Transcriptomic Insight into the Pollen Tube Growth of Olea europaea L. subsp. europaea Reveals Reprogramming and Pollen-Specific Genes Including New Transcription Factors.

The pollen tube is a key innovation of land plants that is essential for successful fertilisation. Its development and growth have been profusely studied in model organisms, but in spite of the economic impact of olive trees, little is known regarding the genome-wide events underlying pollen hydration and growth in this species. To fill this gap, triplicate mRNA samples at 0, 1, 3, and 6 h of in vitro germination of olive cultivar Picual pollen were analysed by RNA-seq. A bioinformatics R workflow called RSeqFlow was developed contemplating the best practices described in the literature, covering from expression data filtering to differential expression and clustering, to finally propose hub genes. The resulting olive pollen transcriptome consisted of 22,418 reliable transcripts, where 5364 were differentially expressed, out of which 173 have no orthologue in plants and up to 3 of them might be pollen-specific transcription factors. Functional enrichment revealed a deep transcriptional reprogramming in mature olive pollen that is also dependent on protein stability and turnover to allow pollen tube emergence, with many hub genes related to heat shock proteins and F-box-containing proteins. Reprogramming extends to the first 3 h of growth, including processes consistent with studies performed in other plant species, such as global down-regulation of biosynthetic processes, vesicle/organelle trafficking and cytoskeleton remodelling. In the last stages, growth should be maintained from persistent transcripts. Mature pollen is equipped with transcripts to successfully cope with adverse environments, even though the in vitro growth seems to induce several stress responses. Finally, pollen-specific transcription factors were proposed as probable drivers of pollen germination in olive trees, which also shows an overall increased number of pollen-specific gene isoforms relative to other plants.

OliveAtlas: A Gene Expression Atlas Tool for Olea europaea.

The olive (Olea europaea L.) is an ancient crop of great importance in the Mediterranean basin due to the production of olive oil and table olives, which are important sources of fat and have benefits for human health. This crop is expanding and increasing its production worldwide and five olive genomes have recently been sequenced, representing a wild olive and important cultivars in terms of olive oil production, intensive agriculture, and adaptation to the East Asian climate. However, few bioinformatic and genomic resources are available to assist olive research and breeding, and there are no platforms to query olive gene expression data. Here, we present OliveAtlas, an interactive gene expression atlas for olive with multiple bioinformatics tools and visualization methods, enabling multiple gene comparison, replicate inspection, gene set enrichment, and data downloading. It contains 70 RNA-seq experiments, organized in 10 data sets representing the main olive plant organs, the pollen germination and pollen tube elongation process, and the response to a collection of biotic and abiotic stresses, among other experimental conditions. OliveAtlas is a web tool based on easyGDB with expression data based on the 'Picual' genome reference and gene annotation.

A comprehensive dataset of the extra virgin olive oil (EVOO) proteome.

Proteins and peptides are minor components of vegetal oils. The presence of these compounds in virgin olive oil was first reported in 2001, but the nature of the olive oil proteome is still a puzzling question for food science researchers. In this paper, we have compiled for a first time a comprehensive proteomic dataset of olive fruit and fungal proteins that are present at low but measurable concentrations in a vegetable oil from a crop of great agronomical relevance as olive (Olea europaea L.). Accurate mass nLC-MS data were collected in high definition direct data analysis (HD-DDA) mode using the ion mobility separation step. Protein identification was performed using the Mascot Server v2.2.07 software (Matrix Science) against an ad hoc database made of olive protein entries. Starting from this proteomic record, the impact of these proteins on olive oil stability and quality could be tested. Moreover, the effect of olive oil proteins on human health and their potential use as functional food components could be also evaluated. In addition, this dataset provides a resource for use in further functional comparisons across other vegetable oils, and also expands the proteomic resources to non-model species, thus also allowing further comparative inter-species studies. The data presented here are related to the research article of Castro et al. [1].

Identification of seed storage proteins as the major constituents of the extra virgin olive oil proteome.

Proteins are minor components of extra virgin olive oil (EVOO), but the nature of the olive oil proteome is still elusive. In this paper, we have uncovered the EVOO proteome for the first time. Seed storage proteins of globulin-type were identified as the most abundant proteins in EVOO, which also contains an active 13-lipoxygenase and several potential allergenic proteins, including the "panallergen" profilin. We validated our proteomic data by Western blotting and enzyme activity assays. Our data also demonstrated that the seed is the main source of proteins in EVOO, while the contribution of the pulp is uncertain and needs further verification. The impact of EVOO proteins on its stability and quality, and on human health is discussed.

Identification of novel superoxide dismutase isoenzymes in the olive (Olea europaea L.) pollen.

BACKGROUND: Among antioxidant enzymes, the superoxide dismutase (SOD) family is a major actor in catalysing the disproportionation of superoxide. Apart from its role as antioxidant, these enzymes have a role in cell signalling, and Cu,Zn-SOD proteins are also major pollen allergens. In order to deepen our understanding of the SOD isoenzymes present in olive pollen and to analyse the molecular variability of the pollen Cu,Zn-SOD family, we carried out biochemical, transcriptomic and localization studies of pollen grains from different olive cultivars and other allergenic species. RESULTS: Olive pollen showed a high rate of total SOD activity in all cultivars assayed, which did not correlate with pollen viability. Mass spectrometry analysis together with activity assays and Western blotting experiments enabled us to identify new forms of Cu,Zn-SOD enzyme (including chloroplastidic and peroxisomal forms) as well as differentially expressed Mn-, Fe- and Cu,Zn-SOD isoenzymes among the pollen of different olive cultivars and allergenic species. Ultrastructural localization of Cu,Zn-SOD revealed its plastidial localization in the pollen grain. We also identified the occurrence of a shorter form of one of the cytosolic Cu,Zn-SOD enzymes, likely as the result of alternative splicing. This shorter enzyme showed lower SOD activity as compared to the full length form. CONCLUSIONS: The presence of multiple SOD isoenzymes in the olive pollen could be related to the need of finely tuning the ROS metabolism during the transition from its quiescent condition at maturity to a highly metabolically active state at germination.

S-nitroso- and nitro- proteomes in the olive (Olea europaea L.) pollen. Predictive versus experimental data by nano-LC-MS.

The data presented here are related to the research article entitled "Generation of nitric oxide by olive (Olea europaea L.) pollen during in vitro germination and assessment of the S-nitroso- and nitro-proteomes by computational predictive methods" doi:10.1016/j.niox.2017.06.005 (Jimenez-Quesada et al., 2017) [1]. Predicted cysteine S-nitrosylation and Tyr-nitration sites in proteins derived from a de novo assembled and annotated pollen transcriptome from olive tree (Olea europaea L.) were obtained after using well-established predictive tools in silico. Predictions were performed using both default and highly restrictive thresholds. Numerous gene products identified with these characteristics are listed here. An experimental validation of the data, consisting in nano-LC-MS (Liquid Chromatography-Mass Spectrometry) determination of olive pollen proteins after immunoprecipitation with antibodies to anti-S-nitrosoCys and anti-3-NT (NitroTyrosine) allowed identification of numerous proteins subjected to these two post-translational modifications, which are listed here together with information regarding their cross-presence among the predictions.

Generation of nitric oxide by olive (Olea europaea L.) pollen during in vitro germination and assessment of the S-nitroso- and nitro-proteomes by computational predictive methods.

Nitric oxide is recognized as a signaling molecule involved in a broad range of physiological processes in plants including sexual reproduction. NO has been detected in the pollen grain at high levels and regulates pollen tube growth. Previous studies demonstrated that NO as well as ROS are produced in the olive reproductive tissues in a stage- and tissue-specific manner. The aim of this study was to assess the production of NO throughout the germination of olive (Olea europaea L.) pollen in vitro. The NO fluorescent probe DAF-2DA was used to image NO production in situ, which was correlated to pollen viability. Moreover, by means of a fluorimetric assay we showed that growing pollen tubes release NO. GSNO -a mobile reservoir of NO, formed by the S-nitrosylation of NO with reduced glutathione (GSH) - was for the first time detected and quantified at different stages of pollen tube growth using a LC-ES/MS analysis. Exogenous NO donors inhibited both pollen germination and pollen tube growth and these effects were partially reverted by the specific NO-scavenger c-PTIO. However, little is known about how NO affects the germination process. With the aim of elucidating the putative relevance of protein S-nitrosylation and Tyr-nitration as important post-translational modifications in the development and physiology of the olive pollen, a de novo assembled and annotated reproductive transcriptome from olive was challenged in silico for the putative capability of transcripts to become potentially modified by S-nitrosylation/Tyr-nitration according to well-established criteria. Numerous gene products with these characteristics were identified, and a broad discussion as regards to their potential role in plant reproduction was built after their functional classification. Moreover, the importance of both S-nitrosylation/Tyr-nitrations was experimentally assessed and validated by using Western blotting, immunoprecipitation and proteomic approaches.

The Pollen Coat Proteome: At the Cutting Edge of Plant Reproduction.

The tapetum is a single layer of secretory cells which encloses the anther locule and sustains pollen development and maturation. Upon apoptosis, the remnants of the tapetal cells, consisting mostly of lipids and proteins, fill the pits of the sculpted exine to form the bulk of the pollen coat. This extracellular matrix forms an impermeable barrier that protects the male gametophyte from water loss and UV light. It also aids pollen adhesion and hydration and retains small signaling compounds involved in pollen-stigma communication. In this study, we have updated the list of the pollen coat's protein components and also discussed their functions in the context of sexual reproduction.

Identification of olive pollen allergens using a fluorescence-based 2D multiplex method.

Olive (Olea europaea L.) pollen is a major health concern in the Mediterranean countries and some olive growing regions in America and Australia. The molecular variability of pollen allergens constitutes a handicap for commercial extract standardization, which is the base of current diagnosis and vaccination procedures. In this paper, we report a time-saving and plant material saving multiplex detection method for the rapid and simultaneous analysis of Ole e 1, Ole e 2, and Ole e 5 allergen polymorphism on a single blot. This method combines high-resolution 2DE techniques with high-sensitive fluorescence-based detection methods. Using this strategy, we were capable to identify a higher number of allergen forms compared with classical 1D approach. The use of fluorescent probes and the increased resolution of 2D blots avoided overlapping effects, and allow estimating the amount of individual allergen forms. In addition, the pattern and identity of the IgE-reactive proteins of either a population or individual patients allergic to olive pollen was also effortlessly determined in a single additional step. This flexible method might be extended to a higher number of olive allergens and cultivars, and is also applicable to other allergogenic plant species and sources.

Chip-based capillary electrophoresis profiling of olive pollen extracts used for allergy diagnosis and immunotherapy.

Standardization of protein extracts for clinical purposes represents an important task in order to maintain adequate reactivity, presence of the relevant allergens, and safety among other factors. The main objective of this work was to explore the potential use of a chip-based automated CE system commercially available to analyze several of the most common forms of allergenic extracts from olive pollen used in allergy clinics. These include experimental extracts prepared from olive pollens, in-house reference extracts, extracts designed for skin prick test assays, and a panel of vaccine variants aimed to specific immunotherapy. As a major conclusion of the study, chip-based CE allowed in all cases to determine accurate protein profiles with different degrees of sensitivity, where several allergens (particularly the major olive pollen allergen Ole e 1) were easily recognized. Moreover, several purified allergens were also analyzed by this method, and proposed as specific standards for different purposes. In the present condition, the method can only provide the protein profile of the extracts with respect to a preestablished standard extract, but not allergen identification. However, these and other future developments and applications are discussed.

Olive seed protein bodies store degrading enzymes involved in mobilization of oil bodies.

The major seed storage reserves in oilseeds are accumulated in protein bodies and oil bodies, and serve as an energy, carbon, and nitrogen source during germination. Here, the spatio-temporal relationships between protein bodies and several key enzymes (phospholipase A, lipase, and lipoxygenase) involved in storage lipid mobilization in cotyledon cells was analysed during in vitro seed germination. Enzyme activities were assayed in-gel and their cellular localization were determined using microscopy techniques. At seed maturity, phospholipase A and triacylglycerol lipase activities were found exclusively in protein bodies. However, after seed imbibition, these activities were shifted to the cytoplasm and the surface of the oil bodies. The activity of neutral lipases was detected by using α-naphthyl palmitate and it was associated mainly with protein bodies during the whole course of germination. This pattern of distribution was highly similar to the localization of neutral lipids, which progressively appeared in protein bodies. Lipoxygenase activity was found in both the protein bodies and on the surface of the oil bodies during the initial phase of seed germination. The association of lipoxygenase with oil bodies was temporally correlated with the appearance of phospholipase A and lipase activities on the surface of oil bodies. It is concluded that protein bodies not only serve as simple storage structures, but are also dynamic and multifunctional organelles directly involved in storage lipid mobilization during olive seed germination.

A protocol for protein extraction from lipid-rich plant tissues suitable for electrophoresis.

Plant tissues contain high levels of nonprotein contaminants such as lipids, phenolic compounds, and polysaccharides among others, which interfere with protein extraction and electrophoretic separation. Preparation of good-quality protein extracts is a critical issue for successful electrophoretic analysis. Here, we describe a three-step method for protein extraction from lipid-rich plant tissues, which is suitable for both 1-D and 2-D electrophoresis and is compatible with downstream applications. The protocol includes prefractionation, filtration, and TCA/acetone precipitation steps prior to protein resolubilization.

Proteomics profiling reveals novel proteins and functions of the plant stigma exudate.

Proteomic analysis of the stigmatic exudate of Lilium longiflorum and Olea europaea led to the identification of 51 and 57 proteins, respectively, most of which are described for the first time in this secreted fluid. These results indicate that the stigmatic exudate is an extracellular environment metabolically active, participating in at least 80 different biological processes and 97 molecular functions. The stigma exudate showed a markedly catabolic profile and appeared to possess the enzyme machinery necessary to degrade large polysaccharides and lipids secreted by papillae to smaller units, allowing their incorporation into the pollen tube during pollination. It may also regulate pollen-tube growth in the pistil through the selective degradation of tube-wall components. Furthermore, some secreted proteins were involved in pollen-tube adhesion and orientation, as well as in programmed cell death of the papillae cells in response to either compatible pollination or incompatible pollen rejection. Finally, the results also revealed a putative cross-talk between genetic programmes regulating stress/defence and pollination responses in the stigma.

Differential expression and sequence polymorphism of the olive pollen allergen Ole e 1 in two Iranian cultivars.

Molecular evidence on the heterogeneity present in the Ole e 1 allergen of the olive pollen is emerging. Such polymorphism is dependent on the cultivar origin of pollen, which also determines wide differences in the expression of this protein. Determination of biochemical and molecular characteristics of Ole e 1 pollen allergen in two Iranian olive cultivars, namely 'Rowghani' and 'Zard' is necessary to assess their allergenicity potential. SDS-PAGE and immunoblotting analysis of pollen extracts showed that both cultivars present high and low expression of Ole e 1, respectively. These protein levels correlated with similarly different levels of transcripts, as determined by RT-PCR. Two-dimensional protein profiles also showed conspicuous differences in the distribution and the level of expression of those spots reacting to an anti-Ole e 1 antibody. Bioinformatic analysis of four Ole e 1 sequences corresponding to 'Rowghani' and two sequences for 'Zard', showed numerous heterogeneities when compared with those Ole e 1 and Ole e 1-like sequences present in databases. Nucleotide substitutions resulted in many cases in changes over the predicted amino acid sequences. A cladistic analysis of the sequences showed Iranian entries in a central position between West-European sequences, and Ole e 1-like sequences from other Oleaceae species. Moreover, amino acid changes affected key epitopes of the protein involved in the recognition of the protein by the human immune system. Putative implications of polymorphism in both the biological role and the allergic reactivity of Ole e 1 are discussed.

New insights into the early steps of oil body mobilization during pollen germination.

In some plants, pollen grains accumulate storage lipids that serve as energy supply during germination. Here, three enzymes involved in early steps of oil body mobilization in the male gametophyte were functionally characterized for the first time. The effect of extracellular sugars on pollen performance and oil body dynamics was also analysed. Olive pollen oil bodies showed phospholipase A, lipase, and lipoxygenase activities on their surface. Enzyme activity levels increased during germination with a maximum after 3h. Removal of extracellular sugars from the germination medium did not affect pollen performance but increased enzyme activity rates and sped up oil body mobilization. Inhibitors seriously hampered pollen germination and pollen tube growth, leading to a characteristic accumulation of oil bodies in the germinative aperture. It can be concluded that storage lipids are sufficient for proper olive pollen germination. A lipase and a lipoxygenase are likely involved in oil body mobilization. Extracellular sugars may modulate their function, while a phospholipase A may promote their access to the storage lipids.

A novel multiplex method for the simultaneous detection and relative quantitation of pollen allergens.

Standardization of pollen protein extracts is essential in order to ensure efficiency and safety in allergy diagnosis and immunotherapy. In this paper, we have optimized a multiplex Western blotting method for the simultaneous detection of four olive pollen allergens (Ole e 1, Ole e 2, Ole e 5, and Ole e 9) on a single blot using a monoclonal antibody from mouse and three polyclonal antibodies raised in rabbit. We utilized unconjugated Fab antibody fragments for blocking rabbit primary antibodies, and fluorescence-based detection. These changes allowed an accurate and reliable comparative quantitation of these allergens among pollen-protein samples from six olive cultivars. In addition, we also tested the IgE-binding capacity of these pollen extracts by reprobing the same blot with a pool of sera from eight patients allergic to olive and detection with enzyme conjugated antibodies. A noticeable variability regarding allergen content and IgE-reactivity was found among the olive cultivars analyzed. Moreover, we could easily confirm the identity of some of the IgE-binding proteins by simply overlapping both fluorescence and chemiluminescence images. This method is versatile since it can be applied to other allergogenic plant species and extended to other allergens.

Rad9B responds to nucleolar stress through ATR and JNK signalling, and delays the G1-S transition.

The complex formed by Rad9, Rad1 and Hus1 (9-1-1) protects against genomic instability by activating DNA damage checkpoint and DNA damage repair pathways, mainly in response to replication fork collapse and UV lesions. Here we compare the role of Rad9A (also known as Rad9) with the human paralogue Rad9B. Unlike Rad9A, overexpression of Rad9B delays cells in G1 phase. Moreover, Rad9B migrates to nucleoli after nucleolar stress in an ATR- and JNK-dependent manner, in a newly described nucleolar domain structure containing p21. Analysis of chimeras of Rad9A and Rad9B demonstrate that localisation to nucleoli and the block in G1 phase upon overexpression crucially depend on the Rad9B C-terminal tail. Taken together, data presented here show a relationship between Rad9B and pathways for checkpoints, stress response and nucleolar function.

Screening of Ole e 1 polymorphism among olive cultivars by peptide mapping and N-glycopeptide analysis.

In the present paper, we have used 2-DE coupled to MS analysis to examine the molecular variability of the Ole e 1 allergen in three olive cultivars (cvs). Our results confirmed that the predicted polymorphism of Ole e 1 at cDNA level is extended to the expressed protein. The profiles of both the Ole e 1 peptides and the N-glycan variants significantly changed among cvs. We observed that Picual and Arbequina cvs presented the highest and lowest degree of Ole e 1 polymorphism, respectively. Some of these peptides and N-glycans were distributed in a cv-specific manner. The putative implications of this molecular polymorphism in the development of the allergy symptoms are discussed.

The herbicide flumioxazin stimulates pathogenesis-related gene expression and enzyme activities in Vitis vinifera.

In this work, the capacity of the soil-applied herbicide flumioxazin (fmx) to trigger defence mechanisms was assessed using 6-week-old in vitro grown Vitis vinifera L. plantlets. Time-course studies demonstrated that the herbicide induced the expression of basic beta-1,3-glucanase (Vvglu), basic chitinase (Vvchit1b) and PR10 (VvPR10.3) genes encoding three pathogenesis-related (PR) proteins involved in grapevine defence against pathogens. Thus, all transcripts accumulated in grapevine tissues to reach maximum values after 24-72 h of herbicide exposure, except for VvPR10.3 gene expression, which was induced in roots and stems but not in leaves. Induction of PR genes was observed to a greater extent in roots and leaves, and its intensity diminished in the stems although still remained noteworthy. The activities of beta-1,3-glucanase and chitinase enzymes significantly increased in the whole plant after herbicide exposure and were still stimulated 21 days after the beginning of treatments. Similarly, the most remarkable effect occurred in roots. However, all enzyme activities tested were stimulated in the upper aerial tissues as well, indicating that fmx or a derived product acts systemically, likely via root uptake.

Olive cultivar origin is a major cause of polymorphism for Ole e 1 pollen allergen.

BACKGROUND: Pollens from different olive (Olea europaea L.) cultivars have been shown to differ significantly in their content in Ole e 1 and in their overall allergenicity. This allergen is, in addition, characterized by a high degree of polymorphism in its sequence. The purpose of this study is to evaluate the putative presence of divergences in Ole e 1 sequences from different olive cultivars. RESULTS: RNA from pollen individually collected from 10 olive cultivars was used to amplify Ole e 1 sequences by RT-PCR, and the sequences were analyzed by using different bioinformatics tools. Numerous nucleotide substitutions were detected throughout the sequences, many of which resulted in amino acid substitutions in the deduced protein sequences. In most cases variability within a single variety was much lower than among varieties. Key amino acid changes in comparison with "canonical" sequences previously described in the literature included: a) the substitution of C19-relevant to the disulphide bond structure of the protein-, b) the presence of an additional N-glycosylation motif, and c) point substitutions affecting regions of Ole e 1 already described like relevant for the immunogenicity/allergenicity of the protein. CONCLUSION: Varietal origin of olive pollen is a major factor determining the diversity of Ole e 1 variants. We consider this information of capital importance for the optimal design of efficient and safe allergen formulations, and useful for the genetic engineering of modified forms of the allergen among other applications.