Genome-Wide Analysis of the PIN Auxin Efflux Carrier Gene Family in Coffee.

Coffee is one of the most popular beverages around the world, which is mainly produced from the allopolyploid Coffea arabica. The genomes of C. arabica and its two ancestors C. canephora and C. eugenioides have been released due to the development of next generation sequencing. However, few studies on C. arabica are related to the PIN-FORMED (PIN) auxin efflux transporter despite its importance in auxin-mediated plant growth and development. In the present study, we conducted a genome-wide analysis of the PIN gene family in the three coffee species. Totals of 17, 9 and 10 of the PIN members were characterized in C. Arabica, C. canephora and C. eugenioides, respectively. Phylogenetic analysis revealed gene loss of PIN1 and PIN2 homologs in C. arabica, as well as gene duplication of PIN5 homologs during the fractionation process after tetraploidy. Furthermore, we conducted expression analysis of PIN genes in C. arabica by in silico and qRT-PCR. The results revealed the existence of gene expression dominance in allopolyploid coffee and illustrated several PIN candidates in regulating auxin transport and homeostasis under leaf rust fungus inoculation and the tissue-specific expression pattern of C. arabica. Together, this study provides the basis and guideline for future functional characterization of the PIN gene family.

Gene expression profiling identifies pathways involved in seed maturation of Jatropha curcas.

BACKGROUND: Jatropha curcas, a tropical shrub, is a promising biofuel crop, which produces seeds with high content of oil and protein. To better understand the maturation process of J. curcas seeds and to improve its agronomic performance, a two-step approach was performed in six different maturation stages of seeds: 1) generation of the entire transcriptome of J. curcas seeds using 454-Roche sequencing of a cDNA library, 2) comparison of transcriptional expression levels using a custom Agilent 8x60K oligonucleotide microarray. RESULTS: A total of 793,875 high-quality reads were assembled into 19,382 unique full-length contigs, of which 13,507 could be annotated with Gene Ontology (GO) terms. Microarray data analysis identified 9111 probes (out of 57,842 probes), which were differentially expressed between the six maturation stages. The expression results were validated for 75 selected transcripts based on expression levels, predicted function, pathway, and length. Result from cluster analyses showed that transcripts associated with fatty acid, flavonoid, and phenylpropanoid biosynthesis were over-represented in the early stages, while those of lipid storage were over-represented in the late stages. Expression analyses of different maturation stages of J. curcas seed showed that most changes in transcript abundance occurred between the two last stages, suggesting that the timing of metabolic pathways during seed maturation in J. curcas occurs in late stages. The co-expression results showed that the hubs (CB5-D, CDR1, TT8, DFR, HVA22) with the highest number of edges, associated with fatty acid and flavonoid biosynthesis, are showing a decrease in their expression during seed maturation. Furthermore, seed development and hormone pathways are significantly well connected. CONCLUSION: The obtained results revealed differentially expressed sequences (DESs) regulating important pathways related to seed maturation, which could contribute to the understanding of the complex regulatory network during seed maturation with the focus on lipid, flavonoid and phenylpropanoid biosynthesis. This study provides detailed information on transcriptional changes during J. curcas seed maturation and provides a starting point for a genomic survey of seed quality traits. The results highlighted specific genes and processes relevant to the molecular mechanisms involved in Jatropha seed maturation. These data can also be utilized regarding other Euphorbiaceae species.

Correction: Virus versus Host Plant MicroRNAs: Who Determines the Outcome of the Interaction?

[This corrects the article DOI: 10.1371/journal.pone.0098263.].

The Pattern and Distribution of Induced Mutations in J. curcas Using Reduced Representation Sequencing.

Mutagenesis in combination with Genotyping by Sequencing (GBS) is a powerful tool for introducing variation, studying gene function and identifying causal mutations underlying phenotypes of interest in crop plant genomes. About 400 million paired-end reads were obtained from 82 ethylmethane sulfonate (EMS) induced mutants and 14 wild-type accessions of Jatropha curcas for the detection of Single Nucleotide Polymorphisms (SNPs) and Insertion/Deletions (InDels) by two different approaches (nGBS and ddGBS) on an Illumina HiSeq 2000 sequencer. Using bioinformatics analyses, 1,452 induced SNPs and InDels were identified in coding regions, which were distributed across 995 genes. The predominantly observed mutations were G/C to A/T transitions (64%), while transversions were observed at a lower frequency (36%). Regarding the effect of mutations on gene function, 18% of the mutations were located in intergenic regions. In fact, mutants with the highest number of heterozygous SNPs were found in samples treated with 0.8% EMS for 3 h. Reconstruction of the metabolic pathways showed that in total 16 SNPs were located in six KEGG pathways by nGBS and two pathways by ddGBS. The most highly represented pathways were ether-lipid metabolism and glycerophospholipid metabolism, followed by starch and sucrose metabolism by nGBS and triterpenoid biosynthesis as well as steroid biosynthesis by ddGBS. Furthermore, high genome methylation was observed in J. curcas, which might help to understand the plasticity of the Jatropha genome in response to environmental factors. At last, the results showed that continuously vegetatively propagated tissue is a fast, efficient and accurate method to dissolve chimeras, especially for long-lived plants like J. curcas. Obtained data showed that allelic variations and in silico analyses of gene functions (gene function prediction), which control important traits, could be identified in mutant populations using nGBS and ddGBS. However, the handling of GBS data is more difficult and more challenging than the traditional TILLING strategy in mutated plants, since the Jatropha genome sequence is incomplete, which makes alignment and variant analysis of target sequence reads challenging to perform and interpret. Therefore, providing a complete Jatropha reference genome sequence with high quality should be a priority for any breeding program.

Editorial: Sustainable production of renewable energy from non-food crops.

Since the world faced the petroleum crisis in the 1970s and people started to realize the limitation of fossil energy resources coupled with concerns over the effects of increasing carbon dioxide in the atmosphere, major efforts were devoted to the search for alternative energy sources.

Geographic origin is not supported by the genetic variability found in a large living collection of Jatropha curcas with accessions from three continents.

Increasing economic interest in Jatropha curcas requires a major research focus on the genetic background and geographic origin of this non-edible biofuel crop. To determine the worldwide genetic structure of this species, amplified fragment length polymorphisms, inter simple sequence repeats, and novel single nucleotide polymorphisms (SNPs) were employed for a large collection of 907 J. curcas accessions and related species (RS) from three continents, 15 countries and 53 regions. PCoA, phenogram, and cophenetic analyses separated RS from two J. curcas groups. Accessions from Mexico, Bolivia, Paraguay, Kenya, and Ethiopia with unknown origins were found in both groups. In general, there was a considerable overlap between individuals from different regions and countries. The Bayesian approach using STRUCTURE demonstrated two groups with a low genetic variation. Analysis of molecular varience revealed significant variation among individuals within populations. SNPs found by in silico analyses of Δ12 fatty acid desaturase indicated possible changes in gene expression and thus in fatty acid profiles. SNP variation was higher in the curcin gene compared to genes involved in oil production. Novel SNPs allowed separating toxic, non-toxic, and Mexican accessions. The present study confirms that human activities had a major influence on the genetic diversity of J. curcas, not only because of domestication, but also because of biased selection.

Impact of sulfur and vitamin C on the allergenicity of Mal d 2 from apple (Malus domestica).

Mal d 2 is a minor allergen from apple which shows a high conformational stability due to its eight conserved disulfide bridges. Chemical reduction of disulfide bridges and linearization of Mal d 2 lead to enhanced IgE reactivity in vitro and indicate a higher potential for allergenicity. Since food preservatives such as sulfur and vitamin C are reducing and denaturing agents, their influence on Mal d 2 allergenicity was verified by simulated food processing conditions. The immunoreactivity of purified Mal d 2 was investigated after different treatments in vitro and in vivo using IgG/IgE Western blotting, mediator-releasing cell assay, and skin prick and oral smear tests. The conformational changes of Mal d 2 upon addition of 1% and 5% vitamin C were also monitored by attenuated total reflectance Fourier transform infrared spectroscopy. The results show no positive skin and oral smear test reactivity to native, heated, or vitamin C-treated purified Mal d 2. Furthermore, the results confirm that sulfur in combination with heat treatment can influence the structural integrity and thus the allergenicity of Mal d 2, while vitamin C is too weak as a reducing agent to change allergenicity.

Virus versus host plant microRNAs: who determines the outcome of the interaction?

Considering the importance of microRNAs (miRNAs) in the regulation of essential processes in plant pathogen interactions, it is not surprising that, while plant miRNA sequences counteract viral attack via antiviral RNA silencing, viruses in turn have developed antihost defense mechanisms blocking these RNA silencing pathways and establish a counter-defense. In the current study, computational and stem-loop Reverse Transcription - Polymerase Chain Reaction (RT-PCR) approaches were employed to a) predict and validate virus encoded mature miRNAs (miRs) in 39 DNA-A sequences of the bipartite genomes of African cassava mosaic virus (ACMV) and East African cassava mosaic virus-Uganda (EACMV-UG) isolates, b) determine whether virus encoded miRs/miRs* generated from the 5'/3' harpin arms have the capacity to bind to genomic sequences of the host plants Jatropha or cassava and c) investigate whether plant encoded miR/miR* sequences have the potential to bind to the viral genomes. Different viral pre-miRNA hairpin sequences and viral miR/miR* length variants occurring as isomiRs were predicted in both viruses. These miRNAs were located in three Open Reading Frames (ORFs) and in the Intergenic Region (IR). Moreover, various target genes for miRNAs from both viruses were predicted and annotated in the host plant genomes indicating that they are involved in biotic response, metabolic pathways and transcription factors. Plant miRs/miRs* from conserved and highly expressed families were identified, which were shown to have potential targets in the genome of both begomoviruses, representing potential plant miRNAs mediating antiviral defense. This is the first assessment of predicted viral miRs/miRs* of ACMV and EACMV-UG and host plant miRNAs, providing a reference point for miRNA identification in pathogens and their hosts. These findings will improve the understanding of host- pathogen interaction pathways and the function of viral miRNAs in Euphorbiaceous crop plants.

Jatropha curcas, a biofuel crop: functional genomics for understanding metabolic pathways and genetic improvement.

Jatropha curcas is currently attracting much attention as an oilseed crop for biofuel, as Jatropha can grow under climate and soil conditions that are unsuitable for food production. However, little is known about Jatropha, and there are a number of challenges to be overcome. In fact, Jatropha has not really been domesticated; most of the Jatropha accessions are toxic, which renders the seedcake unsuitable for use as animal feed. The seeds of Jatropha contain high levels of polyunsaturated fatty acids, which negatively impact the biofuel quality. Fruiting of Jatropha is fairly continuous, thus increasing costs of harvesting. Therefore, before starting any improvement program using conventional or molecular breeding techniques, understanding gene function and the genome scale of Jatropha are prerequisites. This review presents currently available and relevant information on the latest technologies (genomics, transcriptomics, proteomics and metabolomics) to decipher important metabolic pathways within Jatropha, such as oil and toxin synthesis. Further, it discusses future directions for biotechnological approaches in Jatropha breeding and improvement.

Sample preparation and 2-DE procedure for protein expression profiling of black microcolonial fungi.

The ecology and stress adaptation of black rock inhabiting fungi in hot and cold extreme environments are not yet well understood. Two-dimensional gel electrophoresis (2-DE) is a promising tool to study the protein expression profiling and the metabolic status of microorganisms under stress conditions. The sample preparation has been shown to be the bottleneck for high resolution protein separation in 2-DE. For this purpose conditions must be optimized to obtain reliable and reproducible results. In addition, due to a multilayered and strongly melanized cell wall of black microcolonial fungi, special protocols for cell disruption and processing are required. In the present study, the protocol for protein extraction was established and optimized for the black yeast Exophiala jeanselmei MA 2853. The same protocol was successfully examined also for the meristematic fungus Coniosporium perforans MA 1299. Among the three procedures evaluated, trichloroacetic acid (TCA) precipitation, TCA/acetone precipitation, and phenol extraction combined with methanol/ammonium acetate precipitation, the latter showed to be the best method for black yeasts and meristematic fungi. Penicillium chrysogenum was used as reference strain.

Biotechnological approaches to determine the impact of viruses in the energy crop plant Jatropha curcas.

BACKGROUND: Geminiviruses infect a wide range of plant species including Jatropha and cassava both belonging to family Euphorbiaceae. Cassava is traditionally an important food crop in Sub - Saharan countries, while Jatropha is considered as valuable biofuel plant with great perspectives in the future. RESULTS: A total of 127 Jatropha samples from Ethiopia and Kenya and 124 cassava samples from Kenya were tested by Enzyme-Linked Immunosorbent Assay (ELISA) for RNA viruses and polymerase chain reaction for geminiviruses. Jatropha samples from 4 different districts in Kenya and Ethiopia (analyzed by ELISA) were negative for all three RNA viruses tested: Cassava brown streak virus (CBSV), Cassava common mosaic virus, Cucumber mosaic virus, Three cassava samples from Busia district (Kenya) contained CBSV. Efforts to develop diagnostic approaches allowing reliable pathogen detection in Jatropha, involved the amplification and sequencing of the entire DNA A molecules of 40 Kenyan isolates belonging to African cassava mosaic virus (ACMV) and East African cassava mosaic virus - Uganda. This information enabled the design of novel primers to address different questions: a) primers amplifying longer sequences led to a phylogenetic tree of isolates, allowing some predictions on the evolutionary aspects of Begomoviruses in Jatrophia; b) primers amplifying shorter sequences represent a reliable diagnostic tool. This is the first report of the two Begomoviruses in J. curcas. Two cassava samples were co - infected with cassava mosaic geminivirus and CBSV. A Defective DNA A of ACMV was found for the first time in Jatropha. CONCLUSION: Cassava geminiviruses occurring in Jatropha might be spread wider than anticipated. If not taken care of, this virus infection might negatively impact large scale plantations for biofuel production. Being hosts for similar pathogens, the planting vicinity of the two crop plants needs to be handled carefully.

Genome size, karyotyping and FISH physical mapping of 45S and 5S genes in two cherry rootstocks: Prunus subhirtella and Prunus incisa xserrula.

Physical mapping of rDNA genes was used to karyotype two cherry rootstocks, Prunus subhirtella (PAR) and P. incisa xserrula (PIS), since the identification of homologous pairs is hampered by the high degree of similarity and the small size of the chromosomes. The genome size of PAR and PIS was obtained by flow cytometry. Chromosomal landmarks were identified by FISH of 45S rDNA and 5S rDNAs with DIG or Biotin labelled rDNA probes. Double-colour FISH landmarks were located at the terminal regions of chromosomes, differentiating three chromosome groups. Chromosomes 1, 4, 5 and 8 of both species showed no hybridization signals. The chromosomes 2, 3 and 6 of both species carried signals of 45S rDNA. In PAR, chromosomes 2 and 3 carried the 5S rDNA signals, which in PIS was located on chromosome 7. Secondary constrictions were not visible in PAR chromosomes, while PIS chromosome 2 carries one. The results suggest that the degree of chromosomal differentiation among the two Prunus species is low. This study is the first report of using FISH to determine the number and the physical position of rDNAs in PIS and PAR.

Transgene silencing in grapevines transformed with GFLV resistance genes: analysis of variable expression of transgene, siRNAs production and cytosine methylation.

Eight transgenic grapevine lines transformed with the coat protein gene of Grapevine fanleaf virus (GFLV-CP) were analyzed for a correlation between transgene expression, siRNAs production and DNA methylation. Bisulphite genome sequencing was used for a comprehensive analysis of DNA methylation. Methylated cytosine residues of CpG and CpNpG sites were detected in the GFLV-CP transgene, in the T7 terminator and in the 35S promoter of three grapevines without transgene expression, but no detectable level of siRNAs was recorded in these lines. The detailed analysis of 8 lines revealed the complex arrangements of T-DNA and integrated binary vector sequences as crucial factors that influence transgene expression. After inoculation with GFLV, no change in the levels of cytosine methylation was observed, but transgenic and untransformed plants produced short siRNAs (21-22 nt) indicating that the grapevine plants responded to GFLV infection by activating a post-transcriptional gene silencing mechanism.

Bioactive compounds in berries relevant to human health.

Berries contain powerful antioxidants, potential allergens, and other bioactive compounds. Genetic and environmental factors affect production and storage of such compounds. For this reason breeding and biotechnological approaches are currently used to control or to increase the content of specific health-related compounds in fruits. This work reviews the main bioactive compounds determining the nutritional quality of berries, the major factors affecting their content and activity, and the genetic options currently available to achieve new genotypes able to provide, under controlled cultivation conditions, berries with the proper balance of bioactive compounds for improving consumer health.

Detection of phytoplasmas of temperate fruit trees.

Phytoplasmas are associated with hundreds of plant diseases globally. Many fruit tree phytoplasmas are transmitted by insect vectors or grafting, are considered quarantine organisms and a major economic threat to orchards. Diagnosis can be difficult, but immunochemical and molecular methods have been developed.

Expression of calmodulin and lipid transfer protein genes in Prunus incisa x serrula under different stress conditions.

Calcium-binding proteins and pathogenesis-related (PR) proteins are crucial components of the inducible repertoire of plant stress and defence. Considering the important role played by calmodulin (CaM) and lipid transfer protein (LTP) in mediating plant signal transduction, the present study investigated the expression of Ltp and CaM genes in Prunus incisa x serrula (PIS) under various abiotic stress conditions. The aim of this study is to find out whether expression of these proteins is regulated in parallel or independently and to compare the expression profiles of CaM and allergenic proteins like Ltp under different stress conditions. Southern blot analyses indicated that Ltp and CaM are encoded by at least two to four genes, which might be indicative for the expected variability and presence of isoforms. Transcription levels of both genes were analysed in leaves and roots of micropropagated plantlets under low and high temperatures, salicylic acid and wounding stress, harvested after 0, 0.5, 1, 2, 4, 10, 24 and 72 h. Real Time qPCR data showed that both genes respond differently to various stresses. Furthermore, a high variation in transcription levels of both genes was observed in leaf tissues, while in roots both genes were expressed at a lower extent and down-regulated. Western blot analyses indicated that after 24 h the amount of CaM protein is higher, while the amount of LTP is lower in various stresses. Results obtained suggest that CaM and LTP are differentially regulated in response to different stresses in PIS plants, and additionally show tissue-specific expression, hinting at a potential role of different isoforms.

Functional genomics of allergen gene families in fruits.

Fruit consumption is encouraged for health reasons; however, fruits may harbour a series of allergenic proteins that may cause discomfort or even represent serious threats to certain individuals. Thus, the identification and characterization of allergens in fruits requires novel approaches involving genomic and proteomic tools. Since avoidance of fruits also negatively affects the quality of patients' lives, biotechnological interventions are ongoing to produce low allergenic fruits by down regulating specific genes. In this respect, the control of proteins associated with allergenicity could be achieved by fine tuning the spatial and temporal expression of the relevant genes.

Strategies for antiviral resistance in transgenic plants.

Genetic engineering offers a means of incorporating new virus resistance traits into existing desirable plant cultivars. The initial attempts to create transgenes conferring virus resistance were based on the pathogen-derived resistance concept. The expression of the viral coat protein gene in transgenic plants was shown to induce protective effects similar to classical cross protection, and was therefore distinguished as 'coat-protein-mediated' protection. Since then, a large variety of viral sequences encoding structural and non-structural proteins were shown to confer resistance. Subsequently, non-coding viral RNA was shown to be a potential trigger for virus resistance in transgenic plants, which led to the discovery of a novel innate resistance in plants, RNA silencing. Apart from the majority of pathogen-derived resistance strategies, alternative strategies involving virus-specific antibodies have been successfully applied. In a separate section, efforts to combat viroids in transgenic plants are highlighted. In a final summarizing section, the potential risks involved in the introduction of transgenic crops and the specifics of the approaches used will be discussed.

Identification of four IgE-reactive proteins in raspberry (Rubus ideaeus L.).

IgE-reactive proteins in raspberry (Rubus ideaus L.) were identified using PCR, RT-PCR, 2-DE and MS/MS peptide sequencing. Specific polyclonal antibodies and patient sera were used in Western blotting to identify crossreactive epitopes. Initially, two potential allergens Rub i 1 and Rub i 3 were detected using PCR, showing high sequence identity to proteins in Rosaceous species like Mal d 1 and Mal d 3 from apple, Pru av 1 and Pru av 3 from cherry and Pru p 1 and Pru p 3 from peach. Furthermore, de novo identified peptides of a protein band at about 30 kDa reacting with most of the patient sera tested (> 80%) revealed a high sequence homology with class III chitinases. Raspberry chitinase, when subjected to glycoproteomic analysis, showed typical complex plant-type N-glycans with a core alpha1,3 fucose and a beta1,2 xylose at least at one position, indicating the presence of crossreacting carbohydrate determinants (CCDs). Finally, MS/MS analysis revealed an IgE-reactive raspberry cyclophilin, homologous to Bet v 7. Results obtained suggest that the consumption of raspberries might be responsible for adverse reactions in sensitised individuals.

Stress regulated expression of the GUS-marker gene (uidA) under the control of plant calmodulin and viral 35S promoters in a model fruit tree rootstock: Prunus incisa x serrula.

The fact that calmodulin genes (CaM) are tightly associated with the Ca(2+) regulatory pathway, as well as their putative role in plant defence against pathogens, indicate a potential use of alternative plant promoters to express genes of interest in specific tissues or developmental stages. To study the expression level of the apple CaM promoter, 981 bp sequences upstream were fused to the uidA gene, introduced into cherry and compared with a 35S-GUS construct. Transgene copy number and transgenic expression levels were analysed using Southern blot, Western blot and RT-PCR techniques. Transcription levels were assessed by GUS fluorometry, histochemistry and real-time PCR techniques in leaves of plantlets grown in vitro under various abiotic stresses like low- and high-temperature, salicylic acid and wounding, harvested after 0, 0.5, 1, 2, 4, 10, 24 and 72 h. Histochemical analyses showed staining only in veins and petioles of CaM-GUS lines, while in 35S-GUS plants staining extended to the entire leaf. Furthermore, real-time qPCR data indicate that both promoters are differently regulated by various stresses. Obtained results suggest that the selected apple CaM promoter responsible for the expression of a gene in vascular tissues may offer interesting perspectives for plant defense programs.