Protein glycation and drought response of pea (Pisum sativum L.) root nodule proteome: a proteomics approach

Because of ongoing climate change, drought is becoming the major factor limiting productivity of all plants, including legumes. As these protein-rich crops form symbiotic associations with rhizobial bacteria — root nodules — they readily lose their productivity under drought conditions. Understanding the underlying molecular mechanisms might give access to new strategies to preserve the productivity of legume crops under dehydration. As was shown recently, development of drought response is accompanied by alterations in the patterns of protein glycation and formation of advanced glycation end products (AGEs) that might be a part of unknown regulatory mechanisms. Therefore, here we address the effects of moderate drought on protein dynamics and AGE patterns in pea (Pisum sativum) root nodules. For this, plants inoculated with rhizobial culture were subjected to osmotic stress for one week, harvested, the total protein fraction was isolated from root nodules by phenol extraction, analyzed by bottom-up LC-MS-based proteomics, and AGE patterns were characterized. Surprisingly, despite the clear drought-related changes in phenotype and stomatal conductivity, only minimal accompanying expressional changes (14 rhizobial and 14 pea proteins, mostly involved in central metabolism and nitrogen fixation) could be observed. However, 71 pea and 97 rhizobial proteins (mostly transcription factors, ABC transporters and effector enzymes) were glycated, with carboxymethylation being the major modification type. Thereby, the numbers of glycated sites in nodule proteins dramatically decreased upon stress application. It might indicate an impact of glycation in regulation of transport, protein degradation, central, lipid and nitrogen metabolism. The data are available at Proteome Xchange (accession: PXD024042).

Applying a ‘Metabolic Funnel’ for Phenol Production in Escherichia coli

Phenol is an important petrochemical that is conventionally used as a precursor for synthesizing an array of plastics and fine chemicals. As an emerging alternative to its traditional petrochemical production, multiple enzyme pathways have been engineered to date to enable its renewable biosynthesis from biomass feedstocks, each incorporating unique enzyme chemistries and intermediate molecules. Leveraging all three of the unique phenol biosynthesis pathways reported to date, a series of synthetic ‘metabolic funnels’ was engineered, each with the goal of maximizing net precursor assimilation and flux towards phenol via the parallel co-expression of multiple distinct pathways within the same Escherichia coli host. By constructing and evaluating all possible binary and tertiary pathway combinations, one ‘funnel’ was ultimately identified, which supported enhanced phenol production relative to all three individual pathways by 16 to 69%. Further host engineering to increase endogenous precursor availability then allowed for 26% greater phenol production, reaching a final titer of 554 ± 19 mg/L and 28.8 ± 0.34 mg/g yield on glucose. Lastly, using a diphasic culture including dibutyl phthalate for in situ phenol extraction, final titers were further increased to a maximum of 812 ± 145 mg/L at a yield of 40.6 ± 7.2 mg/g. The demonstrated ‘funneling’ pathway holds similar promise in support of phenol production by other, non-E. coli hosts, while this general approach can be readily extended towards a diversity of other value-added bioproducts of interest.

Amino Acid-Based Natural Deep Eutectic Solvents for Extraction of Phenolic Compounds from Aqueous Environments

The environmental pollution of phenol-containing wastewater is an urgent problem with industrial development. Natural deep eutectic solvents provide an environmentally friendly alternation for the solvent extraction of phenol. This study synthesized a series of natural deep eutectic solvents with L-proline and decanoic acid as precursors, characterized by in situ infrared spectrometry, Fourier transform infrared spectrometry, hydrogen nuclear magnetic resonance spectrometry, and differential thermogravimetric analysis. Natural deep eutectic solvents have good thermal stability. The high-efficiency extraction of phenol from wastewater by natural deep eutectic solvents was investigated under mild conditions. The effects of natural deep eutectic solvents, phenol concentration, reaction temperature, and reaction time on phenol extraction were studied. The optimized extraction conditions of phenol with L-prolin/decanoic acid were as follows: molar ratio, 4.2:1; reaction time, 60 min; and temperature, 50 °C. Extraction efficiency was up to 62%. The number of extraction cycles can be up to 6, and extraction rate not less than 57%. The promising results demonstrate that natural deep eutectic solvents are efficient in the field of phenolic compound extraction in wastewater.

Quantification of NAD(P)H in cyanobacterial cells by a phenol extraction method

In photosynthetic organisms, it is recognized that the intracellular redox ratio of NADPH is regulated within an appropriate range for the cooperative function of a wide variety of physiological processes. However, despite its importance, there is large variability in the values of the NADPH fraction [NADPH/(NADPH + NADP+)] quantitatively estimated to date. In the present study, the light response of the NADPH fraction was investigated by applying a novel NADP(H) extraction method using phenol / chloroform / isoamyl alcohol (PCI) in the cyanobacterium Synechocystis sp. PCC 6803. The light response of NADP(H) observed using PCI extraction was qualitatively consistent with the NAD(P)H fluorescence time course measured in vivo. Moreover, the results obtained by PCI extraction and the fluorescence-based methods were also consistent in a mutant lacking the ability to oxidize NAD(P)H in the respiratory chain, and exhibiting a unique NADPH light response. These observations indicate that the PCI extraction method allowed quantitative determination of NADP(H) redox. Notably, the PCI extraction method showed that not all NADP(H) was oxidized or reduced by light–dark transition. Specifically, the fraction of NADPH was 42% in the dark-adapted cell, and saturated at 68% in light conditions.

Protocol Optimization of Proteomic Analysis of Korean Ginseng (Panax ginseng Meyer)

The benefits of ginseng have been mainly attributed to its triterpenoids, called ginsenosides. Recent genome sequencing of the Panax ginseng has paved the way for in-depth proteomic studies of this medicinal plant. The current study was conducted to deepen the proteomic information on the root proteome of Korean ginseng. Proteomic workflow was optimized by testing two different strategies, characterized by the phenol extraction procedure, the presence or the absence of SDS-PAGE fractionation step, and nano-scale liquid chromatographic tandem mass spectrometry (nLC-MS/MS) analysis. The results highlighted an evident improvement of proteome extraction by the combination of phenol extraction with SDS-PAGE before the nLC-MS/MS analysis. In addition, a dramatic impact of the steaming process (the treatment to produce red ginseng from ginseng) on protein properties was observed. Overall, the analyses of Korean ginseng permitted the characterization of a total of 2412 proteins. A large number of identified proteins belonged to the functional categories of protein and carbon/energy metabolism (22.4% and 14.6%, respectively). The primary and secondary metabolisms are major metabolic pathways, which emerged from the proteomic analysis. In addition, a large number of proteins known to play an important role in response to (a)biotic stresses were also identified. The current proteomic study not only confirmed the previous transcriptomic and proteomic reports but also extended proteomic information, including the main metabolic pathways involved in Korean ginseng.

Effect of contacting pattern and various surfactants on phenol extraction efficiency using emulsion liquid membrane

Emulsions prepared using different surfactants, including Span-80, BKC and CTAB, are studied for their stability and phenol remsoval efficiency. The effect of contacting pattern on ELM extraction efficiency is compared in Beaker – Stirrer apparatus and Bubble Flow Recirculation column. The emulsion prepared using Span-80 is relatively more stable than emulsions prepared using other surfactants. The emulsion stability during the extraction process is relatively higher in the Bubble Flow Recirculation column (15 min) than in the Beaker – Stirrer apparatus (10 min). At optimized conditions, the phenol removal efficiency of the emulsion prepared using Span-80 in Beaker – Stirrer apparatus is 96% and in Bubble Flow Recirculation column is 78%. Kinetic studies reveal that the extraction follows zeroth-order kinetics with an average phenol effective diffusivity of 0.0004 s at an initial phenol concentration ranging from 100–500 PPM.

Phenol Extraction from Schistous Tar of Shubarkol Deposit

The process of extracting phenols from schistous tar using extractants based on water solutions of technical ethanol with concentration of 70% is considered in this article. According to chromato-mass-spectrometric (CMS) analysis, initial schistous tar contains 28.29 % of summary phenols, after extraction with 70% alcohol solution this had dropped by 8.76 %. According to results of laboratory studies, the best extraction properties were identified as occurring with 70% water ethanol of schistous tar with the ratio 1:1. As we see from obtained data, after extraction with ethanol, phenol content in tar is decreased by up to 8.76 %. After elimination of phenols, schistous tar might be applied as hydrocarbonic material for future processing into motor or boiler fuel. Keywords: Schistous tar, phenol, extraction

Geographical and within-population variation of constitutive chemical defences in a Mediterranean oak (Quercus ilex)

Aim of study: to assess whether constitutive levels of total phenols and condensed tannins acting as chemical defences in Quercus ilex vary across regions, populations and genotypes.Area of study: acorns from six Spanish regions with natural presence of Q. ilex were collected for later sowing in the greenhouse of the University of Extremadura at Plasencia, western Spain.Materials and methods: 1176 acorns from 12 populations (2 per region) were sown according to a nested design (6 regions x 2 populations/region x 7 genotypes/population). After five months, 588 emerged seedlings were harvested for phenol extraction from their leaves. Quantification of total phenols through the Folin-Ciocalteu method and condensed tannins by Porter's reagent method was carried out.Main results: total phenolics and condensed tannins correlated positively. Significant differences were found for the concentration of total phenolics and condensed tannins among regions and among genotypes, but not among populations within regions. The lowest levels of constitutive defences were found in the northern Iberian Peninsula. Also, the defensive phenolic content was significantly higher in regions with acidic soils. Heritability values (total phenols 0.37±0.08 and condensed tannins 0.48±0.36) were lower than those obtained of total tannins in a previous study.Research highlights: constitutive levels of heritable chemical defences in holm oak significantly depend on their geographical origin and genotype.Keywords: condensed tannins, genetic and latitudinal variability, heritability, total phenolics.