Identification of direct target genes and functional conservation of FaRIF, a key regulator of fruit ripening in strawberry

We have identified FaRIF, a NAC transcription factor (TF) that plays a central role controlling strawberry fruit ripening. Thus, our data showed that FaRIF is involved in multiple ripening-related processes, such as ABA biosynthesis and signalling, cell wall degradation and modification, phenylpropanoid pathway, etc. (Martín-Pizarro at al., 2021). In order to further characterized the mechanism by which FaRIF regulates strawberry fruit ripening, we aimed at identifying direct target genes. For that purpose, we have optimized the Chromatin Immunoprecipitation (ChIP) methodology in strawberry plants using 35S:FaRIF-GFP lines. Our data has allowed us to identify loci directly bound by FaRIF which are in the promoter regions of ripening-related genes. Furthermore, the binding has been confirmed by transactivation assays using Luciferase. FaRIF belongs to a clade of NAC TFs with a role in fruit ripening and senescence in different species, as well as in different fruit and ripening types, suggesting conservation in their ultimate role for seed dispersal. Our results have shown partial complementation of the mutant in Arabidopsis for the closest homolog genes to FaRIF, i.e., the redundant genes NARS1 and NARS2, supporting the conservation of the role for this NAC TFs clade.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Pengaruh Pemberian Terapi Minyak Nigella Sativa Dan Kombinasinya Dengan Seftriakson Terhadap Jumlah Kuman Methicillin Resistant Staphylococcus Aureus (Mrsa) Pada Kultur Limpa Mencit Balb/c

Background: Levels of serum albumin can be used as a predictor of morbidity and mortality in the elderly. Reduced serum albumin concentration can be caused by oxidative modification due to aging or insufficient protein intake. SOD as an enzymatic antioxidant might prevent oxidative stress so that albumin modification process can be inhibited. SOD supplementation was expected to increase serum albumin levels.Aim: To prove the effect of Nigella sativa oil and its combination with ceftriaxone toward MRSA count in BALB/c mice\u27s spleen culture.Methods: This was true laboratory experimental study with post test only control group design. The samples were 20 males BALB/c mice, randomized into 4 groups. Control was given 0,03 ml aquabidest injected intraperitoneally, P1 was given 0,03 ml ceftriaxone injected intraperitoneally, P2 was given 0,3 ml Nigella sativa oil orally and P3 was given the combination of 0,03 ml ceftriaxone and 0,3 ml Nigella sativa oil. 0,2 ml (107 cfu/ml) MRSA was injected intraperitoneally at the hour 0. The treatment was given at the hour 16 and mice were terminated at the hour 24. Kruskall-Wallis Test and Mann-Whitney Test is used to analyze the data.Results: Results of the mean number of MRSA bacteria in spleen culture were K=124x103 160,70x103; P1=24,08x103 26,53x103; P2=0,78x103 1,52x103; P3=6,05x103 13,38x103. The number of MRSA bacteria decreased significantly between control group compared to P2 (p=0,016) and P3 (p=0,046) and P1 compared to P2 (p=0,016) and P3 (p=0,047). There were no significance between control compared to P1 (p=0,674) and P2 toward P3 (p=0,596).Conclusion: Administration of Nigella sativa oil and its combination with ceftriaxone can decrease the number of MRSA bacteria in BALB/c mice\u27s spleen cultures

Metabolic Dynamics During Loquat Fruit Ripening and Postharvest Technologies

Loquat is an important fruit widely cultivated worldwide with high commercial value. During loquat fruit development, ripening, and storage, many important metabolites undergo dramatic changes, resulting in accumulation of a diverse mixture of nutrients. Given the value of loquat fruit, significant progresses have been achieved in understanding the metabolic changes during fruit ripening and storage, as well as postharvest technologies applied in loquat fruit in recent years. The objective of the present review is to summarize currently available knowledge and provide new references for improving loquat fruit quality

Hydrogen Peroxide Acts on Sensitive Mitochondrial Proteins to Induce Death of a Fungal Pathogen Revealed by Proteomic Analysis

How the host cells of plants and animals protect themselves against fungal invasion is a biologically interesting and economically important problem. Here we investigate the mechanistic process that leads to death of Penicillium expansum, a widespread phytopathogenic fungus, by identifying the cellular compounds affected by hydrogen peroxide (H2O2) that is frequently produced as a response of the host cells. We show that plasma membrane damage was not the main reason for H2O2-induced death of the fungal pathogen. Proteomic analysis of the changes of total cellular proteins in P. expansum showed that a large proportion of the differentially expressed proteins appeared to be of mitochondrial origin, implying that mitochondria may be involved in this process. We then performed mitochondrial sub-proteomic analysis to seek the H2O2-sensitive proteins in P. expansum. A set of mitochondrial proteins were identified, including respiratory chain complexes I and III, F1F0 ATP synthase, and mitochondrial phosphate carrier protein. The functions of several proteins were further investigated to determine their effects on the H2O2-induced fungal death. Through fluorescent co-localization and the use of specific inhibitor, we provide evidence that complex III of the mitochondrial respiratory chain contributes to ROS generation in fungal mitochondria under H2O2 stress. The undesirable accumulation of ROS caused oxidative damage of mitochondrial proteins and led to the collapse of mitochondrial membrane potential. Meanwhile, we demonstrate that ATP synthase is involved in the response of fungal pathogen to oxidative stress, because inhibition of ATP synthase by oligomycin decreases survival. Our data suggest that mitochondrial impairment due to functional alteration of oxidative stress-sensitive proteins is associated with fungal death caused by H2O2

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

RNA methylomes reveal the m(6)A-mediated regulation of DNA demethylase gene SlDML2 in tomato fruit ripening

Background Methylation of nucleotides, notably in the forms of 5-methylcytosine (5mC) in DNA and N-6-methyladenosine (m(6)A) in mRNA, carries important information for gene regulation. 5mC has been elucidated to participate in the regulation of fruit ripening, whereas the function of m(6)A in this process and the interplay between 5mC and m(6)A remain uncharacterized. Results Here, we show that mRNA m(6)A methylation exhibits dynamic changes similar to DNA methylation during tomato fruit ripening. RNA methylome analysis reveals that m(6)A methylation is a prevalent modification in the mRNA of tomato fruit, and the m(6)A sites are enriched around the stop codons and within the 3 ' untranslated regions. In the fruit of the ripening-deficient epimutant Colorless non-ripening (Cnr) which harbors DNA hypermethylation, over 1100 transcripts display increased m(6)A levels, while only 134 transcripts show decreased m(6)A enrichment, suggesting a global increase in m(6)A. The m(6)A deposition is generally negatively correlated with transcript abundance. Further analysis demonstrates that the overall increase in m(6)A methylation in Cnr mutant fruit is associated with the decreased expression of RNA demethylase gene SlALKBH2, which is regulated by DNA methylation. Interestingly, SlALKBH2 has the ability to bind the transcript of SlDML2, a DNA demethylase gene required for tomato fruit ripening, and modulates its stability via m(6)A demethylation. Mutation of SlALKBH2 decreases the abundance of SlDML2 mRNA and delays fruit ripening. Conclusions Our study identifies a novel layer of gene regulation for key ripening genes and establishes an essential molecular link between DNA methylation and mRNA m(6)A methylation during fruit ripening

NADPH Oxidase Is Crucial for the Cellular Redox Homeostasis in Fungal Pathogen Botrytis cinerea

During interactions, both plants and pathogens produce reactive oxygen species (ROS). Plants generate ROS for defense induction, while pathogens synthesize ROS for growth, sporulation, and virulence. NADPH oxidase (NOX) complex in the plasma membrane represents a main protein complex for ROS production in pathogens. Although NOX plays a crucial role in pathogenicity of pathogens, the underlying molecular mechanisms of NOX, especially the proteins regulated by NOX, remain largely unknown. Here, we applied an iodoacetyl tandem mass tag-based redox proteomic assay to investigate the protein redox dynamics in deletion mutant of bcnoxR, which encodes a regulatory subunit of NOX in the fungal pathogen Botrytis cinerea. In total, 214 unique peptidyl cysteine (Cys) thiols from 168 proteins were identified and quantified in both the wild type and Delta bcnoxR mutant. The Cys thiols in the Delta bcnoxR mutant were generally more oxidized than those in the wild type, suggesting that BcNoxR is essential for maintaining the equilibrium of the redox state in B. cinerea. Site-specific thiol oxidation analysis indicated that 142 peptides containing the oxidized thiols changed abundance significantly in the Delta bcnoxR mutant. Proteins containing these differential peptides are classified into various functional categories. Functional analysis revealed that one of these proteins, 6-phosphate dehydrogenase, played roles in oxidative stress response and pathogenesis of B. cinerea. These results provide insight into the potential target proteins and the ROS signal transduction pathway regulated by NOX

Reactive oxygen species involved in regulating fruit senescence and fungal pathogenicity

Senescence is a vital aspect of fruit life cycles, and directly affects fruit quality and resistance to pathogens. Reactive oxygen species (ROS), as the primary mediators of oxidative damage in plants, are involved in senescence. Mitochondria are the main ROS and free radical source. Oxidative damage to mitochondrial proteins caused by ROS is implicated in the process of senescence, and a number of senescence-related disorders in a variety of organisms. However, the specific sites of ROS generation in mitochondria remain largely unknown. Recent discoveries have ascertained that fruit senescence is greatly related to ROS and incidental oxidative damage of mitochondrial protein. Special mitochondrial proteins involved in fruit senescence have been identified as the targets of ROS. We focus in discussion on our recent advances in exploring the mechanisms of how ROS regulate fruit senescence and fungal pathogenicity