NMR,HS‐SPME‐GC/MS,and HPLC/MSn Analyses of Phytoconstituents and Aroma Profile of Rosmarinus eriocalyx

In this work, a comprehensive study on the chemical constituents of the aerial parts of Rosmarinus eriocalyx (Lamiaceae), an aromatic shrub traditionally consumed as a food and herbal remedy in Algeria, is presented. The aroma profile was analysed by headspace solid phase microextraction (HS‐SPME) coupled with gas chromatography‐mass spectrometry (GC/MS), whereas the crude extract constituents were analyzed by ¹H‐NMR and by high performance liquid chromatography coupled with mass spectrometry (HPLC/MSⁿ). Thirty‐nine volatile compounds, most of them being monoterpenes, have been identified, with camphor, camphene, and α‐pinene as the most abundant constituents. ¹H‐NMR analysis revealed the presence of phenolic compounds and betulinic acid while HPLC/MSⁿ allowed the identification of glycosilated and aglyconic flavonoids as well as phenylpropanoid derivatives. Some of these constituents, namely as betulinic acid, rosmanol, and cirsimaritin were reported for the first time in R. eriocalyx.     

Isomerization of octadecapentaenoic acid (18:5n‐3) in algal lipid samples under derivatization for GC and GC‐MS analysis

During gas chromatography (GC) analysis of fatty acid (FA) composition of the dinoflagellate Gymnodinium kowalevskii, we found unex‐pectedly low and irreproducible content of all‐cis‐3,6,9,12,15‐octadecapentaenoic acid (18:5n‐3), which is an important chemotaxonomic marker of several classes of microalgae. We compared chromatographic behavior of 18:5n‐3 methyl ester and other GC derivatives obtained using different conventional methods of derivatization. The use of methods based on saponification or base‐catalyzed transesterification resulted in a mixture of double‐bond positional isomers of 18:5. On a SUPELCOWAX 10 column, the equivalent chain length (ECL) value for authentic 18:5n‐3 methyl ester was 20.22, whereas the main component after base‐catalyzed methylation had ECL 20.88. Attempts to prepare N‐acyl pyrrolidides or 4,4‐dimethyloxazoline (DMOX) derivatives of 18:5n‐3 also gave inadequate results. These derivatives also showed a main peak corresponding to isomerized 18:5. Mass spectra for both DMOX and pyrrolidide derivatives of this compound showed the base peak at m/z 139, probably corresponding to 2,6,9,12,15‐18:5 acid. Of all methods tested for methylation, only derivatization with 5% HCl or 1% sulphuric acid in methanol gave satisfactory results. Therefore, GC or GC‐mass spectrometry analyses of algal lipids containing 18:5n‐3 may be inaccurate when base‐catalyzed methods of FA derivatization are applied. The best and simplest way to avoid incorrect GC results is to use standard acid‐catalyzed methylation.     

Chemical profiling of mycosporine‐like amino acids in twenty‐three red algal species

Rhodophyta produce a variety of chemically different mycosporine‐like amino acids (MAAs), compounds that are known as some of the strongest ultraviolet (UV) absorbing molecules in nature. Accordingly, they primarily act as photoprotectants against harmful levels of solar ultraviolet radiation in the UV‐A and UV‐B range. In order to get a deeper understanding of the chemical diversity of MAAs in red algae, pure standards of eleven mycosporine‐like amino acids were isolated from three different species (Agarophyton chilense, Pyropia plicata and Champia novae‐zelandiae) using various chromatographic methods. Their structures were confirmed by nuclear magnetic resonance and mass spectrometry. Four out of the eleven MAAs are reported for the first time in algae. In addition, a new high‐performance liquid chromatography method was developed for the separation of all isolated MAAs and successfully applied for the analysis of twenty‐three red algal species of marine origin. All of them contained MAAs, the most abundant compounds were shinorine, palythine, asterina‐330 and porphyra‐334. For some samples, the direct assignment of MAAs based on their UV spectra was not possible; therefore, the target analytes were enriched by a simple concentration step, followed by liquid chromatography‐mass spectrometry analysis of the extracts. This approach enabled a deeper insight into the MAA pattern of red algae, indicating that not only the four dominant ones are synthesized but also many others, which were often described as unknown compounds in previous studies.     

The diversion of 2‐C‐methyl‐d‐erythritol‐2,4‐cyclodiphosphate from the 2‐C‐methyl‐d‐erythritol 4‐phosphate pathway to hemiterpene glycosides mediates stress responses in Arabidopsis thaliana

2‐C‐Methyl‐d ‐erythritol‐2,4‐cyclodiphosphate (MEcDP) is an intermediate of the plastid‐localized 2‐C‐methyl‐d ‐erythritol‐4‐phosphate (MEP) pathway which supplies isoprenoid precursors for photosynthetic pigments, redox co‐factor side chains, plant volatiles, and phytohormones. The Arabidopsis hds‐3 mutant, defective in the 1‐hydroxy‐2‐methyl‐2‐(E)‐butenyl‐4‐diphosphate synthase step of the MEP pathway, accumulates its substrate MEcDP as well as the free tetraol 2‐C‐methyl‐d ‐erythritol (ME) and glucosylated ME metabolites, a metabolic diversion also occurring in wild type plants. MEcDP dephosphorylation to the free tetraol precedes glucosylation, a process which likely takes place in the cytosol. Other MEP pathway intermediates were not affected in hds‐3. Isotopic labeling, dark treatment, and inhibitor studies indicate that a second pool of MEcDP metabolically isolated from the main pathway is the source of a signal which activates salicylic acid induced defense responses before its conversion to hemiterpene glycosides. The hds‐3 mutant also showed enhanced resistance to the phloem‐feeding aphid Brevicoryne brassicae due to its constitutively activated defense response. However, this MEcDP‐mediated defense response is developmentally dependent and is repressed in emerging seedlings. MEcDP and ME exogenously applied to adult leaves mimics many of the gene induction effects seen in the hds‐3 mutant. In conclusion, we have identified a metabolic shunt from the central MEP pathway that diverts MEcDP to hemiterpene glycosides via ME, a process linked to balancing plant responses to biotic stress.     

Structural basis of siRNA recognition by TRBP double‐stranded RNA binding domains

The accurate cleavage of pre‐micro(mi)RNAs by Dicer and mi/siRNA guide strand selection are important steps in forming the RNA‐induced silencing complex (RISC). The role of Dicer binding partner TRBP in these processes remains poorly understood. Here, we solved the solution structure of the two N‐terminal dsRNA binding domains (dsRBDs) of TRBP in complex with a functionally asymmetric siRNA using NMR, EPR, and single‐molecule spectroscopy. We find that siRNA recognition by the dsRBDs is not sequence‐specific but rather depends on the RNA shape. The two dsRBDs can swap their binding sites, giving rise to two equally populated, pseudo‐symmetrical complexes, showing that TRBP is not a primary sensor of siRNA asymmetry. Using our structure to model a Dicer‐TRBP‐siRNA ternary complex, we show that TRBP's dsRBDs and Dicer's RNase III domains bind a canonical 19 base pair siRNA on opposite sides, supporting a mechanism whereby TRBP influences Dicer‐mediated cleavage accuracy by binding the dsRNA region of the pre‐miRNA during Dicer cleavage.     

Lignin characterization of rice CONIFERALDEHYDE 5‐HYDROXYLASE loss‐of‐function mutants generated with the CRISPR/Cas9 system

The aromatic composition of lignin is an important trait that greatly affects the usability of lignocellulosic biomass. We previously identified a rice (Oryza sativa) gene encoding coniferaldehyde 5‐hydroxylase (OsCAld5H1), which was effective in modulating syringyl (S)/guaiacyl (G) lignin composition ratio in rice, a model grass species. Previously characterized OsCAld5H1‐knockdown rice lines, which were produced via an RNA‐interference approach, showed augmented G lignin units yet contained considerable amounts of residual S lignin units. In this study, to further investigate the effect of suppression of OsCAld5H1 on rice lignin structure, we generated loss‐of‐function mutants of OsCAld5H1 using the CRISPR/Cas9‐mediated genome editing system. Homozygous OsCAld5H1‐knockout lines harboring anticipated frame‐shift mutations in OsCAld5H1 were successfully obtained. A series of wet‐chemical and two‐dimensional NMR analyses on cell walls demonstrated that although lignins in the mutant were predictably enriched in G units all the tested mutant lines produced considerable numbers of S units. Intriguingly, lignin γ‐p‐coumaroylation analysis by the derivatization followed by reductive cleavage method revealed that enrichment of G units in lignins of the mutants was limited to the non‐γ‐p‐coumaroylated units, whereas grass‐specific γ‐p‐coumaroylated lignin units were almost unaffected. Gene expression analysis indicated that no homologous genes of OsCAld5H1 were overexpressed in the mutants. These data suggested that CAld5H is mainly involved in the production of non‐γ‐p‐coumaroylated S lignin units, common in both eudicots and grasses, but not in the production of grass‐specific γ‐p‐coumaroylated S units in rice.     

Metabolic products of [2‐13C]ethanol in the rat brain after chronic ethanol exposure

Most ingested ethanol is metabolized in the liver to acetaldehyde and then to acetate, which can be oxidized by the brain. This project assessed whether chronic exposure to alcohol can increase cerebral oxidation of acetate. Through metabolism, acetate may contribute to long‐term adaptation to drinking. Two groups of adult male Sprague–Dawley rats were studied, one treated with ethanol vapor and the other given room air. After 3 weeks the rats received an intravenous infusion of [2‐¹³C]ethanol via a lateral tail vein for 2 h. As the liver converts ethanol to [2‐¹³C]acetate, some of the acetate enters the brain. Through oxidation the ¹³C is incorporated into the metabolic intermediate α‐ketoglutarate, which is converted to glutamate (Glu), glutamine (Gln), and GABA. These were observed by magnetic resonance spectroscopy and found to be ¹³C‐labeled primarily through the consumption of ethanol‐derived acetate. Brain Gln, Glu, and, GABA ¹³C enrichments, normalized to ¹³C‐acetate enrichments in the plasma, were higher in the chronically treated rats than in the ethanol‐naïve rats, suggesting increased cerebral uptake and oxidation of circulating acetate. Chronic ethanol exposure increased incorporation of systemically derived acetate into brain Gln, Glu, and GABA, key neurochemicals linked to brain energy metabolism and neurotransmission.

     

Introduction of chemically labile substructures into Arabidopsis lignin through the use of LigD,the Cα‐dehydrogenase from Sphingobium sp. strain SYK‐6

Bacteria‐derived enzymes that can modify specific lignin substructures are potential targets to engineer plants for better biomass processability. The Gram‐negative bacterium Sphingobium sp. SYK‐6 possesses a Cα‐dehydrogenase (LigD) enzyme that has been shown to oxidize the α‐hydroxy functionalities in β–O–4‐linked dimers into α‐keto analogues that are more chemically labile. Here, we show that recombinant LigD can oxidize an even wider range of β–O–4‐linked dimers and oligomers, including the genuine dilignols, guaiacylglycerol‐β‐coniferyl alcohol ether and syringylglycerol‐β‐sinapyl alcohol ether. We explored the possibility of using LigD for biosynthetically engineering lignin by expressing the codon‐optimized ligD gene in Arabidopsis thaliana. The ligD cDNA, with or without a signal peptide for apoplast targeting, has been successfully expressed, and LigD activity could be detected in the extracts of the transgenic plants. UPLC‐MS/MS‐based metabolite profiling indicated that levels of oxidized guaiacyl (G) β–O–4‐coupled dilignols and analogues were significantly elevated in the LigD transgenic plants regardless of the signal peptide attachment to LigD. In parallel, 2D NMR analysis revealed a 2.1‐ to 2.8‐fold increased level of G‐type α‐keto‐β–O–4 linkages in cellulolytic enzyme lignins isolated from the stem cell walls of the LigD transgenic plants, indicating that the transformation was capable of altering lignin structure in the desired manner.     

Isolation and characterization of fatty acid methyl ester (FAME)‐producing Streptomyces sp. S161 from sheep (Ovis aries) faeces

An actinomycete producing oil‐like mixtures was isolated and characterized. The strain was isolated from sheep faeces and identified as Streptomyces sp. S161 based on 16S rRNA gene sequence analysis. The strain showed cellulase and xylanase activities. The ¹H nuclear magnetic resonance (NMR) spectra of the mixtures showed that the mixtures were composed of fatty acid methyl esters (52·5), triglycerides (13·7) and monoglycerides (9·1) (mol.%). Based on the gas chromatography–mass spectrometry (GC‐MS) analysis, the fatty acid methyl esters were mainly composed of C14‐C16 long‐chain fatty acids. The results indicated that Streptomyces sp. S161 could produce fatty acid methyl esters (FAME) directly from starch. To our knowledge, this is the first isolated strain that can produce biodiesel (FAME) directly from starch.

Significance and Impact of the Study

Nowadays, production of biodiesel is based on plant oils, animal fats, algal oils and microbial oils. Lipid mostly consists of triacylglycerols (TAG), and conversion of these lipids into fatty acid short‐chain alcohol esters (methanol or ethanol) is the final step in biodiesel production. In this study, an oil‐producing Streptomyces strain was isolated from sheep faeces. The oil was composed of C₁₄‐C₁₆ long‐chain fatty acid methyl esters, triglycerides and monoglycerides. This is the first isolated strain‐producing biodiesel (FAME) directly from starch. Due to showing cellulase and xylanase activities, the strain would be helpful for converting renewable lignocellulose into biodiesel directly.     

Structural determinants in GABARAP required for the selective binding and recruitment of ALFY to LC3B‐positive structures

Several autophagy proteins contain an LC3‐interacting region (LIR) responsible for their interaction with Atg8 homolog proteins. Here, we show that ALFY binds selectively to LC3C and the GABARAPs through a LIR in its WD40 domain. Binding of ALFY to GABARAP is indispensable for its recruitment to LC3B‐positive structures and, thus, for the clearance of certain p62 structures by autophagy. In addition, the crystal structure of the GABARAP‐ALFY‐LIR peptide complex identifies three conserved residues in the GABARAPs that are responsible for binding to ALFY. Interestingly, introduction of these residues in LC3B is sufficient to enable its interaction with ALFY, indicating that residues outside the LIR‐binding hydrophobic pockets confer specificity to the interactions with Atg8 homolog proteins.     

p‐Coumaroyl‐CoA:monolignol transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon

Grass lignins contain substantial amounts of p‐coumarate (pCA) that acylate the side‐chains of the phenylpropanoid polymer backbone. An acyltransferase, named p‐coumaroyl‐CoA:monolignol transferase (OsPMT), that could acylate monolignols with pCA in vitro was recently identified from rice. In planta, such monolignol‐pCA conjugates become incorporated into lignin via oxidative radical coupling, thereby generating the observed pCA appendages; however p‐coumarates also acylate arabinoxylans in grasses. To test the authenticity of PMT as a lignin biosynthetic pathway enzyme, we examined Brachypodium distachyon plants with altered BdPMT gene function. Using newly developed cell wall analytical methods, we determined that the transferase was involved specifically in monolignol acylation. A sodium azide‐generated Bdpmt‐1 missense mutant had no (<0.5%) residual pCA on lignin, and BdPMT RNAi plants had levels as low as 10% of wild‐type, whereas the amounts of pCA acylating arabinosyl units on arabinoxylans in these PMT mutant plants remained unchanged. pCA acylation of lignin from BdPMT‐overexpressing plants was found to be more than three‐fold higher than that of wild‐type, but again the level on arabinosyl units remained unchanged. Taken together, these data are consistent with a defined role for grass PMT genes in encoding BAHD (BEAT, AHCT, HCBT, and DAT) acyltransferases that specifically acylate monolignols with pCA and produce monolignol p‐coumarate conjugates that are used for lignification in planta.     

Qualitative HPLC‐DAD/ESI‐TOF‐MS Analysis,Cytotoxic, and Apoptotic Effects of Croatian Endemic Centaurea ragusina L. Aqueous Extracts

Centaurea ragusina L., an endemic Croatian plant species, revealed a good cytotoxic activity of aqueous extracts (AE) on human bladder (T24) and human glioblastoma (A1235) cancer cell lines. The chemical constituents were tentatively identified using high performance liquid chromatography HPLC‐DAD/ESI‐TOF‐MS in negative ionization mode. The main compounds of herba extract were sesquiterpene lactones: solstitialin A 3,13‐diacetate and epoxyrepdiolide; organic acid: quinic acid. The main compounds of flower extract were organic acids: quinic acid, citric acid, and malic acid; sesquiterpene lactone: cynaropicrin; phenolic compounds: chlorogenic acid and phenylpropanoid: syringin. The AE of C. ragusina were investigated for correlation of their effects on human bladder (T24) and human glioblastoma (A1235) cancer cell lines using the MTT assay. Although both extracts showed significant dose‐ and time‐dependent cytotoxic activity against both cancer cell lines, the flower extract exhibited slightly higher activity. In order to determine type of cell death induced by treatment, cell lines were exposed subsequently to a treatment with both flower and herba AE. The majority of the cells died by induced apoptosis treatment. Flower AE (26.25%), compared to a leaf AE (22.15%) showed slightly higher percentage of an apoptosis in T24 cells, when compared to a non‐treated cells (0.04%).