Genome of wild olive and the evolution of oil biosynthesis.

Here we present the genome sequence and annotation of the wild olive tree (Olea europaea var. sylvestris), called oleaster, which is considered an ancestor of cultivated olive trees. More than 50,000 protein-coding genes were predicted, a majority of which could be anchored to 23 pseudochromosomes obtained through a newly constructed genetic map. The oleaster genome contains signatures of two Oleaceae lineage-specific paleopolyploidy events, dated at ∼28 and ∼59 Mya. These events contributed to the expansion and neofunctionalization of genes and gene families that play important roles in oil biosynthesis. The functional divergence of oil biosynthesis pathway genes, such as FAD2, SACPD, EAR, and ACPTE, following duplication, has been responsible for the differential accumulation of oleic and linoleic acids produced in olive compared with sesame, a closely related oil crop. Duplicated oleaster FAD2 genes are regulated by an siRNA derived from a transposable element-rich region, leading to suppressed levels of FAD2 gene expression. Additionally, neofunctionalization of members of the SACPD gene family has led to increased expression of SACPD2, 3, 5, and 7, consequently resulting in an increased desaturation of steric acid. Taken together, decreased FAD2 expression and increased SACPD expression likely explain the accumulation of exceptionally high levels of oleic acid in olive. The oleaster genome thus provides important insights into the evolution of oil biosynthesis and will be a valuable resource for oil crop genomics.

Isolation, molecular characterization and functional analysis of OeMT2, an olive metallothionein with a bioremediation potential.

Metallothioneins are essential in plants for metal detoxification in addition to their other roles in plant life cycle. This study reports the characterization of an olive (Olea europaea L. cv. Ayvalik) metallothionein with respect to molecular and functional properties. A cDNA encoding a type 2 metallothionein from olive was isolated from a leaf cDNA library, characterized and named OeMT2 after its molecular and functional properties. OeMT2 was expressed in Escherichia coli, and a single protein band was confirmed by protein gel blot analysis. Metal tolerance ability of bacterial cells expressing OeMT2 was determined against 0.2 mM CdCl2, 0.4 mM CdCl2 and 1 mM CuSO4 in the growth medium. Metal ion contents of bacterial cells expressing OeMT2 were measured by ICP. Metal tolerance assays and ICP measurements suggested that OeMT2 effectively binds Cu and Cd. Molecular analysis of OeMT2 revealed two introns, three exons, a short 3' UTR and a long 5' UTR. Comparing the genomic sequences from 14 olive cultivars revealed OeMT2 had both intron and exon polymorphisms dividing the cultivars into three groups. Real-time PCR analysis demonstrated that OeMT2 expresses more or less the same amounts in all tissues of the olive tree examined. The genomic copy number of OeMT2 was also determined employing real-time PCR which suggested a single copy gene in the olive genome while three other MT2 members were determined from the draft olive genome sequences of Ayvalik cultivar and that of wild olive. This is the first report on molecular and functional characterization of an olive metallothionein and shows that OeMT2 expressed in E. coli has the capability of effectively binding toxic heavy metals. This may suggest that OeMT2 plays an important role in metal homeostasis in addition to a good potential for environmental and industrial usage.

Isolation and expression analysis of cDNAs that are associated with alternate bearing in Olea europaea L. cv. Ayvalik.

BACKGROUND: Olive cDNA libraries to isolate candidate genes that can help enlightening the molecular mechanism of periodicity and / or fruit production were constructed and analyzed. For this purpose, cDNA libraries from the leaves of trees in "on year" and in "off year" in July (when fruits start to appear) and in November (harvest time) were constructed. Randomly selected 100 positive clones from each library were analyzed with respect to sequence and size. A fruit-flesh cDNA library was also constructed and characterized to confirm the reliability of each library's temporal and spatial properties. RESULTS: Quantitative real-time RT-PCR (qRT-PCR) analyses of the cDNA libraries confirmed cDNA molecules that are associated with different developmental stages (e. g. "on year" leaves in July, "off year" leaves in July, leaves in November) and fruits. Hence, a number of candidate cDNAs associated with "on year" and "off year" were isolated. Comparison of the detected cDNAs to the current EST database of GenBank along with other non - redundant databases of NCBI revealed homologs of previously described genes along with several unknown cDNAs. Of around 500 screened cDNAs, 48 cDNA elements were obtained after eliminating ribosomal RNA sequences. These independent transcripts were analyzed using BLAST searches (cutoff E-value of 1.0E-5) against the KEGG and GenBank nucleotide databases and 37 putative transcripts corresponding to known gene functions were annotated with gene names and Gene Ontology (GO) terms. Transcripts in the biological process were found to be related with metabolic process (27%), cellular process (23%), response to stimulus (17%), localization process (8.5%), multicellular organismal process (6.25%), developmental process (6.25%) and reproduction (4.2%). CONCLUSIONS: A putative P450 monooxigenase expressed fivefold more in the "on year" than that of "off year" leaves in July. Two putative dehydrins expressed significantly more in "on year" leaves than that of "off year" leaves in November. Homologs of UDP - glucose epimerase, acyl - CoA binding protein, triose phosphate isomerase and a putative nuclear core anchor protein were significant in fruits only, while a homolog of an embryo binding protein / small GTPase regulator was detected in "on year" leaves only. One of the two unknown cDNAs was specific to leaves in July while the other was detected in all of the libraries except fruits. KEGG pathway analyses for the obtained sequences correlated with essential metabolisms such as galactose metabolism, amino sugar and nucleotide sugar metabolisms and photosynthesis. Detailed analysis of the results presents candidate cDNAs that can be used to dissect further the genetic basis of fruit production and / or alternate bearing which causes significant economical loss for olive growers.

Genome-wide identification of alternate bearing-associated microRNAs (miRNAs) in olive (Olea europaea L.).

BACKGROUND: Alternate bearing is a widespread phenomenon among crop plants, defined as the tendency of certain fruit trees to produce a high-yield crop one year ("on-year"), followed by a low-yield or even no crop the following year ("off-year"). Several factors may affect the balance between such developmental phase-transition processes. Among them are the microRNA (miRNA), being gene-expression regulators that have been found to be involved as key determinants in several physiological processes. RESULTS: Six olive (Olea europaea L. cv. Ayvalik variety) small RNA libraries were constructed from fruits (ripe and unripe) and leaves ("on year" and "off year" leaves in July and in November, respectively) and sequenced by high-throughput Illumina sequencing. The RNA was retrotranscribed and sequenced using the high-throughput Illumina platform. Bioinformatics analyses of 93,526,915 reads identified 135 conserved miRNA, belonging to 22 miRNA families in the olive. In addition, 38 putative novel miRNAs were discovered in the datasets. Expression of olive tree miRNAs varied greatly among the six libraries, indicating the contribution of diverse miRNA in balancing between reproductive and vegetative phases. Predicted targets of miRNA were categorized into 108 process ontology groups with significance abundance. Among those, potential alternate bearing-associated processes were found, such as development, hormone-mediated signaling and organ morphogenesis. The KEGG analyses revealed that the miRNA-targeted genes are involved in seven main pathways, belonging to carbohydrate metabolism and hormone signal-transduction pathways. CONCLUSION: A comprehensive study on olive miRNA related to alternate bearing was performed. Regulation of miRNA under different developmental phases and tissues indicated that control of nutrition and hormone, together with flowering processes had a noteworthy impact on the olive tree alternate bearing. Our results also provide significant data on the miRNA-fruit development interaction and advance perspectives in the miRNA profile of the olive tree.

Identification of conserved micro-RNAs and their target transcripts in opium poppy (Papaver somniferum L.).

Micro-RNAs (miRNA) are regulatory non-coding class of small RNAs functioning in many organisms. Using computational approaches we have identified 20 conserved opium poppy (Papaver somniferum L.) miRNAs belonging to 16 miRNA families in Expressed Sequence Tags (EST) database. The existence of ESTs suggested that the miRNAs were expressed in P. somniferum. Lengths of mature miRNAs varied from 20 to 23 nucleotides located at the different positions of precursor RNAs. Uracil was found to be a dominant nucleotide in both poppy pre-miRNA sequences (31.28 +/- 7.06% of total nucleotide composition) and in the first position at the 5' end of the mature poppy miRNAs. We have applied quantitative real-time PCR (qRT-PCR) assays to compare and validate expression levels of selected P. somniferum miRNAs and their target transcripts. As a result, some of the predicted miRNAs and their target genes were found to be differentially expressed in P. somniferum leaf and root tissues. A meaningful correlation between three of the four analyzed pairs of miRNAs and their target transcript expression levels was detected. Additionally, using these predicted miRNAs as queries, 41 potential target mRNAs were found in National Center for Biotechnology Information (NCBI) protein-coding nucleotide (mRNA) database of all plant species. Some of the target mRNAs were found to be transcription factors regulating plant development, morphology, and flowering time. Other target mRNAs of identified conserved miRNAs involve in metabolic processes, signal transduction, and stress responses. This study reports the first identification of opium poppy miRNAs.

BAT1, a bidirectional amino acid transporter in Arabidopsis.

The Arabidopsis thaliana At2g01170 gene is annotated as a putative gamma amino butyric acid (GABA) permease based on its sequence similarity to a yeast GABA transporting gene (UGA4). A cDNA of At2g01170 was expressed in yeast and analyzed for amino acid transport activity. Both direct measurement of amino acid transport and yeast growth experiments demonstrated that the At2g01170 encoded-protein exhibits transport activity for alanine, arginine, glutamate and lysine, but not for GABA or proline. Significantly, unlike other amino acid transporters described in plants to date, At2g01170 displayed both export and import activity. Based on that observation, it was named bidirectional amino acid transporter 1 (BAT1). Sequence comparisons show BAT1 is not a member of any previously defined amino acid transporter family. It does share, however, several conserved protein domains found in a variety of prokaryotic and eukaryotic amino acid transporters, suggesting membership in an ancient family of transporters. BAT1 is a single copy gene in the Arabidopsis genome, and its mRNA is ubiquitously expressed in all organs. A transposon--GUS gene-trap insert in the BAT1 gene displays GUS localization in the vascular tissues (Dundar in Ann Appl Biol, 2009) suggesting BAT1 may function in amino acid export from the phloem into sink tissues.

Phenolics profiles of olive fruits (Olea europaea L.) and oils from Ayvalik, Domat and Gemlik varieties at different ripening stages.

Phenolic compounds in olive fruit and oils obtained from Ayvalik, Domat and Gemlik olive varieties collected at different ripening periods were evaluated by High Performance Liquid Chromatography (HPLC). Gallic acid and p-cumaric acid were identified for Ayvalik and Domat at each period of ripening, respectively. In addition, gallic acid, p-cumaric acid, sinapinic and apigenin acids were detected in Gemlik olive fruit. Hydroxytyrosol, rutin, oleoropein, luteolin, tyrosol, vanilic acid and gallic acid in Ayvalik olive fruit in all ripening periods were determined. The tyrasol contents varied between 0.18 to 1.57mg/kg. Luteolin contents of olive oils ranged at the levels between 0.12 to 2.28mg/kg. In contrast, oils had the lowest syringic, p-cumaric, chlorogenic and ferulic acids. Vanillic acid contents of oils ranged between 0.08 to 2.38mg/kg.

Nutrition metabolism plays an important role in the alternate bearing of the olive tree (Olea europaea L.).

The olive tree (Olea europaea L.) is widely known for its strong tendency for alternate bearing, which severely affects the fruit yield from year to year. Microarray based gene expression analysis using RNA from olive samples (on-off years leaves and ripe-unripe fruits) are particularly useful to understand the molecular mechanisms influencing the periodicity in the olive tree. Thus, we carried out genome wide transcriptome analyses involving different organs and temporal stages of the olive tree using the NimbleGen Array containing 136,628 oligonucleotide probe sets. Cluster analyses of the genes showed that cDNAs originated from different organs could be sorted into separate groups. The nutritional control had a particularly remarkable impact on the alternate bearing of olive, as shown by the differential expression of transcripts under different temporal phases and organs. Additionally, hormonal control and flowering processes also played important roles in this phenomenon. Our analyses provide further insights into the transcript changes between "on year" and "off year" leaves along with the changes from unrpipe to ripe fruits, which shed light on the molecular mechanisms underlying the olive tree alternate bearing. These findings have important implications for the breeding and agriculture of the olive tree and other crops showing periodicity. To our knowledge, this is the first study reporting the development and use of an olive array to document the gene expression profiling associated with the alternate bearing in olive tree.

Determination of tocopherol contents of some olive varieties harvested at different ripening periods.

The tocopherol contents of oils obtained from Ayvalik, Domat and Gemlik olive varieties harvested at different ripening periods were evaluated by high-performance liquid chromatography. α-Tocopherol was the major tocol detected in all the studied olive oil samples. The oils extracted from olive fruits composed of 130.54-180.43 mg kg⁻¹ α-tocopherol, 0.73-1.61 mg kg⁻¹ ß-tocopherol and 0.53-2.28 mg kg⁻¹ γ-tocopherol for Ayvalik oil. The α-, ß- and γ-tocopherol contents of Domat oil are in the ranges 95.60-125.56, 0.71-2.70 and 0.49-1.25 mg kg⁻¹ at different harvesting periods. The α-, ß- and γ-tocopherol contents of Gemlik olive oil are in the ranges 112.59-168.19, 0.94-1.21 and 0.85-2.40 mg kg⁻¹, respectively. There were significant differences between the oils from cultivars grown in different environments.

Crimean-Congo hemorrhagic fever virus in various ixodid tick species from a highly endemic area.

Ticks are major vectors of numerous diseases affecting animals and humans. Presence of various tick-borne pathogens such as Crimean-Congo hemorrhagic fever virus (CCHFV) in various tick species was documented. CCHF is a severe tick-borne illness caused by the CCHFV which is a member of the Nairovirus genus (family: Bunyaviridae). Presence of CCHFV was shown in the most prevalent ixodid tick species such as Hyalomma marginatum and Rhiphicephalus bursa in Turkey. In the present study, prevalence and species diversity of ixodid ticks carrying CCHFV in Tokat province where CCHF is highly endemic were determined by using real-time reverse transcription-polymerase chain reaction (RRT-PCR). As a result, 15 out 745 ticks from various hosts (2%) were found to be CCHFV positive. The CCHFV positive ticks were Haemaphysalis concinna, Hyalomma anatolicum, Hyalomma detritum, Hyalomma marginatum, Hyalomma turanicum, Rhipicephalus bursa, and Rhiphicephalus turanicus indicating that multiple ixodid tick species may contribute to transmission of CCHFV to humans and animals in Turkey.

Large scale structure of wheat, rice and potato starch revealed by ultra small angle X-ray diffraction.

Rice, wheat, and potato starches were investigated using ultra-small angle X-Ray diffraction (USXRD) in the range of 100-58,000 A. The results showed trends consistent with the known sizes of starches. However, the observed Rg values for the scattering substances lie in the 100-300 nm range, very much in the low end of the known starch granule size distributions (and below the resolution of the light microscope) suggesting different, perhaps interesting, structures than those observed by light microscopy. Thus what were detected may possibly be the sizes of the crystalline regions postulated to occur in individual starch granules.