienone, curcumol, germacrone, curzerene, furanodiene and beta-elemene, in rhizomes of three species of Curcuma. The analysis was performed on an ODS C18 column. The mobile phase consisted of (A) water and (B) acetonitrile using a gradient elution. The peaks were monitored at both 214 nm and 256 nm. All calibration curves showed good linearity (r2 > 0.9996) within test ranges. This method showed good repeatability for the quantification of these eleven components in three species Curcuma rhizomes with intra- and inter-day variations of less than 1.57% and 1.98%, respectively. The validated method was successfully applied to quantify 11 investigated components in eighteen samples of three species of Curcuma, which is helpful to control their quality.

Screening for new hydroxynitrilases from plants.

We established a simple HPLC method to determine the activity and stereochemistry of the chiral mandelonitrile synthesized from benzaldehyde and cyanide, and applied it to screen for hydroxynitrile lyase (HNL) activity of plant origin. A total of 163 species of plants among 74 families were examined for (R)- and (S)-HNL activities using the method. We discovered that homogenate of leaves of Baliospermum montanum shows (S)-HNL activity, while leaves and seeds from Passiflora edulis, and seeds from Eriobotrya japonica, Chaenomles sinensis, Sorbus aucuparia, Prunus mume, and Prunus persica show (R)-HNL activity. Partially purified (R)-HNLs from Passiflora edulis and Eriobotrya japonica acted not only on benzaldehyde but also on aliphatic ketone. The enantiomeric excess of (R)-methylpropylketone cyanohydrin synthesized from 2-pentanone using homogenate from leaves of Passiflora edulis was 87.0%, and that of (R)-mandelonitrile synthesized by homogenate from seeds of Eriobotrya japonica was 85.0%.

Reaction of beta-alkannin (shikonin) with reactive oxygen species: detection of beta-alkannin free radicals.

beta-Alkannin (shikonin), a compound isolated from the root of Lithospermum erythrorhizon Siebold Zucc., has been used as a purple dye in ancient Japan and is known to exert an anti-inflammatory activity. This study aimed to understand the biological activity in terms of physico-chemical characteristics of beta-alkannin. Several physico-chemical properties including proton dissociation constants, half-wave potentials and molecular orbital energy of beta-alkannin were elucidated. This compound shows highly efficient antioxidative activities against several types of reactive oxygen species (ROS), such as singlet oxygen ((1)O2). superoxide anion radical (.O2), hydroxyl radical (.OH) and tert-butyl peroxyl radical (BuOO.) as well as iron-dependent microsomal lipid peroxidation. During the reactions of beta-alkannin with 1O2, .O2- and BuOO., intermediate organic radicals due to beta-alkannin were detectable by ESR spectrometry. Compared with the radicals due to naphthazarin, the structural skeleton of beta-alkannin, the beta-alkannin radical observed as an intermediate in the reactions with (1)O2, and .O2- was concluded to be a semiquinone radical. On the other hand, during the reactions of beta-alkannin and naphthazarin with BuOO., ESR spectra different from the semiquinone radical were observed, and proposed to result from the abstraction of hydrogen atoms from phenolic hydroxyl groups of beta-alkannin by BuOO.. Based on the ROS-scavenging abilities of beta-alkannin, the compound was concluded to react directly with ROS and exhibits antioxidative activity, which in turn exerts anti-inflammatory activity.