Proteomic analysis of Arabidopsis thaliana (L.) Heynh responses to a generalist sucking pest (Myzus persicae Sulzer)

Herbivorous insects can cause severe cellular changes to plant foliage following infestations, depending on feeding behaviour. Here, a proteomic study was conducted to investigate the influence of green peach aphid (Myzus persicae Sulzer) as a polyphagous pest on the defence response of Arabidopsis thaliana (L.) Heynh after aphid colony establishment on the host plant (3 days). Analysis of about 574 protein spots on 2‐DE gels revealed 31 differentially expressed protein spots. Twenty out of these 31 differential proteins were selected for analysis by mass spectrometry. In 12 of the 20 analysed spots, we identified seven and nine proteins using MALDI‐TOF‐MS and LC‐ESI‐MS/MS, respectively. Of the analysed spots, 25% contain two proteins. Different metabolic pathways were modulated in Arabidopsis leaves according to aphid feeding: most corresponded to carbohydrate, amino acid and energy metabolism, photosynthesis, defence response and translation. This paper has established a survey of early alterations induced in the proteome of Arabidopsis by M. persicae aphids. It provides valuable insights into the complex responses of plants to biological stress, particularly for herbivorous insects with sucking feeding behaviour.     

UHPLC/HR‐ESI‐MS/MS Profiling of Phenolics from Tunisian Lycium arabicum Boiss. Antioxidant and Anti‐lipase Activities’ Evaluation

This study was performed in the aim to evaluate nine different extracts from Tunisian Lycium arabicum for their total phenolic and total flavonoid contents, phytochemical analyses as well as their antioxidant and anti‐lipase activities. The in vitro antioxidant property was investigated using three complementary methods (DPPH, ferric reducing antioxidant power (FRAP), and β‐carotene‐linoleic acid bleaching assays) while anti‐lipase activity was evaluated using 4‐methylumbelliferyl oleate method. From all of the tested extracts the most potent found to be the polar MeOH extracts especially those of stems and leaves. In order to investigate the chemical composition of these extracts and possible correlation of their constituents with the observed activities, an UHPLC/HR‐ESI‐MS/MS analysis was performed. Several compounds belonging to different chemical classes were tentatively identified such as rutin and kampferol rutinoside, the major constituents of the leaves, and N‐caffeoyltyramine, lyciumide A, N‐dihydrocaffeoyltyramine as well as fatty acids: trihydroxyoctadecadienoic acid and hydroxyoctadecadienoic acid isomers were detected abundantly in the stems. These results showed that the MeOH extracts of stems and leaves of L. arabicum can be considered as a potential source of biological active compounds.     

Chemical Diversity and Antimicrobial Activity of Volatile Compounds from Zanthoxylum zanthoxyloides Lam. according to Compound Classes,Plant Organs and Senegalese Sample Locations

The chemical diversity of Zanthoxylum zanthoxyloides growing wild in Senegal was studied according to volatile compound classes, plant organs and sample locations. The composition of fruit essential oil was investigated using an original targeted approach based on the combination of gas chromatography (GC) and liquid chromatography (LC) both coupled with mass spectrometry (MS). The volatile composition of Z. zanthoxyloides fruits exhibited relative high amounts of hydrocarbon monoterpenes (24.3 – 55.8%) and non‐terpenic oxygenated compounds (34.5 – 63.1%). The main components were (E)‐β‐ocimene (12.1 – 39%), octyl acetate (11.6 – 21.8%) and decanol (9.7 – 15.4%). The GC and GC/MS profiling of fruit essential oils showed a chemical variability according to geographical locations of plant material. The LC/MS/MS analysis of fruit oils allowed the detection of seven coumarins in trace content. The chemical composition of fruit essential oils was compared with volatile fractions of leaves and barks (root and trunk) from the same plant station. Hexadecanoic acid, germacrene D and decanal were identified as the major constituents of leaves whereas the barks (root and trunk) were dominated by pellitorine (85.8% and 57%, respectively), an atypic linear compound with amide group. The fruit essential oil exhibited interesting antimicrobial activities against Staphylococcus aureus and Candida albicans, particularly the alcohol fraction of the oil.     

Secretome Analysis of Hypoxia‐Induced 3T3‐L1 Adipocytes Uncovers Novel Proteins Potentially Involved in Obesity

In the obese state, as adipose tissue expands, adipocytes become hypoxic and dysfunctional, leading to changes in the pattern of adipocyte‐secreted proteins. To better understand the role of hypoxia in the mechanisms linked to obesity, we comparatively analyzed the secretome of murine differentiated 3T3‐L1 adipocytes exposed to normoxia or hypoxia for 24 h. Proteins secreted into the culture media were precipitated by trichloroacetic acid and then digested with trypsin. The peptides were labeled with dimethyl labeling and analyzed by reversed phase nanoscale liquid chromatography coupled to a quadrupole Orbitrap mass spectrometer. From a total of 1508 identified proteins, 109 were differentially regulated, of which 108 were genuinely secreted. Factors significantly downregulated in hypoxic conditions included adiponectin, a known adipokine implicated in metabolic processes, as well as thrombospondin‐1 and ‐2, and matrix metalloproteinase‐11, all multifunctional proteins involved in extracellular matrix (ECM) homeostasis. Findings were validated by Western blot analysis. Expression studies of the relative genes were performed in parallel experiments in vitro, in differentiated 3T3‐L1 adipocytes, and in vivo, in fat tissues from obese versus lean mice. Our observations are compatible with the concept that hypoxia may be an early trigger for both adipose cell dysfunction and ECM remodeling.     

Bacterial iturins mediate biocontrol activity of Bacillus sp. against postharvest pear fruit‐rotting fungi

A potential antagonist, Bacillus sp. LYLB4 isolated from pear fruits, was tested for its antifungal activity against postharvest pear pathogens. LYLB4 had a remarkable antifungal effect on Botryosphaeria dothidea. Although it showed a weak inhibition effect on the growth of Rhizopus stolonifer on potato dextrose agar (PDA) plates, LYLB4 almost completely destroyed R. stolonifer during direct contact in potato dextrose broth (PDB). LYLB4 treatment was able to significantly reduce disease incidence (by 68.9% and 100%, respectively) and lesion diameter (by 68.7% and 100%, respectively) of ring rot caused by B. dothidea and soft rot caused by R. stolonifer in pears. LYLB4 also suppressed several other phytopathogens in vitro, suggesting a broad‐spectrum antagonistic activity against fungal pathogens. 16S rRNA and gyrA sequence analysis indicated that LYLB4 is closely related to B. velezensis. Genome mining indicated that LYLB4 had 11 secondary metabolites encoding clusters, but that the surfactin and fengycin gene clusters may not be functional because of a large deletion. Matrix‐assisted laser desorption ionization‐time of flight mass spectra (MALDI‐TOF‐MS) demonstrated that iturins were the major lipopeptides, and that C₁₆/C₁₇ Bacillomycin D synthesis was stimulated when LYLB4 was co‐cultured with B. dothidea compared to the control. Overall, these results demonstrate that the main biocontrol mechanism adopted by LYLB4 could be through the production of toxic metabolites and direct contact with pathogens.     

Metabolomic Tools to Assess the Chemistry and Bioactivity of Endophytic Aspergillus Strain

Endophytic fungi associated with medicinal plants are a potential source of novel chemistry and biology that may find applications as pharmaceutical and agrochemical drugs. In this study, a combination of metabolomics and bioactivity‐guided approaches were employed to isolate secondary metabolites with cytotoxicity against cancer cells from an endophytic Aspergillus aculeatus. The endophyte was isolated from the Egyptian medicinal plant Terminalia laxiflora and identified using molecular biological methods. Metabolomics and dereplication studies were accomplished by utilizing the MZmine software coupled with the universal Dictionary of Natural Products database. Metabolic profiling, with aid of multivariate data analysis, was performed at different stages of the growth curve to choose the optimized method suitable for up‐scaling. The optimized culture method yielded a crude extract abundant with biologically‐active secondary metabolites. Crude extracts were fractionated using different high‐throughput chromatographic techniques. Purified compounds were identified by HR‐ESI‐MS, 1D‐ and 2D‐NMR. This study introduced a new method of dereplication utilizing both high‐resolution mass spectrometry and NMR spectroscopy. The metabolites were putatively identified by applying a chemotaxonomic filter. We also present a short review on the diverse chemistry of terrestrial endophytic strains of Aspergillus, which has become a part of our dereplication work and this will be of wide interest to those working in this field.     

Utilization of UPLC/Q‐TOF‐MS‐Based Metabolomics and AFLP‐Based Marker‐Assisted Selection to Facilitate/Assist Conventional Breeding of Polygala tenuifolia

As one of the most important traditional Chinese medicine, the quality of Polygala tenuifolia is difficult to control and a new method must be established to facilitate/assist the breeding of P. tenuifolia. In this study, UPLC/Q‐TOF‐MS‐based metabolomics analysis was performed to determine the chemical composition and screen metabolite biomarkers according to agronomic traits. A total of 29 compounds and 18 metabolite biomarkers were found. AFLP‐based marker‐assisted selection (MAS) was used to identify molecular marker bands and screen characteristic bands associated with specific agronomic traits. 184 bands and 76 characteristic AFLP bands were found. The correlation network between compounds and characteristic AFLP bands was built, so we may directly breed certain P. tenuifolia herbs with special agronomic traits (or characteristic AFLP bands), which exhibit specific pharmacological functions depending on the content of the active compounds. The proposed method of metabolomics coupled with MAS could facilitate/assist the breeding of P. tenuifolia.     

Determination of DNA methylation associated with Acer rubrum (red maple) adaptation to metals: analysis of global DNA modifications and methylation‐sensitive amplified polymorphism

Red maple (Acer rubum), a common deciduous tree species in Northern Ontario, has shown resistance to soil metal contamination. Previous reports have indicated that this plant does not accumulate metals in its tissue. However, low level of nickel and copper corresponding to the bioavailable levels in contaminated soils in Northern Ontario causes severe physiological damages. No differentiation between metal‐contaminated and uncontaminated populations has been reported based on genetic analyses. The main objective of this study was to assess whether DNA methylation is involved in A. rubrum adaptation to soil metal contamination. Global cytosine and methylation‐sensitive amplified polymorphism (MSAP) analyses were carried out in A. rubrum populations from metal‐contaminated and uncontaminated sites. The global modified cytosine ratios in genomic DNA revealed a significant decrease in cytosine methylation in genotypes from a metal‐contaminated site compared to uncontaminated populations. Other genotypes from a different metal‐contaminated site within the same region appear to be recalcitrant to metal‐induced DNA alterations even ≥30 years of tree life exposure to nickel and copper . MSAP analysis showed a high level of polymorphisms in both uncontaminated (77%) and metal‐contaminated (72%) populations. Overall, 205 CCGG loci were identified in which 127 were methylated in either outer or inner cytosine. No differentiation among populations was established based on several genetic parameters tested. The variations for nonmethylated and methylated loci were compared by analysis of molecular variance (AMOVA). For methylated loci, molecular variance among and within populations was 1.5% and 13.2%, respectively. These values were low (0.6% for among populations and 5.8% for within populations) for unmethylated loci. Metal contamination is seen to affect methylation of cytosine residues in CCGG motifs in the A. rubrum populations that were analyzed.     

Lipidomic analysis can distinguish between two morphologically similar strains of Nannochloropsis oceanica

The two morphologically similar microalgae NMBluh014 and NMBluh‐X belong to two different strains of Nannochloropsis oceanica. They possess obviously different feeding effects on bivalves, but are indistinguishable by 18S rRNA and morphological features. In this work, lipidomic analysis followed by principal component analysis and orthogonal projections to latent structures discriminant analysis provided a clear distinction between these strains. Metabolites that definitively contribute to the classification were selected as potential biomarkers. The most important difference in polar lipids were sulfoquinovosyldiacylglycerol (containing 18:1/16:0 and 18:3/16:0) and monogalactosyldiacylglycerol (containing 18:3/16:3 and 20:5/14:0), which were detected only in NMBluh‐X. Additionally, an exhaustive qualitative and quantitative profiling of the neutral lipid triacylglycerol (TAG) in the two strains was carried out. The predominant species of TAG containing 16:1/16:1/16:1 acyl groups was detected only in NMBluh‐X with a content of ~93.67 ± 11.85 nmol · mg^?1 dry algae at the onset of stationary phase. Meanwhile, TAG containing 16:0/16:0/16:0 was the main TAG in NMBluh014 with a content of 40.25 ± 3.92 nmol · mg^?1. These results provided the most straightforward evidence for differentiating the two species. The metabolomic profiling indicated that NMBluh‐X underwent significant chemical and physiological changes during the growth process, whereas NMBluh014 did not show such noticeable time‐dependent metabolite change. This study is the first using Ultra Performance Liquid Chromatography coupled with Electrospray ionization‐Quadrupole‐Time of Flight Mass Spectrometry (UPLC‐Q‐TOF‐MS) for lipidomic profiling with multivariate statistical analysis to explore lipidomic differences of plesiomorphous microalgae. Our results demonstrate that lipidomic profiling is a valid chemotaxonomic tool in the study of microalgal systematics.     

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.     

Phenolic Profile of Artemisia alba Turra

The aim of this study was to evaluate flower and leaf methanol extracts of Artemisia alba Turra for their total phenolic and flavonoid contents, antioxidant capacity and to investigate their phenolic composition. The flower extract was richer in total phenolics and flavonoids and possessed higher antioxidant activity through DPPH and ABTS assays. The UHPLC‐PDA‐MS analysis of the flower and leaf methanol extracts revealed similar phenolic profile and allowed identification of 31 phenolic compounds (flavonoids, coumarins, and phenolic acids) by comparison with the respective reference compounds or tentatively characterized by their chromatographic behavior, UV patterns, and MS fragmentations. The presence of hispidulin, jaceosidin, desmethoxycentaureidin, and dicaffeoyl esters of quinic acid in A. alba is reported herein for the first time. The distribution of flavonoids in A. alba from different origins was discussed from chemotaxonomic point of view.     

One‐pot sample preparation approach for profiling spatial distribution of gibberellins in a single shoot of germinating cereal seeds

Sample preparation remains a bottleneck in the rapid and reliable quantification of gibberellins (GAs) for obtaining an insight into the physiological processes mediated by GAs. The challenges arise from not only the extremely low content of GAs in complex plant matrices, but the poor detectability of GAs by mass spectrometry (MS) in negative ion mode. In an effort to solve these urgent difficulties, we present a spatial‐resolved analysis method to investigate the distribution of GAs in tiny plant tissues based on a simplified one‐pot sample preparation approach coupled with ultrahigh‐performance liquid chromatography‐tandem MS. By integrating extraction and derivatization into one step, target GAs were effectively extracted from plant materials and simultaneously reacted with N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide, the sample preparation time was largely shortened, the probability of sample loss was minimized and the detection sensitivity of MS was also greatly improved compared with underivatized GAs. Under optimal conditions, the method was validated from the quantification linearity, limits of detection and limits of quantification in the presence of plant matrices, recoveries, and precision. With the proposed method, 15 endogenous GAs were detected and, among these, 11 GAs could be quantified in 0.50 mg fresh weight (FW) wheat shoot samples, and five GAs were quantified in only 0.15 mg FW developing seed samples of Arabidopsis thaliana. The distribution patterns of GAs along both the non‐13‐hydroxylation pathway and the early 13‐hydroxylation pathway in a single shoot of germinating wheat, rice and maize seeds were finally profiled with a spatial resolution down to approximately 1 mm².     

Matrix‐assisted laser desorption/ionization mass spectrometry imaging: a powerful tool for probing the molecular topology of plant cutin polymer

The cutin polymers of different fruit cuticles (tomato, apple, nectarine) were examined using matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) after in situ release of the lipid monomers by alkaline hydrolysis. The mass spectra were acquired from each coordinate with a lateral spatial resolution of approximately 100 μm. Specific monomers were released at their original location in the tissue, suggesting that post‐hydrolysis diffusion can be neglected. Relative quantification of the species was achieved by introducing an internal standard, and the collection of data was subjected to non‐supervised and supervised statistical treatments. The molecular images obtained showed a specific distribution of ions that could unambiguously be ascribed to cutinized and suberized regions observed at the surface of fruit cuticles, thus demonstrating that the method is able to probe some structural changes that affect hydrophobic cuticle polymers. Subsequent chemical assignment of the differentiating ions was performed, and all of these ions could be matched to cutin and suberin molecular markers. Therefore, this MALDI‐MSI procedure provides a powerful tool for probing the surface heterogeneity of plant lipid polymers. This method should facilitate rapid investigation of the relationships between cuticle phenotypes and the structure of cutin within a large population of mutants.     

Athenolide A,a New Steroidal Lactone from the Leaves of Athenaea martiana (Solanaceae) Determined by Means of HPLC‐HR‐MS‐SPE‐NMR Analysis

Athenaea (Solanaceae) is an endemic genus belonging to the Brazilian Atlantic Rainforest. Recently, botanical investigations suggested the re‐evaluation of the generic status of the genus Athenaea as a synonym of Aureliana. In this study, the first investigation of the Athenaea genus performed on Athenaea martiana by means of HPLC‐HR‐MS‐SPE‐NMR combined with high‐resolution radical scavenging profile led to identification of several phenolic acids as radical scavengers: protocatechuic acid ( 1 ), 4‐hydroxybenzoic acid ( 2 ), caffeic acid ( 3 ), vanillic acid ( 4 ), and ferulic acid ( 6 ). Additional analysis revealed a new steroidal lactone, named athenolide A ( 9 ). Their structures were elucidated by extensive use of NMR spectroscopy as well as HR‐MS. Chemotaxonomic considerations based on these results supported the chemical relationships between the Athenaea and Aureliana genera, in agreement with the recent botanical findings.     

Comparison of Polyphenol Profile and Antimutagenic and Antioxidant Activities in Two Species Used as Source of Solidaginis herba – Goldenrod

European Pharmacopoeia accepts two equivalent species Solidago canadensis L. and S. gigantea Aiton as goldenrod (Solidaginis herba). We compared phytochemical profile of both species from invasive populations in Poland. Further, we compared in vitro antimutagenic and antioxidant activities of solvent extracts from aerial (AP) and underground parts (UP). In S. gigantea, flavonoid profile was dominated by quercetin glycosides, with quercitrin as the major compound. In S. canadensis, quercetin and kaempferol rutinosides were two major constituents. Caffeoylquinic acids (CQAs) were less diverse with 5‐CQA as a main compound. In UP, over 20 putative diterpenoids were detected, mostly unidentified. Several CQAs were present in higher amounts than in AP. Antioxidant and antimutagenic activities were different between species and organs, with the strongest inhibition of lipid peroxidation by Et₂O and AcOEt fractions from AP of both species (IC₅₀ 13.33 – 16.89 μg/mL) and BuOH fraction from S. gigantea UP (IC₅₀ = 13.32 μg/mL). Chemical mutagenesis was completely inhibited by non‐polar fractions, but oxidative mutagenesis was inhibited up to 35% only by S. canadensis. No clear relationship was found between chemical profiles and antimutagenic activity. In conclusion, both species have diverse activity and their phytochemical profiles should be considered in quality evaluation. UP of these weeds can also provide potential chemopreventive substances for further studies.