Structural Insight into the Complex of Ferredoxin and [FeFe] Hydrogenase from Chlamydomonas reinhardtii

The transfer of photosynthetic electrons by the ferredoxin PetF to the [FeFe] hydrogenase HydA1 in the microalga Chlamydomonas reinhardtii is a key step in hydrogen production. Electron delivery requires a specific interaction between PetF and HydA1. However, because of the transient nature of the electron‐transfer complex, a crystal structure remains elusive. Therefore, we performed protein–protein docking based on new experimental data from a solution NMR spectroscopy investigation of native and gallium‐substituted PetF. This provides valuable information about residues crucial for complex formation and electron transfer. The derived complex model might help to pinpoint residue substitution targets for improved hydrogen production.     

Advances in the Function and Regulation of Hydrogenase in the Cyanobacterium Synechocystis PCC6803

In order to use cyanobacteria for the biological production of hydrogen, it is important to thoroughly study the function and the regulation of the hydrogen-production machine in order to better understand its role in the global cell metabolism and identify bottlenecks limiting H₂ production. Most of the recent advances in our understanding of the bidirectional [Ni-Fe] hydrogenase (Hox) came from investigations performed in the widely-used model cyanobacterium Synechocystis PCC6803 where Hox is the sole enzyme capable of combining electrons with protons to produce H₂ under specific conditions. Recent findings suggested that the Hox enzyme can receive electrons from not only NAD(P)H as usually shown, but also, or even preferentially, from ferredoxin. Furthermore, plasmid-encoded functions and glutathionylation (the formation of a mixed-disulfide between the cysteines residues of a protein and the cysteine residue of glutathione) are proposed as possible new players in the function and regulation of hydrogen production.     

Identification and Expression Analysis of Candidate Genes Associated with Defense Responses to Phytophthora capsici in Pepper Line “PI 201234”

Phytophthora capsici (Leonian), classified as an oomycete, seriously threatens the production of pepper (Capsicum annuum). Current understanding of the defense responses in pepper to P. capsici is limited. In this study, RNA-sequencing analysis was utilized to identify differentially expressed genes in the resistant line “PI 201234”, with 1220 differentially expressed genes detected. Of those genes, 480 were up-regulated and 740 were down-regulated, with 211 candidate genes found to be involved in defense responses based on the gene annotations. Furthermore, the expression patterns of 12 candidate genes were further validated via quantitative real-time PCR (qPCR). These genes were found to be significantly up-regulated at different time points post-inoculation (6 hpi, 24 hpi, and 5 dpi) in the resistant line “PI 201234” and susceptible line “Qiemen”. Seven genes were found to be involved in cell wall modification, phytoalexin biosynthesis, symptom development, and phytohormone signaling pathways, thus possibly playing important roles in combating exogenous pathogens. The genes identified herein will provide a basis for further gene cloning and functional verification studies and will aid in an understanding of the regulatory mechanism of pepper resistance to P. capsici.     

A Clickable Photosystem I,Ferredoxin, and Ferredoxin NADP+ Reductase Fusion System for Light-Driven NADPH Regeneration**

Photosynthetic organisms like plants, algae, and cyanobacteria use light for the regeneration of dihydronicotinamide dinucleotide phosphate (NADPH). The process starts with the light-driven oxidation of water by photosystem II (PSII) and the released electrons are transferred via the cytochrome b₆f complex towards photosystem I (PSI). This membrane protein complex is responsible for the light-driven reduction of the soluble electron mediator ferredoxin (Fd), which passes the electrons to ferredoxin NADP⁺ reductase (FNR). Finally, NADPH is regenerated by FNR at the end of the electron transfer chain. In this study, we established a clickable fusion system for in vitro NADPH regeneration with PSI−Fd and PSI−Fd−FNR, respectively. For this, we fused immunity protein 7 (Im7) to the C-terminus of the PSI−PsaE subunit in the cyanobacterium Synechocystis sp. PCC 6803. Furthermore, colicin DNase E7 (E7) fusion chimeras of Fd and FNR with varying linker domains were expressed in Escherichia coli. Isolated Im7−PSI was coupled with the E7−Fd or E7−Fd−FNR fusion proteins through high-affinity binding of the E7/Im7 protein pair. The corresponding complexes were tested for NADPH regeneration capacity in comparison to the free protein systems demonstrating the general applicability of the strategy.     

Response of Different Genotypes of Faba Bean Plant to Drought Stress

Drought stress is one of the major abiotic stresses that are a threat to crop production worldwide. Drought stress impairs the plants growth and yield. Therefore, the aim of the present experiment was to select the tolerant genotype/s on the basis of moprpho-physiological and biochemical characteristics of 10 Vicia faba genotypes (Zafar 1, Zafar 2, Shebam, Makamora, Espan, Giza Blanka, Giza 3, C4, C5 and G853) under drought stress. We studied the effect of different levels of drought stress i.e., (i) normal irrigation (ii) mild stress (iii) moderate stress, and (iv) severe stress on plant height (PH) plant⁻¹, fresh weight (FW) and dry weight (DW) plant⁻¹, area leaf⁻¹, leaf relative water content (RWC), proline (Pro) content, total chlorophyll (Total Chl) content, electrolyte leakage (EL), malondialdehyde (MDA), hydrogen peroxide (H₂O₂) content, and activities of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) of genotypes of faba bean. Drought stress reduced all growth parameters and Total Chl content of all genotypes. However, the deteriorating effect of drought stress on the growth performance of genotypes “C5” and “Zafar 1” were relatively low due to its better antioxidant enzymes activities (CAT, POD and SOD), and accumulation of Pro and Total Chl, and leaf RWC. In the study, genotype “C5” and “Zafar 1” were found to be relatively tolerant to drought stress and genotypes “G853” and “C4” were sensitive to drought stress.     

Direct electrochemistry of dinuclear CuA fragment from cytochrome c oxidase of Thermus thermophilus at surfactant modified glassy carbon electrode

Cytochrome c oxidase is ubiquitous enzyme involved in the terminal step of respiratory electron transfer process. The unique binuclear copper center containing bis-dithiolato bridges form a valance delocalized [Cu^1.5+-Cu^1.5+] state of the metal center located at the subunit II of cytochrome c oxidase. This metal center acts as the electron entry site of the enzyme and accepts electrons from cytochrome c. Direct electrochemistry of this binuclear copper center containing the water soluble protein obtained by genetically truncating the membrane bound part of the subunit II from Thermus thermophilus was achieved by favorable orientation of the protein on glassy carbon electrode surface promoting efficient electron transfer in the presence of various surfactants. Very reproducible, Nernstian responses are obtained with Cu_A. The redox potential and the electrochemical response were enhanced prominently in case of cationic surfactant CTAB indicating that the nature of the surfactant has a significant effect on the microenvironment of the protein-electrode interface. The results have been used to understand the mechanism of electron transfer from cytochrome c to the copper center during the enzymatic reaction.     

Kinetic Model for Signal Binding to the Quorum Sensing Regulator LasR

We propose a kinetic model for the activation of the las regulon in the opportunistic pathogen Pseudomonas aeruginosa. The model is based on in vitro data and accounts for the LasR dimerization and consecutive activation by binding of two OdDHL signal molecules. Experimentally, the production of the active LasR quorum-sensing regulator was studied in an Escherichia coli background as a function of signal molecule concentration. The functional activity of the regulator was monitored via a GFP reporter fusion to lasB expressed from the native lasB promoter. The new data shows that the active form of the LasR dimer binds two signal molecules cooperatively and that the timescale for reaching saturation is independent of the signal molecule concentration. This favors a picture where the dimerized regulator is protected against proteases and remains protected as it is activated through binding of two successive signal molecules. In absence of signal molecules, the dimerized regulator can dissociate and degrade through proteolytic turnover of the monomer. This resolves the apparent contradiction between our data and recent reports that the fully protected dimer is able to “degrade” when the induction of LasR ceases.     

Genotypic Variation under Fe Deficiency Results in Rapid Changes in Protein Expressions and Genes Involved in Fe Metabolism and Antioxidant Mechanisms in Tomato Seedlings (Solanum lycopersicum L.)

To investigate Fe deficiency tolerance in tomato cultivars, quantification of proteins and genes involved in Fe metabolism and antioxidant mechanisms were performed in “Roggusanmaru” and “Super Doterang”. Fe deficiency (Moderate, low and –Fe) significantly decreased the biomass, total, and apoplastic Fe concentration of “Roggusanmaru”, while a slight variation was observed in “Super Doterang” cultivar. The quantity of important photosynthetic pigments such as total chlorophyll and carotenoid contents significantly decreased in “Roggusanmaru” than “Super Doterang” cultivar. The total protein profile in leaves and roots determines that “Super Doterang” exhibited an optimal tolerance to Fe deficiency compared to “Roggusanmaru” cultivar. A reduction in expression of PSI (photosystem I), PSII (photosystem II) super-complexes and related thylakoid protein contents were detected in “Roggusanmaru” than “Super Doterang” cultivar. Moreover, the relative gene expression of SlPSI and SlPSII were well maintained in “Super Doterang” than “Roggusanmaru” cultivar. The relative expression of genes involved in Fe-transport (SlIRT1 and SlIRT2) and Fe(III) chelates reductase oxidase (SlFRO1) were relatively reduced in “Roggusanmaru”, while increased in “Super Doterang” cultivar under Fe deficient conditions. The H⁺-ATPase relative gene expression (SlAHA1) in roots were maintained in “Super Doterang” compared to “Roggusanmaru”. Furthermore, the gene expressions involved in antioxidant defense mechanisms (SlSOD, SlAPX and SlCAT) in leaves and roots showed that these genes were highly increased in “Super Doterang”, whereas decreased in “Roggusanmaru” cultivar under Fe deficiency. The present study suggested that “Super Doterang” is better tomato cultivar than “Roggusanmaru” for calcareous soils.     

Constituents and Pharmacological Activities of Myrcia (Myrtaceae): A Review of an Aromatic and Medicinal Group of Plants

Myrcia is one of the largest genera of the economically important family Myrtaceae. Some of the species are used in folk medicine, such as a group known as “pedra-hume-caá” or “pedra-ume-caá” or “insulina vegetal” (insulin plant) that it is used for the treatment of diabetes. The species are an important source of essential oils, and most of the chemical studies on Myrcia describe the chemical composition of the essential oils, in which mono- and sesquiterpenes are predominant. The non-volatile compounds isolated from Myrcia are usually flavonoids, tannins, acetophenone derivatives and triterpenes. Anti-inflammatory, antinociceptive, antioxidant, antimicrobial activities have been described to Myrcia essential oils, while hypoglycemic, anti-hemorrhagic and antioxidant activities were attributed to the extracts. Flavonoid glucosides and acetophenone derivatives showed aldose reductase and α-glucosidase inhibition, and could explain the traditional use of Myrcia species to treat diabetes. Antimicrobial and anti-inflammatory are some of the activities observed for other isolated compounds from Myrcia.     

CrWSKP1, an SKP1-like Gene,Is Involved in the Self-Incompatibility Reaction of “Wuzishatangju” (Citrus reticulata Blanco)

Plant S-phase kinase-associated protein 1 (SKP1) genes play crucial roles in plant development and differentiation. However, the role of SKP1 in citrus is unclear. Herein, we described a novel SKP1-like gene, designated as CrWSKP1, from “Wuzishatangju” (Citrus reticulata Blanco). The cDNA sequence of CrWSKP1 is 779 base pairs (bp) and contains an open reading frame (ORF) of 477 bp. The genomic sequence of the CrWSKP1 gene is 1296 bp with two exons and one intron. CrWSKP1 has high identity with SKP1-like genes from other plant species within two conserved regions. Approximately 85% of pollen tubes of self-pollinated CrWSKP1 transgenic tobaccos became twisted at four days after self-pollination. Pollen tube numbers of self-pollinated CrWSKP1 transformants entering into ovules were significantly fewer than that of the control. Seed number of self-pollinated CrWSKP1 transformants was significantly reduced. These results suggested that the CrWSKP1 is involved in the self-incompatibility (SI) reaction of “Wuzishatangju”.     

Effects of Aqueous Dispersions of C60, C70 and Gd@C82 Fullerenes on Genes Involved in Oxidative Stress and Anti-Inflammatory Pathways

Background: Fullerenes and metallofullerenes can be considered promising nanopharmaceuticals themselves and as a basis for chemical modification. As reactive oxygen species homeostasis plays a vital role in cells, the study of their effect on genes involved in oxidative stress and anti-inflammatory responses are of particular importance. Methods: Human fetal lung fibroblasts were incubated with aqueous dispersions of C₆₀, C₇₀, and Gd@C₈₂ in concentrations of 5 nM and 1.5 µM for 1, 3, 24, and 72 h. Cell viability, intracellular ROS, NOX4, NFκB, PRAR-γ, NRF2, heme oxygenase 1, and NAD(P)H quinone dehydrogenase 1 expression have been studied. Results & conclusion: The aqueous dispersions of C₆₀, C₇₀, and Gd@C₈₂ fullerenes are active participants in reactive oxygen species (ROS) homeostasis. Low and high concentrations of aqueous fullerene dispersions (AFD) have similar effects. C₇₀ was the most inert substance, C₆₀ was the most active substance. All AFDs have both “prooxidant” and “antioxidant” effects but with a different balance. Gd@C₈₂ was a substance with more pronounced antioxidant and anti-inflammatory properties, while C₇₀ had more pronounced “prooxidant” properties.     

Overexpression of Ferredoxin,PETF, Enhances Tolerance to Heat Stress in Chlamydomonas reinhardtii

Reactive oxygen species (ROS) produced by plants in adverse environments can cause damage to organelles and trigger cell death. Removal of excess ROS can be achieved through the ascorbate scavenger pathway to prevent plant cell death. The amount of this scavenger can be regulated by ferredoxin (FDX). Chloroplastic FDXs are electron transfer proteins that perform in distributing photosynthetic reducing power. In this study, we demonstrate that overexpression of the endogenous photosynthetic FDX gene, PETF, in Chlamydomonas reinhardtii could raise the level of reduced ascorbate and diminish H₂O₂ levels under normal growth conditions. Furthermore, the overexpressing PETF transgenic Chlamydomonas lines produced low levels of H₂O₂ and exhibited protective effects that were observed through decreased chlorophyll degradation and increased cell survival under heat-stress conditions. The findings of this study suggest that overexpression of PETF can increase the efficiency of ROS scavenging in chloroplasts to confer heat tolerance. The roles of PETF in the downregulation of the ROS level offer a method for potentially improving the tolerance of crops against heat stress.     

Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect l-Lysine Production in Corynebacterium glutamicum

Corynebacterium glutamicum is widely used for amino acid production. In the present study, 543 genes showed a significant change in their mRNA expression levels in l-lysine-producing C. glutamicum ATCC21300 than that in the wild-type C. glutamicum ATCC13032. Among these 543 differentially expressed genes (DEGs), 28 genes were up- or downregulated. In addition, 454 DEGs were functionally enriched and categorized based on BLAST sequence homologies and gene ontology (GO) annotations using the Blast2GO software. Interestingly, NCgl0071 (bioB, encoding biotin synthase) was expressed at levels ~20-fold higher in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain. Five other genes involved in biotin metabolism or transport—NCgl2515 (bioA, encoding adenosylmethionine-8-amino-7-oxononanoate aminotransferase), NCgl2516 (bioD, encoding dithiobiotin synthetase), NCgl1883, NCgl1884, and NCgl1885—were also expressed at significantly higher levels in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain, which we determined using both next-generation RNA sequencing and quantitative real-time PCR analysis. When we disrupted the bioB gene in C. glutamicum ATCC21300, l-lysine production decreased by approximately 76%, and the three genes involved in biotin transport (NCgl1883, NCgl1884, and NCgl1885) were significantly downregulated. These results will be helpful to improve our understanding of C. glutamicum for industrial amino acid production.     

An Efficient Agrobacterium-Mediated Transformation System for Poplar

Poplar is a model system for the regeneration and genetic transformation of woody plants. To shorten the time required for studies of transgenic poplar, efforts have been made to optimize transformation methods that use Agrobacterium tumefaciens. In this study, an Agrobacterium infective suspension was treated at 4 °C for at least 10 h before infecting explants. By transforming the Populus hybrid clone “Nanlin895” (Populus deltoides × P. euramericana) with Agrobacterium harboring the PBI121:CarNAC6 binary vector, we showed that the transformation efficiency was improved significantly by multiple independent factors, including an Agrobacterium infective suspension with an OD₆₀₀ of 0.7, an Agrobacterium infection for 120 min, an Agrobacterium infective suspension at a pH of 5.0, an acetosyringone concentration of 200 µM, a cocultivation at 28 °C, a cocultivation for 72 h and a sucrose concentration of 30 g/L in the cocultivation medium. We also showed that preculture of wounded leaf explants for two days increased the regeneration rate. The integration of the desired gene into transgenic poplars was detected using selective medium containing kanamycin, followed by southern blot analysis. The expression of the transgene in the transgenic lines was confirmed by northern blot analysis.     

Salinity-Induced Variation in Biochemical Markers Provides Insight into the Mechanisms of Salt Tolerance in Common (Phaseolus vulgaris) and Runner (P. coccineus) Beans

The evaluation of biochemical markers is important for the understanding of the mechanisms of tolerance to salinity of Phaseolus beans. We have evaluated several growth parameters in young plants of three Phaseolus vulgaris cultivars subjected to four salinity levels (0, 50, 100, and 150 mM NaCl); one cultivar of P. coccineus, a closely related species reported as more salt tolerant than common bean, was included as external reference. Biochemical parameters evaluated in leaves of young plants included the concentrations of ions (Na⁺, K⁺, and Cl⁻), osmolytes (proline, glycine betaine, and total soluble sugars), and individual soluble carbohydrates. Considerable differences were found among cultivars, salinity levels, and in their interaction for most traits. In general, the linear component of the salinity factor for the growth parameters and biochemical markers was the most important. Large differences in the salinity response were found, with P. vulgaris cultivars “The Prince” and “Maxidor” being, respectively, the most susceptible and tolerant ones. Our results support that salt stress tolerance in beans is mostly based on restriction of Na⁺ (and, to a lesser extent, also of Cl⁻) transport to shoots, and on the accumulation of myo-inositol for osmotic adjustment. These responses to stress during vegetative growth appear to be more efficient in the tolerant P. vulgaris cultivar “Maxidor”. Proline accumulation is a reliable marker of the level of salt stress affecting Phaseolus plants, but does not seem to be directly related to stress tolerance mechanisms. These results provide useful information on the responses to salinity of Phaseolus.     

Reconstructing a Flavodoxin Oxidoreductase with Early Amino Acids

Primitive proteins are proposed to have utilized organic cofactors more frequently than transition metals in redox reactions. Thus, an experimental validation on whether a protein constituted solely by early amino acids and an organic cofactor can perform electron transfer activity is an urgent challenge. In this paper, by substituting “late amino acids (C, F, M, T, W, and Y)” with “early amino acids (A, L, and V)” in a flavodoxin, we constructed a flavodoxin mutant and evaluated its characteristic properties. The major results showed that: (1) The flavodoxin mutant has structural characteristics similar to wild-type protein; (2) Although the semiquinone and hydroquinone flavodoxin mutants possess lower stability than the corresponding form of wild-type flavodoxin, the redox potential of double electron reduction E_m,7 (fld) reached −360 mV, indicating that the flavodoxin mutant constituted solely by early amino acids can exert effective electron transfer activity.