Genome-Wide Identification and Spatial Expression Analysis of Histone Modification Gene Families in the Rubber Dandelion Taraxacum kok-saghyz.

Taraxacum kok-saghyz (Tks), also known as the Russian dandelion, is a recognized alternative source of natural rubber quite comparable, for quality and use, to the one obtained from the so-called rubber tree, Hevea brasiliensis. In addition to that, Tks roots produce several other compounds, including inulin, whose use in pharmaceutical and dietary products is quite extensive. Histone-modifying genes (HMGs) catalyze a series of post-translational modifications that affect chromatin organization and conformation, which, in turn, regulate many downstream processes, including gene expression. In this study, we present the first analysis of HMGs in Tks. Altogether, we identified 154 putative Tks homologs: 60 HMTs, 34 HDMs, 42 HATs, and 18 HDACs. Interestingly, whilst most of the classes showed similar numbers in other plant species, including M. truncatula and A. thaliana, HATs and HMT-PRMTs were indeed more abundant in Tks. Composition and structure analysis of Tks HMG proteins showed, for some classes, the presence of novel domains, suggesting a divergence from the canonical HMG model. The analysis of publicly available transcriptome datasets, combined with spatial expression of different developmental tissues, allowed us to identify several HMGs with a putative role in metabolite biosynthesis. Overall, our work describes HMG genomic organization and sets the premises for the functional characterization of epigenetic modifications in rubber-producing plants.

Functional Characterization of MtrGSTF7, a Glutathione S-Transferase Essential for Anthocyanin Accumulation in Medicago truncatula.

Flavonoids are essential compounds widespread in plants and exert many functions such as defence, definition of organ colour and protection against stresses. In Medicago truncatula, flavonoid biosynthesis and accumulation is finely regulated in terms of tissue specificity and induction by external factors, such as cold and other stresses. Among flavonoids, anthocyanin precursors are synthesised in the cytoplasm, transported to the tonoplast, then imported into the vacuole for further modifications and storage. In the present work, we functionally characterised MtrGSTF7, a phi-class glutathione S-transferase involved in anthocyanin transport to the tonoplast. The mtrgstf7 mutant completely lost the ability to accumulate anthocyanins in leaves both under control and anthocyanin inductive conditions. On the contrary, this mutant showed an increase in the levels of soluble proanthocyanidins (Pas) in their seeds with respect to the wild type. By complementation and expression data analysis, we showed that, differently from A. thaliana and similarly to V. vinifera, transport of anthocyanin and proanthocyanidins is likely carried out by different GSTs belonging to the phi-class. Such functional diversification likely results from the plant need to finely tune the accumulation of diverse classes of flavonoids according to the target organs and developmental stages.

Genome-Wide Identification of Histone Modification Gene Families in the Model Legume Medicago truncatula and Their Expression Analysis in Nodules.

Histone methylation and acetylation are key processes in the epigenetic regulation of plant growth, development, and responses to environmental stimuli. The genes encoding for the enzymes that are responsible for these chromatin post-translational modifications, referred to as histone modification genes (HMGs), have been poorly investigated in Leguminosae species, despite their importance for establishment and activity of nitrogen-fixing nodules. In silico analysis of Medicago truncatula HMGs identified 81 histone methyltransferases, 46 histone demethylases, 64 histone acetyltransferases, and 15 histone deacetylases. MtHMGs were analyzed for their structure and domain composition, and some combinations that were not yet reported in other plant species were identified. Genes have been retrieved from M. truncatula A17 and R108 genotypes as well as M. sativa CADL and Zhongmu No.1; the gene number and distribution were compared with Arabidopsis thaliana. Furthermore, by analyzing the expression data that were obtained at various developmental stages and in different zones of nitrogen-fixing nodules, we identified MtHMG loci that could be involved in nodule development and function. This work sets a reference for HMG genomic organization in legumes which will be useful for functional investigation that is aimed at elucidating HMGs involvement in nodule development and symbiotic nitrogen fixation.

Correction: Transcript Analysis and Regulative Events during Flower Development in Olive (Olea europaea L.).

[This corrects the article DOI: 10.1371/journal.pone.0152943.].

Cryptochromes and the Circadian Clock: The Story of a Very Complex Relationship in a Spinning World.

Cryptochromes are flavin-containing blue light photoreceptors, present in most kingdoms, including archaea, bacteria, plants, animals and fungi. They are structurally similar to photolyases, a class of flavoproteins involved in light-dependent repair of UV-damaged DNA. Cryptochromes were first discovered in Arabidopsis thaliana in which they control many light-regulated physiological processes like seed germination, de-etiolation, photoperiodic control of the flowering time, cotyledon opening and expansion, anthocyanin accumulation, chloroplast development and root growth. They also regulate the entrainment of plant circadian clock to the phase of light-dark daily cycles. Here, we review the molecular mechanisms by which plant cryptochromes control the synchronisation of the clock with the environmental light. Furthermore, we summarise the circadian clock-mediated changes in cell cycle regulation and chromatin organisation and, finally, we discuss a putative role for plant cryptochromes in the epigenetic regulation of genes.

Comparative transcriptomics between high and low rubber producing Taraxacum kok-saghyz R. plants.

BACKGROUND: Taraxacum kok-saghyz R. (Tks) is a promising alternative species to Hevea brasiliensis for production of high quality natural rubber (NR). A comparative transcriptome analysis of plants with differential production of NR will contribute to elucidate which genes are involved in the synthesis, regulation and accumulation of this natural polymer and could help to develop Tks into a rubber crop. RESULTS: We measured rubber content in the latex of 90 individual Tks plants from 9 accessions, observing a high degree of variability. We carried out de novo root transcriptome sequencing, assembly, annotation and comparison of gene expression of plants with the lower (LR plants) and the higher rubber content (HR plants). The transcriptome analysis also included one plant that did not expel latex, in principle depleted of latex transcripts. Moreover, the transcription of some genes well known to play a major role in rubber biosynthesis, was probed by qRT-PCR. Our analysis showed a high modulation of genes involved in the synthesis of NR between LR and HR plants, and evidenced that genes involved in sesquiterpenoids, monoterpenoids and phenylpropanoid biosynthesis are upregulated in LR plants. CONCLUSIONS: Our results show that a higher amount of rubber in the latex in HR plants is positively correlated with high expression levels of a number of genes directly involved in rubber synthesis showing that NR production is highly controlled at transcriptional level. On the other hand, lower amounts of rubber in LR plants is related with higher expression of genes involved in the synthesis of other secondary metabolites that, we hypothesize, may compete towards NR biosynthesis. This dataset represents a fundamental genomic resource for the study of Tks and the comprehension of the synthesis of NR and other biochemically and pharmacologically relevant compounds in the Taraxacum genus.

Omics approaches on fresh-cut lettuce reveal global molecular responses to sodium hypochlorite and peracetic acid treatment.

BACKGROUND: Lettuce is a leafy vegetable that is extensively commercialized as a ready-to-eat product because of its widespread use in human nutrition as salad. It is well known that washing treatments can severely affect the quality and shelf-life of ready-to-eat vegetables. The study presented here evaluated the effect of two washing procedures on fresh-cut lettuce during storage. RESULTS: An omics approach was applied to reveal global changes at molecular level induced by peracetic acid washing in comparison with sodium hypochlorite treatment. Microbiological analyses were also performed to quantify total bacterial abundance and composition. The study revealed wide metabolic alterations induced by the two sanitizers. In particular, transcriptomic and proteomic analyses pointed out a number of transcripts and proteins differentially accumulated in response to peracetic acid washing, mainly occurring on the first day of storage. In parallel, different microbiota composition and significant reduction in total bacterial load following washing were also observed. CONCLUSION: The results provide useful information for the fresh-cut industry to select an appropriate washing procedure preserving fresh-like attributes as much as possible during storage of the end product. Molecular evidence indicated peracetic acid to be a valid alternative to sodium hypochlorite as sanitizer solution. © 2017 Society of Chemical Industry.

The transcriptional response to the olive fruit fly (Bactrocera oleae) reveals extended differences between tolerant and susceptible olive (Olea europaea L.) varieties.

The olive fruit fly Bactrocera oleae (Diptera: Tephritidae) is the most devastating pest of cultivated olive (Olea europaea L.). Intraspecific variation in plant resistance to B. oleae has been described only at phenotypic level. In this work, we used a transcriptomic approach to study the molecular response to the olive fruit fly in two olive cultivars with contrasting level of susceptibility. Using next-generation pyrosequencing, we first generated a catalogue of more than 80,000 sequences expressed in drupes from approximately 700k reads. The assembled sequences were used to develop a microarray layout with over 60,000 olive-specific probes. The differential gene expression analysis between infested (i.e. with II or III instar larvae) and control drupes indicated a significant intraspecific variation between the more tolerant and susceptible cultivar. Around 2500 genes were differentially regulated in infested drupes of the tolerant variety. The GO annotation of the differentially expressed genes implies that the inducible resistance to the olive fruit fly involves a number of biological functions, cellular processes and metabolic pathways, including those with a known role in defence, oxidative stress responses, cellular structure, hormone signalling, and primary and secondary metabolism. The difference in the induced transcriptional changes between the cultivars suggests a strong genetic role in the olive inducible defence, which can ultimately lead to the discovery of factors associated with a higher level of tolerance to B. oleae.

Correction: Pyrosequencing Unveils Cystic Fibrosis Lung Microbiome Differences Associated with a Severe Lung Function Decline.

[This corrects the article DOI: 10.1371/journal.pone.0156807.].

Pyrosequencing Unveils Cystic Fibrosis Lung Microbiome Differences Associated with a Severe Lung Function Decline.

Chronic airway infection is a hallmark feature of cystic fibrosis (CF) disease. In the present study, sputum samples from CF patients were collected and characterized by 16S rRNA gene-targeted approach, to assess how lung microbiota composition changes following a severe decline in lung function. In particular, we compared the airway microbiota of two groups of patients with CF, i.e. patients with a substantial decline in their lung function (SD) and patients with a stable lung function (S). The two groups showed a different bacterial composition, with SD patients reporting a more heterogeneous community than the S ones. Pseudomonas was the dominant genus in both S and SD patients followed by Staphylococcus and Prevotella. Other than the classical CF pathogens and the most commonly identified non-classical genera in CF, we found the presence of the unusual anaerobic genus Sneathia. Moreover, the oligotyping analysis revealed the presence of other minor genera described in CF, highlighting the polymicrobial nature of CF infection. Finally, the analysis of correlation and anti-correlation networks showed the presence of antagonism and ecological independence between members of Pseudomonas genus and the rest of CF airways microbiota, with S patients showing a more interconnected community in S patients than in SD ones. This population structure suggests a higher resilience of S microbiota with respect to SD, which in turn may hinder the potential adverse impact of aggressive pathogens (e.g. Pseudomonas). In conclusion, our findings shed a new light on CF airway microbiota ecology, improving current knowledge about its composition and polymicrobial interactions in patients with CF.

Transcript Analysis and Regulative Events during Flower Development in Olive (Olea europaea L.).

The identification and characterization of transcripts involved in flower organ development, plant reproduction and metabolism represent key steps in plant phenotypic and physiological pathways, and may generate high-quality transcript variants useful for the development of functional markers. This study was aimed at obtaining an extensive characterization of the olive flower transcripts, by providing sound information on the candidate MADS-box genes related to the ABC model of flower development and on the putative genetic and molecular determinants of ovary abortion and pollen-pistil interaction. The overall sequence data, obtained by pyrosequencing of four cDNA libraries from flowers at different developmental stages of three olive varieties with distinct reproductive features (Leccino, Frantoio and Dolce Agogia), included approximately 465,000 ESTs, which gave rise to more than 14,600 contigs and approximately 92,000 singletons. As many as 56,700 unigenes were successfully annotated and provided gene ontology insights into the structural organization and putative molecular function of sequenced transcripts and deduced proteins in the context of their corresponding biological processes. Differentially expressed genes with potential regulatory roles in biosynthetic pathways and metabolic networks during flower development were identified. The gene expression studies allowed us to select the candidate genes that play well-known molecular functions in a number of biosynthetic pathways and specific biological processes that affect olive reproduction. A sound understanding of gene functions and regulatory networks that characterize the olive flower is provided.

The coffee genome provides insight into the convergent evolution of caffeine biosynthesis.

Coffee is a valuable beverage crop due to its characteristic flavor, aroma, and the stimulating effects of caffeine. We generated a high-quality draft genome of the species Coffea canephora, which displays a conserved chromosomal gene order among asterid angiosperms. Although it shows no sign of the whole-genome triplication identified in Solanaceae species such as tomato, the genome includes several species-specific gene family expansions, among them N-methyltransferases (NMTs) involved in caffeine production, defense-related genes, and alkaloid and flavonoid enzymes involved in secondary compound synthesis. Comparative analyses of caffeine NMTs demonstrate that these genes expanded through sequential tandem duplications independently of genes from cacao and tea, suggesting that caffeine in eudicots is of polyphyletic origin.

Tomato plants overexpressing cryptochrome 2 reveal altered expression of energy and stress-related gene products in response to diurnal cues.

In order to sense and respond to the fluctuating light conditions, higher plants possess several families of photoreceptors, such as phytochromes (PHYs), cryptochromes (CRYs) and phototropins. CRYs are responsible for photomorphogenesis and play a role in circadian, developmental and adaptive growth regulation of plants. In tomato (Solanum lycopersicum), CRY2 controls vegetative development, flowering time, fruit antioxidant content as well as the diurnal transcription of several other photoreceptor genes. We applied large-scale molecular approaches to identify altered transcripts and proteins in tomato wild-type (WT) versus a CRY2 overexpressing transgenic genotype, under a diurnal rhythm. Our results showed that tomato CRY2 profoundly affects both gene and protein expression in response to daily light cycle. Particularly altered molecular pathways are related to biotic/abiotic stress, photosynthesis, including components of the light and dark reactions and of starch and sucrose biosynthesis, as well as to secondary metabolism, such as phenylpropanoid, phenolic and flavonoid/anthocyanin biosynthesis pathways. One of the most interesting results is the coordinated up-regulation, in the transgenic genotype, of a consistent number of transcripts and proteins involved in photorespiration and photosynthesis. It is conceivable that light modulates the energetic metabolism of tomato through a fine CRY2-mediated transcriptional control.

First survey of the wheat chromosome 5A composition through a next generation sequencing approach.

Wheat is one of the world's most important crops and is characterized by a large polyploid genome. One way to reduce genome complexity is to isolate single chromosomes using flow cytometry. Low coverage DNA sequencing can provide a snapshot of individual chromosomes, allowing a fast characterization of their main features and comparison with other genomes. We used massively parallel 454 pyrosequencing to obtain a 2x coverage of wheat chromosome 5A. The resulting sequence assembly was used to identify TEs, genes and miRNAs, as well as to infer a virtual gene order based on the synteny with other grass genomes. Repetitive elements account for more than 75% of the genome. Gene content was estimated considering non-redundant reads showing at least one match to ESTs or proteins. The results indicate that the coding fraction represents 1.08% and 1.3% of the short and long arm respectively, projecting the number of genes of the whole chromosome to approximately 5,000. 195 candidate miRNA precursors belonging to 16 miRNA families were identified. The 5A genes were used to search for syntenic relationships between grass genomes. The short arm is closely related to Brachypodium chromosome 4, sorghum chromosome 8 and rice chromosome 12; the long arm to regions of Brachypodium chromosomes 4 and 1, sorghum chromosomes 1 and 2 and rice chromosomes 9 and 3. From these similarities it was possible to infer the virtual gene order of 392 (5AS) and 1,480 (5AL) genes of chromosome 5A, which was compared to, and found to be largely congruent with the available physical map of this chromosome.

Strawberry proteome characterization and its regulation during fruit ripening and in different genotypes.

Strawberry is worldwide appreciated for its unique flavour and as a source of macronutrients and high levels of antioxidants which are closely related to fruit ripening. We report the investigation of the complex physiological processes of strawberry fruit ripening at proteomic level. Multiple approaches were used to investigate strawberry fruit proteome. In particular, a proteome reference map of strawberry fruit from Queen Elisa élite genotype was achieved by 2-D analyses of proteins extracted from berries at immature, turning and red stages to isolate a set of proteins commonly present in fruit during ripening. In addition, several hundreds of proteins were identified by a combination of multidimensional liquid chromatography/tandem mass spectrometry and one dimensional SDS-PAGE coupled with nano-liquid chromatography/tandem mass spectrometry. DIGE technology was also used to identify differentially accumulated proteins during ripening and to correlate fruit protein expression with quality traits of the reference variety Queen Elisa and its parental genotypes. A number of constitutive or differentially accumulated proteins were found. Generally, the pattern of protein expression as well as the putative function of identified proteins argues for a role in major fruit physiological developmental and ripening processes. The role of some of the identified proteins is discussed in relation to strawberry fruit ripening and to quality traits. Consequently, this study provides the first characterization of the strawberry fruit proteome and the time course of variation during maturation by using multiple approaches.

Diurnal and circadian rhythms in the tomato transcriptome and their modulation by cryptochrome photoreceptors.

BACKGROUND: Circadian clocks are internal molecular time-keeping mechanisms that provide living organisms with the ability to adjust their growth and physiology and to anticipate diurnal environmental changes. Circadian clocks, without exception, respond to light and, in plants, light is the most potent and best characterized entraining stimulus. The capacity of plants to respond to light is achieved through a number of photo-perceptive proteins including cryptochromes and phytochromes. There is considerable experimental evidence demonstrating the roles of photoreceptors in providing light input to the clock. METHODOLOGY: In order to identify genes regulated by diurnal and circadian rhythms, and to establish possible functional relations between photoreceptors and the circadian clock in tomato, we monitored the temporal transcription pattern in plants entrained to long-day conditions, either by large scale comparative profiling, or using a focused approach over a number of photosensory and clock-related genes by QRT-PCR. In parallel, focused transcription analyses were performed in cry1a- and in CRY2-OX tomato genotypes. CONCLUSIONS: We report a large series of transcript oscillations that shed light on the complex network of interactions among tomato photoreceptors and clock-related genes. Alteration of cryptochrome gene expression induced major changes in the rhythmic oscillations of several other gene transcripts. In particular, over-expression of CRY2 had an impact not only on day/night fluctuations but also on rhythmicity under constant light conditions. Evidence was found for widespread diurnal oscillations of transcripts encoding specific enzyme classes (e.g. carotenoid biosynthesis enzymes) as well as for post-transcriptional diurnal and circadian regulation of the CRY2 transcript.

CRY-DASH gene expression is under the control of the circadian clock machinery in tomato.

Recently a new member of the blue-light photoreceptor family, CRY-DASH, was reported in Arabidopsis, though its distinctive biological functions are still unclear. We characterized the CRY-DASH gene of tomato and evidenced that its mRNA is expressed in both seeds and adult organs showing diurnal and circadian fluctuations. Moreover, the CRY-DASH transcription pattern is altered in both in a cry1a mutant and in a transgenic CRY2 overexpressor suggesting that CRY-DASH regulation must be mediated at least partially by an interaction of CRY1a and CRY2 with the timekeeping mechanism.

A novel additional group II intron distinguishes the mitochondrial rps3 gene in gymnosperms.

Comparative analysis of the ribosomal protein S3 gene (rps3) in the mitochondrial genome of Cycas with newly sequenced counterparts from Magnolia and Helianthus and available sequences from higher plants revealed that the positional clustering with the genes for ribosomal protein S19 (rps19) and L16 (rpl16) is preserved in gymnosperms. However, in contrast to the other land plant species, the rps3 gene in Cycas mitochondria is unique in possessing a second intron: rps3i2. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the transcripts generated from the rps19-rps3-rpl16 cluster in Cycas mitochondria demonstrated that the genes are cotranscribed and extensively modified by RNA editing and that both introns are efficiently spliced. Despite remarkable size heterogeneity, the Cycas rps3i1 can be shown to be homologous to the group IIA introns present within the rps3 gene of algae and land plants, including Magnolia and Helianthus. Conversely, sequences similar to the rps3i2 have not been reported previously. On the basis of conserved primary and secondary structure the second intervening sequence interrupting the Cycas rps3 gene has been classified as a group II intron. The close relationship of the rps3i2 to a group of different plant mitochondrial introns is intriguing and suggestive of a mitochondrial derivation for this novel intervening sequence. Interestingly, the rps3i2 appears to be conserved at the same gene location in other gymnosperms. Furthermore, the pattern of the rps3i2 distribution among algae and land plants provides evidence for the evolutionary acquisition of this novel intron in gymnosperms via intragenomic transposition or retrotransposition.

Striking differences in RNA editing requirements to express the rps4 gene in magnolia and sunflower mitochondria.

The ribosomal protein S4 gene (rps4) has been identified as a single copy sequence in the mitochondrial genomes of two distant higher plants, Magnolia and Helianthus. Sequence analysis revealed that the rps4 genes present in the magnolia and sunflower mitochondrial genomes encode S4 polypeptides of 352 and 331 amino acids, respectively, longer than their counterparts in liverwort and bacteria. Expression of the rps4 genes in the investigated higher plant mitochondria was confirmed by Western blot analysis. In Helianthus, one of two short nucleotide insertions at the 3'-end introduces in the coding region a premature termination codon. Northern hybridizations and reverse transcription-polymerase chain reaction analysis demonstrated that the monocistronic RNA transcripts generated from the rps4 locus in Magnolia and Helianthus mitochondria are modified by RNA editing at 28 and 13 positions, respectively. Although evolutionarily conserved, RNA editing requirements of the rps4 appear more extensive in Magnolia than in Helianthus and in the other higher plants so far investigated. Furthermore, our analysis also suggests that selection of editing sites is RNA sequence-specific in a duplicated sequence context.