Isoenzymes of the Flavonoid and Phenylpropanoid Pathways Show Organ-Specific Regulation during Apple Fruit Development.

Elucidating the molecular mechanisms controlling fruit development is a primary target for the improvement of new apple (Malus × domestica Borkh.) cultivars. The first two weeks of development following pollination are crucial to determine fruit characteristics. During this period, a lot of changes take place in apple fruit, going from rapid cell division to the production of important metabolites. In this work, attention was focused on the phenylpropanoid and flavonoid pathways responsible for the production of numerous compounds contributing to fruit quality, such as flavonols, catechins, dihydrochalcones and anthocyanins. A total of 17 isoenzymes were identified, belonging to seven classes of the phenylpropanoid and flavonoid pathways that, despite showing more than 80% sequence identity, showed differential expression regulation during the first two weeks of apple fruit development. This feature seems to be quite common for most of the enzymes of both pathways. Differential regulation of isoenzymes was shown to be present in both 'Golden Delicious' and a wild relative (Malus mandshurica), even though differences were also present. Each isoenzyme showed a specific pattern of expression in the flower and fruit organs, suggesting that genes coding for enzymes with the same function may control different aspects of plant biology. Finally, promoter analysis was performed in order to highlight differences in the number and type of regulatory motifs. Overall, our results indicate that the control of the expression of genes involved in the phenylpropanoid and flavonoid pathways may be very complex as not only enzymes belonging to the same class, but even putative isoenzymes, can have different roles for the plant. Such genes may represent an important regulatory mechanism, as they would allow the plant to fine-tune the processing of metabolic intermediates towards different branches of the pathway, for example, in an organ-specific way.

Exploration of alternative splicing events in ten different grapevine cultivars.

BACKGROUND: The complex dynamics of gene regulation in plants are still far from being fully understood. Among many factors involved, alternative splicing (AS) in particular is one of the least well documented. For many years, AS has been considered of less relevant in plants, especially when compared to animals, however, since the introduction of next generation sequencing techniques the number of plant genes believed to be alternatively spliced has increased exponentially. RESULTS: Here, we performed a comprehensive high-throughput transcript sequencing of ten different grapevine cultivars, which resulted in the first high coverage atlas of the grape berry transcriptome. We also developed findAS, a software tool for the analysis of alternatively spliced junctions. We demonstrate that at least 44% of multi-exonic genes undergo AS and a large number of low abundance splice variants is present within the 131.622 splice junctions we have annotated from Pinot noir. CONCLUSIONS: Our analysis shows that ~70% of AS events have relatively low expression levels, furthermore alternative splice sites seem to be enriched near the constitutive ones in some extent showing the noise of the splicing mechanisms. However, AS seems to be extensively conserved among the 10 cultivars.

Application of High-Throughput Sequencing for Comprehensive Virome Profiling in Grapevines Shows Yellows in Iran.

A comprehensive study on the whole spectrum of viruses and viroids in five Iranian grapevine cultivars was carried out using sRNA libraries prepared from phloem tissue. A comparison of two approaches to virus detection from sRNAome data indicated a significant difference in the results and performance of the aligners in viral genome reconstruction. The results showed a complex virome in terms of viral composition, abundance, and richness. Thirteen viruses and viroids were identified in five Iranian grapevine cultivars, among which the grapevine red blotch virus and grapevine satellite virus were detected for the first time in Iranian vineyards. Grapevine leafroll-associated virus 1 (GLRaV1) and grapevine fanleaf virus (GFLV) were highly dominant in the virome. However, their frequency and abundance were somewhat different among grapevine cultivars. The results revealed a mixed infection of GLRaV1/grapevine yellow speckle viroid 1 (GYSVd1) and GFLV/GYSVd1 in grapevines that exhibited yellows and vein banding. We also propose a threshold of 14% of complete reconstruction as an appropriate threshold for detection of grapevine viruses that can be used as indicators for reliable grapevine virome profiling or in quarantine stations and certification programs.

The MADS-Box Gene MdDAM1 Controls Growth Cessation and Bud Dormancy in Apple.

Apple trees require a long exposure to chilling temperature during winter to acquire competency to flower and grow in the following spring. Climate change or adverse meteorological conditions can impair release of dormancy and delay bud break, hence jeopardizing fruit production and causing substantial economic losses. In order to characterize the molecular mechanisms controlling bud dormancy in apple we focused our work on the MADS-box transcription factor gene MdDAM1. We show that MdDAM1 silencing is required for the release of dormancy and bud break in spring. MdDAM1 transcript levels are drastically reduced in the low-chill varieties 'Anna' and 'Dorsett Golden' compared to 'Golden Delicious' corroborating its role as a key genetic factor controlling the release of bud dormancy in Malus species. The functional characterization of MdDAM1 using RNA silencing resulted in trees unable to cease growth in winter and that displayed an evergrowing, or evergreen, phenotype several years after transgenesis. These trees lost their capacity to enter in dormancy and produced leaves and shoots regardless of the season. A transcriptome study revealed that apple evergrowing lines are a genocopy of 'Golden Delicious' trees at the onset of the bud break with the significant gene repression of the related MADS-box gene MdDAM4 as a major feature. We provide the first functional evidence that MADS-box transcriptional factors are key regulators of bud dormancy in pome fruit trees and demonstrate that their silencing results in a defect of growth cessation in autumn. Our findings will help producing low-chill apple variants from the elite commercial cultivars that will withstand climate change.

A multi-omics study of the grapevine-downy mildew (Plasmopara viticola) pathosystem unveils a complex protein coding- and noncoding-based arms race during infection.

Fungicides are applied intensively to prevent downy mildew infections of grapevines (Vitis vinifera) with high impact on the environment. In order to develop alternative strategies we sequenced the genome of the oomycete pathogen Plasmopara viticola causing this disease. We show that it derives from a Phytophthora-like ancestor that switched to obligate biotrophy by losing genes involved in nitrogen metabolism and γ-Aminobutyric acid catabolism. By combining multiple omics approaches we characterized the pathosystem and identified a RxLR effector that trigger an immune response in the wild species V. riparia. This effector is an ideal marker to screen novel grape resistant varieties. Our study reveals an unprecedented bidirectional noncoding RNA-based mechanism that, in one direction might be fundamental for P. viticola to proficiently infect its host, and in the other might reduce the effects of the infection on the plant.

Plant microRNAs as novel immunomodulatory agents.

An increasing body of literature is addressing the immuno-modulating functions of miRNAs which include paracrine signaling via exosome-mediated intercellular miRNA. In view of the recent evidence of intake and bioavailability of dietary miRNAs in humans and animals we explored the immuno-modulating capacity of plant derived miRNAs. Here we show that transfection of synthetic miRNAs or native miRNA-enriched fractions obtained from a wide range of plant species and organs modifies dendritic cells ability to respond to inflammatory agents by limiting T cell proliferation and consequently dampening inflammation. This immuno-modulatory effect appears associated with binding of plant miRNA on TLR3 with ensuing impairment of TRIF signaling. Similarly, in vivo, plant small RNAs reduce the onset of severity of Experimental Autoimmune Encephalomyelities by limiting dendritic cell migration and dampening Th1 and Th17 responses in a Treg-independent manner. Our results indicate a potential for therapeutic use of plant miRNAs in the prevention of chronic-inflammation related diseases.

Functional characterization of three Coffea arabica L. monoterpene synthases: insights into the enzymatic machinery of coffee aroma.

The chemical composition of the coffee beverage is extremely complex, being made up of hundreds of volatile and non-volatile compounds, many of which are generated in the thermal reactions that occur during the roasting process. However, in the raw coffee bean there are also compounds that survive roasting and are therefore extracted into the beverage. Monoterpenes are an example of this category, as their presence has been reported in the coffee flower, fruit, seed, roasted bean and in the beverage aroma. The present work describes the isolation, heterologous expression and functional characterization of three Coffea arabica cDNAs coding for monoterpene synthases. RNA was purified from C. arabica (cv. Catuai Red) flowers, seeds and fruits at 4 successive ripening stages. Degenerate primers were designed on the most conserved regions of the monoterpene synthase gene family, and then used to isolate monoterpene synthase-like sequences from the cDNA libraries. After 5'- and 3'-RACE, the complete transcripts of 4 putative C. arabica monoterpene synthases (CofarTPS) were obtained. Gene expression in different tissues and developmental stages was analysed. After heterologous expression in Escherichia coli, enzyme activity and substrate specificity were evaluated in vitro by incubation of the recombinant proteins with geranyl pyrophosphate (GPP), geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP), precursors respectively of mono-, di- and sesquiterpenes. The reaction products were characterized by HS-SPME GC-MS. CofarTPS1 was classified as a limonene synthase gene, while CofarTPS2 and 3 showed lower activity with the production of linalool and ß-myrcene.

Differential responses of Coffea arabica L. leaves and roots to chemically induced systemic acquired resistance.

Coffea arabica is susceptible to several pests and diseases, some of which affect the leaves and roots. Systemic acquired resistance (SAR) is the main defence mechanism activated in plants in response to pathogen attack. Here, we report the effects of benzo(1,2,3)thiadiazole-7-carbothioic acid-S-methyl ester (BTH), a SAR chemical inducer, on the expression profile of C. arabica. Two cDNA libraries were constructed from the mRNA isolated from leaves and embryonic roots to create 1587 nonredundant expressed sequence tags (ESTs). We developed a cDNA microarray containing 1506 ESTs from the leaves and embryonic roots, and 48 NBS-LRR (nucleotide-binding site leucine-rich repeat) gene fragments derived from 2 specific genomic libraries. Competitive hybridization between untreated and BTH-treated leaves resulted in 55 genes that were significantly overexpressed and 16 genes that were significantly underexpressed. In the roots, 37 and 42 genes were over and underexpressed, respectively. A general shift in metabolism from housekeeping to defence occurred in the leaves and roots after BTH treatment. We observed a systemic increase in pathogenesis-related protein synthesis, in the oxidative burst, and in the cell wall strengthening processes. Moreover, responses in the roots and leaves varied significantly.