Varying the expression pattern of the strigolactone receptor gene DAD2 results in phenotypes distinct from both wild type and knockout mutants.

The action of the petunia strigolactone (SL) hormone receptor DAD2 is dependent not only on its interaction with the PhMAX2A and PhD53A proteins, but also on its expression patterns within the plant. Previously, in a yeast-2-hybrid system, we showed that a series of a single and double amino acid mutants of DAD2 had altered interactions with these binding partners. In this study, we tested the mutants in two plant systems, Arabidopsis and petunia. Testing in Arabidopsis was enabled by creating a CRISPR-Cas9 knockout mutant of the Arabidopsis strigolactone receptor (AtD14). We produced SL receptor activity in both systems using wild type and mutant genes; however, the mutants had functions largely indistinguishable from those of the wild type. The expression of the wild type DAD2 from the CaMV 35S promoter in dad2 petunia produced plants neither quite like the dad2 mutant nor the V26 wild type. These plants had greater height and leaf size although branch number and the plant shape remained more like those of the mutant. These traits may be valuable in the context of a restricted area growing system such as controlled environment agriculture.

Is mushroom polysaccharide extract a better fat replacer than dried mushroom powder for food applications?

Introduction: ß-glucans found in the cell walls of mushrooms can be a beneficial food additive in replacing fat in commercial food products. Methods: Four commonly consumed mushroom species in Singapore, i.e., Pleurotus ostreatus spp., Lentinus edodes, Agaricus bisporus, and Flammulina velutipes were profiled for the ß-glucan content in the lyophilized form and ultrasonicated assisted extracted form. Both forms were added into chicken patties, which were characterized for the moisture, cooking loss, texture, color, and chemically analyzed for the protein, crude fat, and fatty acid profiles with gas chromatography mass spectrometry (GC-MS). Results and discussion: Pleurotus Ostreatus spp. had the highest ß-glucan of 29.8 ± 0.7 g/100 g in the pure powder form and 15.9 ± 0.3 g/100 g from the extract. Crude fat in 100% fat substituted patties was lowest in Flammulina velutipes extract enriched patties and least in A. bisporus pure powder patties. Additionally, fat replacement with A. bisporus extract and powder forms resulted in the highest polyunsaturated fatty acid profile of 49.6 ± 1.9 mg/100 g patty and 79.9 ± 4.5 mg/100 g patty, respectively. Chicken patties with added mushroom extract were notable in retaining moisture, cooking yield and its structure. Fat substitution with mushroom powder was also conducted, satisfactory results indicated a possibility as a better fat replacer that is easily processed and an efficient alternative to ß-glucan extract. With increasing demand for low fat foods with acceptable organoleptic properties, our study demonstrates that the inclusion of dry mushroom powder has the ability to mimic the "fattiness" of chicken patties.

A Rapid qPCR for the Detection of Verticillium nonalfalfae MLST2 - A Highly Pathogenic Fungus on Kiwifruit.

A highly pathogenic fungus characterized as Verticillium nonalfalfae multilocus sequence type 2 (MLST2) is an emerging fungal pathogen causing Verticillium wilt on kiwifruit. Although V. nonalfalfae MLST2 has not been reported outside Chile, there is a risk that this pathogen could spread through the global movement of germplasms to other countries. Current diagnostic methods for this fungus rely on a laborious and time-consuming plating assay for morphological identification and DNA sequence analysis. In this study, we describe the development and validation of a novel quantitative polymerase chain reaction (qPCR) assay for rapid and specific detection of V. nonalfalfae MLST2 in plant tissues. The assay targets the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene and was shown to detect all tested isolates of V. nonalfalfae MLST2 with a detection limit of approximately 2 pg of pathogen genomic DNA. There was no cross-reaction with V. nonalfalfae MLST1, other Verticillium species, or non-target fungal species found on kiwifruit. This assay was duplexed with a plant internal control for simultaneous detection of the pathogen and cytochrome oxidase gene from the host plant. This new specific and sensitive qPCR assay is a valuable molecular diagnostic tool for rapid screening of imported plant material and would also be useful for testing samples collected from field surveillance activities to monitor the presence of V. nonalfalfae MLST2.

Physicochemical and functional properties of red lentil protein isolates from three origins at different pH.

Red lentils (Lens culinaris) present an attractive raw material for meat mimics due to its red-coloured proteins, abundance, high protein and low cost. However, data on its functional properties at various pH remain scarce. In this study, the physicochemical and functional properties of red lentil proteins (RLP) from three origins (USA, Nepal and Turkey), isolated by isoelectric precipitation, were evaluated. Amino acid profiles, water holding (ranging from 3.1 to 3.5 g/g) and oil absorption (ranging from 5.8 to 7.3 g/g) capacities of RLP samples were significantly different (p < 0.05). RLP consisted of legumin and vicilin, and comprised predominantly glutamine/glutamic acid (ranging from 8.72 to 10.55 g/100 g). Surface charge, protein solubility, foaming and emulsifying properties were the lowest and poorest at pH 5.2 (isoelectric point). Overall, good functional properties of RLP under high acidity and alkalinity conditions make it a promising protein for mimicking a wide range of meats.

Nutritional metabolites in Brassica rapa subsp. chinensis var. parachinensis (choy sum) at three different growth stages: Microgreen, seedling and adult plant.

Choy sum is a commonly consumed Asian green leafy brassica vegetable. A comprehensive spectrum of nutritional important metabolites, including amino acids, plant sugars, essential minerals, vitamins (A, B9, E, and K1) and glucosinolates were systematically quantified using LC-QQQ-MS, GC-QQQ-MS and ICP-MS. Significant metabolic profile shifts were observed during the three major developmental stages (microgreen, seedling and adult) studied. Primary metabolites, especially essential amino acids decreased while most plant sugars increased from microgreens to seedlings. Carotenoids, such as violaxanthin, neoxanthin, together with vitamin K1 were higher in the seedlings whereas CHO-folate vitamers and ß-cryptoxanthin were much lower in adult plants. Most essential minerals were concentrated in the microgreens, while sodium increased in adult plants. Aliphatic glucosinolates in microgreens were converted to indolic glucosinolates in the seedlings and further to aromatic glucosinolates in the adults. Overall findings reveal that most of the nutritional metabolites were concentrated either in the microgreens or seedlings.

Datasets on spatial and temporal distribution of heavy metals concentration in recent sediment at merang river system, Terengganu, Malaysia.

Heavy metal pollution in an aquatic environment has become of the main concern to the world due to their non-biodegradable properties, toxicity, persistence, and their ability to adsorb into food chains. With rapid industrialization and development nowadays, heavy metals are introduced continuously into the estuaries and coastal region through rivers, runoff, and land-based point sources. These heavy metals may degrade the aquatic environment and harm the living organisms and toward human indirection through secondary contact. The dataset provided is to give an overview of the spatial and temporal distribution of the heavy metals concentration in Merang River surficial sediment collected from September 2017 to July 2018, subsequently every two months dataset. Sediment samples were collected in 44 stations along the river and 20 stations in the coastal area, which total up to 64 stations. Teflon Bomb closed digestion method with mixed acid was used to digest the sediments. The concentration of heavy metals in the sediment were analysed by using Inductively Coupled Plasma Mass Spectrometer (ICP-MS). The spatial distribution of heavy metals shows the effect of monsoon and wet and dry seasons in the sampling area. Thus, this dataset reveals six months of information on natural and anthropogenic sources intrusion at the Merang River and may also help in monitoring the pollution in the area.

Flexibility of the petunia strigolactone receptor DAD2 promotes its interaction with signaling partners.

Strigolactones (SLs) are terpenoid-derived plant hormones that regulate various developmental processes, particularly shoot branching, root development, and leaf senescence. The SL receptor has an unusual mode of action. Upon binding SL, it hydrolyzes the hormone, and then covalently binds one of the hydrolytic products. These initial events enable the SL receptor DAD2 (in petunia) to interact with the F-box protein PhMAX2A of the Skp-Cullin-F-box (SCF) complex and/or a repressor of SL signaling, PhD53A. However, it remains unclear how binding and hydrolysis structurally alters the SL receptor to enable its engagement with signaling partners. Here, we used mutagenesis to alter DAD2 and affect SL hydrolysis or DAD2's ability to interact with its signaling partners. We identified three DAD2 variants whose hydrolytic activity had been separated from the receptor's interactions with PhMAX2A or PhD53A. Two variants, DAD2N242I and DAD2F135A, having substitutions in the core α/ß hydrolase-fold domain and the hairpin, exhibited hormone-independent interactions with PhMAX2A and PhD53A, respectively. Conversely, the DAD2D166A variant could not interact with PhMAX2A in the presence of SL, but its interaction with PhD53A remained unaffected. Structural analyses of DAD2N242I and DAD2D166A revealed only small differences compared with the structure of the WT receptor. Results of molecular dynamics simulations of the DAD2N242I structure suggested that increased flexibility is a likely cause for its SL-independent interaction with PhMAX2A. Our results suggest that PhMAX2A and PhD53A have distinct binding sites on the SL receptor and that its flexibility is a major determinant of its interactions with these two downstream regulators.

Chemical synthesis and characterization of a new quinazolinedione competitive antagonist for strigolactone receptors with an unexpected binding mode.

Strigolactones (SLs) are multifunctional plant hormones regulating essential physiological processes affecting growth and development. In vascular plants, SLs are recognized by α/ß hydrolase-fold proteins from the D14/DAD2 (Dwarf14/Decreased Apical Dominance 2) family in the initial step of the signaling pathway. We have previously discovered that N-phenylanthranilic acid derivatives (e.g. tolfenamic acid) are potent antagonists of SL receptors, prompting us to design quinazolinone and quinazolinedione derivatives (QADs and QADDs, respectively) as second-generation antagonists. Initial in silico docking studies suggested that these compounds would bind to DAD2, the petunia SL receptor, with higher affinity than the first-generation compounds. However, only one of the QADs/QADDs tested in in vitro assays acted as a competitive antagonist of SL receptors, with reduced affinity and potency compared with its N-phenylanthranilic acid 'parent'. X-ray crystal structure analysis revealed that the binding mode of the active QADD inside DAD2's cavity was not that predicted in silico, highlighting a novel inhibition mechanism for SL receptors. Despite a ∼10-fold difference in potency in vitro, the QADD and tolfenamic acid had comparable activity in planta, suggesting that the QADD compensates for lower potency with increased bioavailability. Altogether, our results establish this QADD as a novel lead compound towards the development of potent and bioavailable antagonists of SL receptors.

Characterization of Plant Volatiles Reveals Distinct Metabolic Profiles and Pathways among 12 Brassicaceae Vegetables.

Plants emit characteristic organic volatile compounds (VOCs) with diverse biological/ecological functions. However, the links between plant species/varieties and their phytochemical emission profiles remain elusive. Here, we developed a direct headspace solid-phase microextraction (HS-SPME) technique and combined with non-targeted gas chromatography‒high-resolution mass spectrometry (GC-HRMS) platform to investigate the VOCs profiles of 12 common Brassicaceae vegetables (watercress, rocket, Brussels sprouts, broccoli, kai lan, choy sum, pak choi, cabbage, Chinese cabbage, cauliflower, radish and cherry radish). The direct HS-SPME sampling approach enabled reproducible capture of the rapid-emitting VOCs upon plant tissue disruption. The results revealed extensive variation in VOCs profiles among the 12 Brassicaceae vegetables. Furthermore, principal component analysis (PCA) showed that the VOC profiles could clearly distinguish the 12 Brassicaceae vegetables, and that these profiles well reflected the classical morphological classification. After multivariate statistical analysis, 44 VOCs with significant differences among the Brassicaceae vegetables were identified. Pathway analysis showed that three secondary metabolism pathways, including the fatty acid pathway, methylerythritol phosphate (MEP) pathway and glucosinolate (GLS) pathway, behave distinctively in these vegetables. These three pathways are responsible for the generation and emission of green leaf volatiles (GLVs), terpenes and isothiocyanates (ITCs), respectively. Correlation analysis further showed that volatile metabolites formed via the common pathway had significantly positive correlations, whereas metabolites from different pathways had either non-significant or significantly negative correlations. Genetic influences on these metabolites across various vegetable types were also evaluated. These findings extend our phytochemical knowledge of the 12 edible Brassicaceae vegetables and provide useful information on their secondary metabolism.

Profiling of Phenolic Compounds and Antioxidant Activity of 12 Cruciferous Vegetables.

The phenolic profiles of 12 cruciferous vegetables (pakchoi, choysum, Chinese cabbage, kailan, Brussels sprout, cabbage, cauliflower, broccoli, rocket salad, red cherry radish, daikon radish, and watercress) were studied with UHPLC-MS/MS. Antioxidant activity and total phenolic content (TPC) were also evaluated. A total of 74 phenolic compounds were identified, including 16 hydroxycinnamic acids and derivatives, and 58 flavonoids and derivatives. The main flavonoids identified were glycosylated quercetin, kaempferol and isorhamnetin, and the main hydroxycinnamic acids were ferulic, sinapic, caffeic and p-coumaric acids. Principal component analysis (PCA) revealed that the distribution of phenolic compounds in different genera of cruciferous vegetables was in accordance with their conventional taxonomy. The DPPH, ORAC and TPC values ranged from 1.11 to 9.54 µmoles Trolox equivalent/g FW, 5.34 to 32.92 µmoles Trolox equivalent/g FW, and 0.16 to 1.93 mg gallic acid equivalent/g FW respectively. Spearman's correlation showed significant (p < 0.05) positive correlations between TPC, flavonoids and antioxidant activity.

Inhibition of strigolactone receptors by N-phenylanthranilic acid derivatives: Structural and functional insights.

The strigolactone (SL) family of plant hormones regulates a broad range of physiological processes affecting plant growth and development and also plays essential roles in controlling interactions with parasitic weeds and symbiotic fungi. Recent progress elucidating details of SL biosynthesis, signaling, and transport offers many opportunities for discovering new plant-growth regulators via chemical interference. Here, using high-throughput screening and downstream biochemical assays, we identified N-phenylanthranilic acid derivatives as potent inhibitors of the SL receptors from petunia (DAD2), rice (OsD14), and Arabidopsis (AtD14). Crystal structures of DAD2 and OsD14 in complex with inhibitors further provided detailed insights into the inhibition mechanism, and in silico modeling of 19 other plant strigolactone receptors suggested that these compounds are active across a large range of plant species. Altogether, these results provide chemical tools for investigating SL signaling and further define a framework for structure-based approaches to design and validate optimized inhibitors of SL receptors for specific plant targets.

Simultaneous Quantification of 22 Glucosinolates in 12 Brassicaceae Vegetables by Hydrophilic Interaction Chromatography-Tandem Mass Spectrometry.

Glucosinolates, which are unique to Brassicaceae vegetables, have diverse biological activities, including antimicrobial, antioxidant, and anticancer actions. In this study, we applied hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS) to the simultaneous quantification of 22 glucosinolates in 12 Brassicaceae vegetables, including pak choi, choy sum, Chinese cabbage, cauliflower, cabbage, broccoli, Kai Lan, Brussels sprouts, rocket salad, daikon radish, red cherry radish, and watercress. Significant differences in concentration and composition of glucosinolates were observed among these vegetables. Cabbage had the highest level of total glucosinolates (µg/g dry weight: 19 551.2 ± 1317.7), whereas Kai Lan had the lowest level (7611.3 ± 868.4). Aliphatic and indole glucosinolates were the major components in the 12 vegetables ranging from 76 to 100%, except watercress (37%). On the basis of the content of glucosinolates, the 12 vegetables were well distinguishable and classified according to their morphological taxonomy. This study presents a HILIC-MS/MS approach for quantification of glucosinolates, and demonstrates the potential of glucosinolate profiles for Brassicaceae species identification.

Antimicrobial efficacy of Cinnamomum javanicum plant extract against Listeria monocytogenes and its application potential with smoked salmon.

Extracts from medicinal plants have been reported to possess good antimicrobial properties, but a majority of them remain unexplored. This study aimed at identifying a novel plant extract with antimicrobial activity, to validate its efficacy in food model, and to elucidate its composition and antimicrobial mechanism. A total of 125 plant extracts were screened, and Cinnamomum javanicum leaf and stem extract showed potential antimicrobial activity against Listeria monocytogenes (MIC=0.13mg/mL). Total phenolic content of the extract was 78.3mg GAE/g extract and its antioxidant activity was 57.2-326.5mg TE/g extract. When applied on cold smoked salmon, strong strain-dependent antimicrobial effectiveness was observed, with L. monocytogenes LM2 (serotype 4b) and LM8 (serotype 3a) being more resistant compared to SSA81 (serotype 1/2a). High extract concentration (16mg/mL) was needed to inhibit or reduce the growth of L. monocytogenes on smoked salmon, which resulted in surface color change. GC-MS revealed that eucalyptol (25.54 area%) was the most abundant compound in the crude extract. Both crude extract and eucalyptol induced significant membrane damages in treated L. monocytogenes. These results suggest anti-L. monocytogenes activity of C. javanicum plant extract, identified its major volatile components, and elucidated its membrane-damaging antimicrobial mechanisms.

Dissecting structural transitions in the HIV-1 dimerization initiation site RNA using 2-aminopurine fluorescence.

A highly conserved 35 nucleotide RNA stem-loop, the dimerization initiation site (DIS), in the 5' untranslated region (UTR) of the human immunodeficiency virus type I (HIV-1) genome has been identified as the sequence primarily responsible for initiation of viral genome dimerization. The DIS initiates viral genome dimerization through a loop-loop 'kissing' interaction and is converted from an intermediate 'kissing' to a more thermodynamically stable extended duplex dimer in a conformational rearrangement that is chaperoned by the HIV-1 nucleocapsid protein (NCp7). Here we describe fluorescence methods designed to probe local RNA dynamics and structural transitions associated with the DIS dimer formation and its NCp7 chaperoned structural conversion. These methods take advantage of the exquisite sensitivity of the quantum yield of the fluorescent nucleotide base analog, 2-aminopurine (2-AP), to its immediate structural and dynamic environment. The 2-AP fluorescence methods described allow a detailed kinetic and thermodynamic examination of this type of RNA-RNA interaction, as well as an analysis of the molecular mechanism of NCp7 chaperone activity.