Main conclusion The DNA-binding protein WHIRLY1, sharing structural similarities with ferritin, plays a role in the formation of iron cofactor proteins within chloroplasts. Abstract Previous studies indicated that barley plants with a knockdown of HvWHIRLY1 containing a minimal amount of the protein are compromised in chloroplast development and photosynthesis, and get chlorotic leaves when grown at high irradiance. Thereby, the leaves display signs of iron deficiency. Metal determination revealed, however, that leaves of WHIRLY1-deficient plants had a regular iron content. Nevertheless, WHIRLY1-deficiency affected the functionality of photosystem II less than that of photosystem I, which has a higher demand for iron. Immunological analyses revealed that components of both photosystems had reduced levels. Additionally, the levels of other chloroplast proteins containing different classes of iron cofactors were lower in the WHIRLY1-deficient plants compared to the wild type. In contrast, the level of the iron sequestering protein ferritin increased in WHIRLY1-deficient lines, whereby high irradiance intensified this effect. RNA analyses showed that the upregulation of ferritin coincided with an enhanced expression of the corresponding gene, reflecting an apparent overload of chloroplasts with free iron. Ferritin and WHIRLY proteins are known to share the same oligomeric structure. Therefore, the high abundance of ferritin in WHIRLY1-deficient plants might be a compensation for the reduced abundance of WHIRLY1. Enhanced expression levels of genes encoding photosynthesis proteins and iron cofactor proteins indicate a demand for protein formation or assembly of protein complexes. The results support a general role of WHIRLY1 in assembly and/or stabilization of chloroplast proteins and, moreover, suggest a specific function in sequestering and supply of iron in chloroplasts.

Piper nigrum L. (black and white peppercorn) is one of the most common culinary spices used worldwide. The current study aims to dissect pepper metabolome using 1H-NMR targeting of its major primary and secondary metabolites. Eighteen metabolites were identified with piperine detected in black and white pepper at 20.2 and 23.9 mg mg−1, respectively. Aroma profiling using HS-SPME coupled to GC-MS analysis and in the context of autoclave treatment led to the detection of a total of 52 volatiles with an abundance of b-caryophyllene at 82% and 59% in black and white pepper, respectively. Autoclaving of black and white pepper revealed improvement of pepper aroma as manifested by an increase in oxygenated compounds\' level. In vitro remote antimicrobial activity against food-borne Gram-positive and Gram-negative bacteria revealed the highest activity against P. aeruginosa (VP-MIC 16.4 and 12.9 mg mL−1) and a direct effect against Enterobacter cloacae at ca. 11.6 mg mL−1 for both white and black pepper.

Balanites aegyptiaca (L.) Delile (Zygophyllaceae), also known as the desert date, is an edible fruit-producing tree popular for its nutritional and several health benefits. In this study, multi-targeted comparative metabolic profiling and fingerprinting approaches were conducted for the assessment of the nutrient primary and secondary metabolite heterogeneity in different parts, such as leaves, stems, seeds, unripe, and ripe fruits of B. aegyptiaca using nuclear magnetic resonance (NMR), ultra-performance liquid chromatography (UPLC-MS), and gas chromatography mass-spectrometry (GC-MS) based metabolomics coupled to multivariate analyses and in relation to its cytotoxic activities. NMR-based metabolomic study identified and quantified 15 major primary and secondary metabolites belonging to alkaloids, saponins, flavonoids, sugars, and amino and fatty acids. Principal component analysis (PCA) of the NMR dataset revealed α-glucose, sucrose, and isorhamnetin as markers for fruit and stem and unsaturated fatty acids for predominated seeds. Orthogonal projections to latent structure discriminant analysis (OPLS-DA) revealed trigonelline as a major distinctive metabolite in the immature fruit and isorhamnetin as a major distinct marker in the mature fruit. UPLC-MS/MS analysis using feature-based molecular networks revealed diverse chemical classes viz. steroidal saponins, N-containing metabolites, phenolics, fatty acids, and lipids as the constitutive metabolome in Balanites. Gas chromatography-mass spectroscopy (GC-MS) profiling of primary metabolites led to the detection of 135 peaks belonging to sugars, fatty acids/esters, amino acids, nitrogenous, and organic acids. Monosaccharides were detected at much higher levels in ripe fruit and disaccharides in predominate unripe fruits, whereas B. aegyptiaca vegetative parts (leaves and stem) were rich in amino acids and fatty acids. The antidiabetic compounds, viz, nicotinic acid, and trigonelline, were detected in all parts especially unripe fruit in addition to the sugar alcohol D-pinitol for the first time providing novel evidence for B. aegyptiaca use in diabetes. In vitro cytotoxic activity revealed the potential efficacy of immature fruit and seeds as cytotoxic agents against human prostate cancer (PC3) and human colorectal cancer (HCT-116) cell lines. Collectively, such detailed profiling of parts provides novel evidence for B. aegyptiaca medicinal uses.

The tree species Eucalyptus camaldulensis shows exceptionally high tolerance against aluminum, a widespread toxic metal in acidic soils. In the roots of E. camaldulensis, aluminum is detoxified via the complexation with oenothein B, a hydrolyzable tannin. In our approach to elucidate the biosynthesis of oenothein B, we here report on the identification of E. camaldulensis enzymes that catalyze the formation of gallate, which is the phenolic constituent of hydrolyzable tannins. By systematical screening of E. camaldulensis dehydroquinate dehydratase/shikimate dehydrogenases (EcDQD/SDHs), we found two enzymes, EcDQD/SDH2 and 3, catalyzing the NADP+-dependent oxidation of 3-dehydroshikimate to produce gallate. Based on extensive in vitro assays using recombinant EcDQD/SDH2 and 3 enzymes, we present for the first time a detailed characterization of the enzymatic gallate formation activity, including the cofactor preferences, pH optima, and kinetic constants. Sequence analyses and structure modeling suggest the gallate formation activity of EcDQD/SDHs is based on the reorientation of 3-dehydroshikimate in the catalytic center, which facilitates the proton abstraction from the C5 position. Additionally, EcDQD/SDH2 and 3 maintain DQD and SDH activities, resulting in a 3-dehydroshikimate supply for gallate formation. In E. camaldulensis, EcDQD/SDH2 and 3 are co-expressed with UGT84A25a/b and UGT84A26a/b involved in hydrolyzable tannin biosynthesis. We further identified EcDQD/SDH1 as a “classical” bifunctional plant shikimate pathway enzyme and EcDQD/SDH4a/b as functional quinate dehydrogenases of the NAD+/NADH-dependent clade. Our data indicate that in E. camaldulensis the enzymes EcDQD/SDH2 and 3 provide the essential gallate for the biosynthesis of the aluminum-detoxifying metabolite oenothein B.

Herbs of the Umbelliferae family are popular spices valued worldwide for their many nutritional and health benefits. Herein, five chief umbelliferous fruits viz., cumin, fennel, anise, coriander and caraway were assessed for its secondary metabolites diversity along with compositional changes incurring upon roasting as analyzed via ultra-high performance liquid chromatography coupled to photodiode array and electrospray ionization mass detectors UHPLC-qToF/MS. A total of 186 metabolites were annotated, according to metabolomics society guidelines, belonging mainly to flavonoids, fatty acids and phenolic acids. Multivariate models viz., PCA, HCA and OPLS-DA were further employed to assess fruits\' heterogeneity in an untargeted manner and determine mechanistic changes in bioactive makeup post roasting viz., glycosidic cleavage, lipid degradation and Maillard reaction. Finally, the fruits\' antioxidant activity showed decline upon roasting and in correlation with its total phenolic content. This study presents the first complete map of umbelliferous fruit metabolome, compositional differences and its roasting effect.

Main conclusionSolanum tuberosum tropinone reductase I reduced tropinone in vivo. Suppression of tropinone reductase II strongly reduced calystegines in sprouts. Overexpression of putrescine N -methyltransferase did not alter calystegine accumulation.Calystegines are hydroxylated alkaloids formed by the tropane alkaloid pathway. They accumulate in potato (Solanum tuberosum L., Solanaceae) roots and sprouting tubers. Calystegines inhibit various glycosidases in vitro due to their sugar-mimic structure, but functions of calystegines in plants are not understood. Enzymes participating in or competing with calystegine biosynthesis, including putrescine N-methyltransferase (PMT) and tropinone reductases (TRI and TRII), were altered in their activity in potato plants by RNA interference (RNAi) and by overexpression. The genetically altered potato plants were investigated for the accumulation of calystegines and for intermediates of their biosynthesis. An increase in N-methylputrescine provided by DsPMT expression was not sufficient to increase calystegine accumulation. Overexpression and gene knockdown of StTRI proved that S. tuberosum TRI is a functional tropinone reductase in vivo, but no influence on calystegine accumulation was observed. When StTRII expression was suppressed by RNAi, calystegine formation was severely compromised in the transformed plants. Under phytochamber and green house conditions, the StTRII RNAi plants did not show phenotypic alterations. Further investigation of calystegines function in potato plants under natural conditions is enabled by the calystegine deprived StTRII RNAi plants.

A methodology that brings together sugar and steroid scaffolds linked by a selenium atom is discussed in this work. A series of 6β and 3α glycoconjugated steroids were achieved by stereoselective nucleophilic substitution of cholesterol, pregnenolone, stigmasterol and sitosterol with different seleno-pyranosides and furanosides.

The anticancer drug cisplatin (CP) is loaded into SBA-15 mesoporous silica (SBA-15|CP) and its release from the nanomaterial is studied. The CP-loaded SBA-15 is tested against four tumor cell lines: mouse malignant melanoma B16F10, human adenocarcinoma HeLa, colon HT-29 and prostate PC3. Most importantly, the superiority of this novel material in comparison to CP arises from the fact that the CP-grafted nanomaterial SBA-15 (→SBA-15|CP) is enhancing cessation of proliferation along with induction of senescence in B16F10 in approximately 3.5 times lower concentration. The control material loaded with therapeutically inactive K2[PtCl4] (→SBA-15|TC) showed no antitumor activity. To a large extent, SBA-15|CP-induced senescence might present a safe approach in tumor treatment. Such cells can be cleared by immune cells resulting in efficient tumor regression. So far only apoptotic agents are being exploited in clinics, thus an understanding of the chemotherapeutic-induced senescence will allow oncologists to explore this essential tumor suppressor mechanism.

A methodology that brings together sugar and steroid scaffolds linked by a selenium atom is discussed in this work. A series of 6β and 3α glycoconjugated steroids were achieved by stereoselective nucleophilic substitution of cholesterol, pregnenolone, stigmasterol and sitosterol with different seleno-pyranosides and furanosides.

The anticancer drug cisplatin (CP) is loaded into SBA-15 mesoporous silica (SBA-15|CP) and its release from the nanomaterial is studied. The CP-loaded SBA-15 is tested against four tumor cell lines: mouse malignant melanoma B16F10, human adenocarcinoma HeLa, colon HT-29 and prostate PC3. Most importantly, the superiority of this novel material in comparison to CP arises from the fact that the CP-grafted nanomaterial SBA-15 (→SBA-15|CP) is enhancing cessation of proliferation along with induction of senescence in B16F10 in approximately 3.5 times lower concentration. The control material loaded with therapeutically inactive K2[PtCl4] (→SBA-15|TC) showed no antitumor activity. To a large extent, SBA-15|CP-induced senescence might present a safe approach in tumor treatment. Such cells can be cleared by immune cells resulting in efficient tumor regression. So far only apoptotic agents are being exploited in clinics, thus an understanding of the chemotherapeutic-induced senescence will allow oncologists to explore this essential tumor suppressor mechanism.