Molecular and metabolic mechanisms associated with fleshy fruit quality

Funding to AF was provided by the Portuguese Foundation for Science and Technology (FCT Investigator IF/00169/2015, PEst-OE/BIA/UI4046/2014). Research in the AG lab was supported by the EC H2020 Program:TRADITOM-634561 and TOMGEM679796 and networking activities by COST FA1106.Fortes, AM.; Granell Richart, A.; Pezzotti, M.; Bouzayen, M. (2017). Molecular and metabolic mechanisms associated with fleshy fruit quality. Frontiers in Plant Science. 8:6-10. https://doi.org/10.3389/fpls.2017.01236S6108Agudelo-Romero, P., Erban, A., Sousa, L., Pais, M. S., Kopka, J., & Fortes, A. M. (2013). Search for Transcriptional and Metabolic Markers of Grape Pre-Ripening and Ripening and Insights into Specific Aroma Development in Three Portuguese Cultivars. PLoS ONE, 8(4), e60422. doi:10.1371/journal.pone.0060422Fortes, A. M., & Gallusci, P. (2017). Plant Stress Responses and Phenotypic Plasticity in the Epigenomics Era: Perspectives on the Grapevine Scenario, a Model for Perennial Crop Plants. Frontiers in Plant Science, 08. doi:10.3389/fpls.2017.00082Fortes, A., Teixeira, R., & Agudelo-Romero, P. (2015). Complex Interplay of Hormonal Signals during Grape Berry Ripening. Molecules, 20(5), 9326-9343. doi:10.3390/molecules20059326Liu, R., How-Kit, A., Stammitti, L., Teyssier, E., Rolin, D., Mortain-Bertrand, A., … Gallusci, P. (2015). A DEMETER-like DNA demethylase governs tomato fruit ripening. Proceedings of the National Academy of Sciences, 112(34), 10804-10809. doi:10.1073/pnas.150336211

Comparison of normalization methods for differential gene expression analysis in RNA-Seq experiments: A matter of relative size of studied transcriptomes

In recent years, RNA-Seq technologies became a powerful tool for transcriptome studies. However, computational methods dedicated to the analysis of high-throughput sequencing data are yet to be standardized. In particular, it is known that the choice of a normalization procedure leads to a great variability in results of differential gene expression analysis. The present study compares the most widespread normalization procedures and proposes a novel one aiming at removing an inherent bias of studied transcriptomes related to their relative size. Comparisons of the normalization procedures are performed on real and simulated data sets. Real RNA-Seq data sets analyses, performed with all the different normalization methods, show that only 50% of significantly differentially expressed genes are common. This result highlights the influence of the normalization step on the differential expression analysis. Real and simulated data sets analyses give similar results showing 3 different groups of procedures having the same behavior. The group including the novel method named “Median Ratio Normalization” (MR N) gives the lower number of false discoveries. Within this group the MR N method is less sensitive to the modification of parameters related to the relative size of transcriptomes such as the number of down- and upregulated genes and the gene expression levels. The newly proposed MR N method efficiently deals with intrinsic bias resulting from relative size of studied transcriptomes. Validation with real and simulated data sets confirmed that MR N is more consistent and robust than existing methods

Influence of oxygen deficiency on optical and dielectric properties of La0.75Ba0.10Sr0.15FeO2.875-δ compounds

Using the conventional sol-gel process, the series of non-stoichiometric oxygen lanthanum ferrites with the formula 0.750.100.152.875 ― (δ=0.00, 0.125 and 0.25) were prepared. X-ray diffraction analysis confirmed the formation of the orthorhombic structure with Pnma space group. In the present work, we confirm the potential of 0.750.100.15 2.875 ― (δ=0.00, 0.125 and 0.25) as an efficient dielectric material. All samples show high absorbance in the visible region with wavelength above 300 nm. The optical band gaps are found to increase from 3.25 to 4.1 with increasing oxygen vacancy concentration. The dielectric constant, dielectric loss and loss factor were carried out as a function of oxygen vacancy concentration and frequency (102 -106 Hz) at room temperature. The variation of dielectric constant with frequency indicates dispersive behavior and giant dielectric response (more than 104 ) at low frequencies for compounds deficient in oxygen. Such value of makes ′ these samples an interesting material to be used in applications namely the reduction of electronic components size. To summarize, incorporating vacancy oxygen enhances the dielectric properties. Thus, the interesting dielectric constant and weak loss strengthen the use for potential applications.publishe

Genome-wide analysis of the AP2/ERF superfamily in apple and transcriptional evidence of ERF involvement in scab pathogenesis.

The APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) superfamily of transcriptional regulators is involved in several growth, development and stress responses processes in higher plants. Currently, the available information on the biological roles of AP2/ERF genes is derived from Arabidopsis thaliana. In the present work, we have investigated genomic and transcriptional aspects of AP2/ERF genes in the economically important perennial species, Malus × domestica. We have identified 259 sequences containing at least one ERF domain in apple genome. The vast majority of the putative proteins display predicted nuclear localization, compatible with a biological role in transcription regulation. The AP2 and ERF families are greatly expanded in apple. Wholegenome analyses in other plant species have identified a single genomic sequence with divergent ERF, whereas in apple seven soloists are present. In the apple genome, the most noteworthy expansion occurred in subgroups V, VIII and IX of the ERF family. Expression profiling analyses have revealed the association of ripeninginvolved ERF genes to scab (Venturia inequalis) pathogenesis in the susceptible Gala cultivar, indicating that gene expansion processes were accompanied by functional divergence. The presented analyses of AP2/ERF genes in apple provide evidences of shared ethylenemediated signaling pathways in ripening and disease responses

The mitochondrial elongation factor LeEF-Tsmt is regulated during tomato fruit ripening and upon wounding and ethylene treatment.

A gene encoding an elongation factor LeEF-Tsmt that participates in the protein synthesis process in mitochondria shows strong expression in ripening fruit as compared to other organs. It is strongly up-regulated during the first stages of the ripening process in parallel with the climacteric rise in respiration. LeEF-Tsmt expression is stimulated by ethylene, wounding and high temperature but ethylene-insensitive mutants exhibit normal expression. Transgenic fruit have been generated in which LeEF-Tsmt has been constitutively up- and down-regulated. Surprisingly, altering the expression of the gene by genetic transformation with antisense and sense LeEF-Tsmt constructs did not affect the pattern of respiration and ethylene production during ripening and upon wounding. In addition, expression of the alternative oxidase gene which is known to play an important role in respiratory climacteric was not affected. Possible reasons for the absence of effect on respiration of variations of LeEF-Tsmt gene expression are discussed

A conserved phosphorylation site regulates the transcriptional function of ETHYLENE-INSENSITIVE3-like1 in tomato

ETHYLENE-INSENSITIVE3/ETHYLENE-INSENSITIVE3-like (EIN3/EIL) transcription factors are important downstream components of the ethylene transduction pathway known to regulate the transcription of early ethylene-responsive genes in plants. Previous studies have shown that phosphorylation can repress their transcriptional activity by promoting protein degradation. The present study identifies a new phosphorylation region named EPR1 (EIN3/EIL phosphorylation region 1) in tomato EIL1 proteins. The functional significance of EPR1 was tested by introducing mutations in this region of the Sl-EIL1 gene and by expressing these mutated versions in transgenic tomato plants. Transient expression data and phenotypic analysis of the transgenic lines indicated that EPR1 is essential for the transcriptional activity of Sl-EIL1. Moreover, mutation in the EPR1 site that prevents phosphorylation abolishes ethylene constitutive responses normally displayed by the Sl-EIL1-overexpressing lines. Bimolecular fluorescence complementation (BiFC) studies showed that the presence of a functional phosphorylation site within EPR1 is instrumental in the dimerization of Sl-EIL1 proteins. The results illuminate a new molecular mechanism for the control of EIN3/EIL activity and propose a model where phosphorylation within the EPR1 promotes the dimerization process allowing the initiation of EIL-mediated transcription of early ethylene-regulated genes

Climacteric fruit ripening: Ethylene-dependent and independent regulation of ripening pathways in melon fruit

Cantaloupe melons have a typical climacteric behaviour with ethylene playing a major role in the regulation of the ripening process and affecting the ripening rate. Crossing of Cantaloupe Charentais melon with a non-climacteric melon indicated that the climacteric character is genetically dominant and conferred by two duplicated loci only. However, other experiments made by crossing two non-climacteric melons have generated climacteric fruit, indicating that different and complex genetic regulation exists for the climacteric character. Suppression of ethylene production by antisense ACC oxidase RNA in Charentais melon has shown that, while many ripening pathways were regulated by ethylene (synthesis of aroma volatiles, respiratory climacteric and degreening of the rind), some were ethylene-independent (initiation of climacteric, sugar accumulation, loss of acidity and coloration of the pulp). Softening of the flesh comprised both ethylene-dependent and independent components that were correlated with differential regulation of cell wall degrading genes. These results indicate that climacteric (ethylene-dependent) and non-climacteric (ethylene-independent) regulation coexist during climacteric fruit ripening. In addition, ethylenesuppressed melons allowed demonstrating that the various ethylene-dependent events exhibited differential sensitivity to ethylene and that ethylene was promoting sensitivity to chilling injury. Throughout this review, the data generated with melon are compared with those obtained with tomato and other fruit

Functional characterization of a melon alcohol acyl-transferase gene family involved in the biosynthesis of ester volatiles. Identification of the crucial role of a threonine residue for enzyme activity

Volatile esters, a major class of compounds contributing to the aroma of many fruit, are synthesized by alcohol acyl-transferases (AAT). We demonstrate here that, in Charentais melon (Cucumis melo var. cantalupensis), AAT are encoded by a gene family of at least four members with amino acid identity ranging from 84% (Cm-AAT1/Cm-AAT2) and 58% (Cm-AAT1/Cm-AAT3) to only 22% (Cm-AAT1/Cm-AAT4). All encoded proteins, except Cm-AAT2, were enzymatically active upon expression in yeast and show differential substrate preferences. Cm-AAT1 protein produces a wide range of short and long-chain acyl esters but has strong preference for the formation of E-2-hexenyl acetate and hexyl hexanoate. Cm-AAT3 also accepts a wide range of substrates but with very strong preference for producing benzyl acetate. Cm-AAT4 is almost exclusively devoted to the formation of acetates, with strong preference for cinnamoyl acetate. Site directed mutagenesis demonstrated that the failure of Cm-AAT2 to produce volatile esters is related to the presence of a 268-alanine residue instead of threonine as in all active AAT proteins. Mutating 268-A into 268-T of Cm-AAT2 restored enzyme activity, while mutating 268-T into 268-A abolished activity of Cm-AAT1. Activities of all three proteins measured with the prefered substrates sharply increase during fruit ripening. The expression of all Cm-AAT genes is up-regulated during ripening and inhibited in antisense ACC oxidase melons and in fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. The data presented in this work suggest that the multiplicity of AAT genes accounts for the great diversity of esters formed in melon

Two highly divergent alcohol dehydrogenases of melon exhibit fruit ripening-specific expression and distinct biochemical characteristics

Alcohol dehydrogenases (ADH) participate in the biosynthetic pathway of aroma volatiles in fruit by interconverting aldehydes to alcohols and providing substrates for the formation of esters. Two highly divergent ADH genes (15% identity at the amino acid level) of Cantaloupe Charentais melon (Cucumis melo var. Cantalupensis) have been isolated. Cm-ADH1 belongs to the medium-chain zinc-binding type of ADHs and is highly similar to all ADH genes expressed in fruit isolated so far. Cm-ADH2 belongs to the short-chain type of ADHs. The two encoded proteins are enzymatically active upon expression in yeast. Cm-ADH1 has strong preference for NAPDH as a co-factor, whereas Cm-ADH2 preferentially uses NADH. Both Cm-ADH proteins are much more active as reductases with Kms 10–20 times lower for the conversion of aldehydes to alcohols than for the dehydrogenation of alcohols to aldehydes. They both show strong preference for aliphatic aldehydes but Cm-ADH1 is capable of reducing branched aldehydes such as 3-methylbutyraldehyde, whereas Cm-ADH2 cannot. Both Cm-ADH genes are expressed specifically in fruit and up-regulated during ripening. Gene expression as well as total ADH activity are strongly inhibited in antisense ACC oxidase melons and in melon fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. These data suggest that each of the Cm-ADH protein plays a specific role in the regulation of aroma biosynthesis in melon fruit

Identification, Structure Analyses and Expression Pattern of the ERF Transcription Factor Family in Coffea arabica.

Members of the ERF Family of Transcription Factors play an important role in plant development and gene expression that regulates responses to biotic and abiotic stress. This work identified 36 ERF family genes in Coffea arabica within the AP2/ERF full domain, using the EST-based genomic resource of the Brazilian Coffee Genome Project. The ERF family genes were classified into nine of the ten existing groups through phylogenetic analysis of the deduced amino acid sequences and comparison with the sequences of the ERF family genes in Arabidopsis. In addition to the AP2 domain, other conserved domains were identified, typical of members of each group. The in silico analysis and expression profiling showed high levels of expression for libraries derived from tissues of fruits, leaves and flowers as well as for libraries subjected to water stress. These results suggest the participation of the ERF family genes of C. arabica in distinct biological functions, such as control of development, maturation, and responses to water stress. The results of this work imply in the selection of promising genes for further functional characterizations that will provide a better understanding of the complex regulatory networks related to plant development and responses to stress, opening up opportunities for coffee breeding programs