Transcriptomic and metabolomic analysis reveals that symbiotic nitrogen fixation enhances drought resistance in common bean.

Common bean (Phaseolus vulgaris L.), one of the most important legume crops, uses atmospheric nitrogen through symbiosis with soil rhizobia, reducing the need for nitrogen fertilization. However, this legume is particularly sensitive to drought conditions, prevalent in arid regions where this crop is cultured. Therefore, studying the response to drought is important to sustain crop productivity. We have used integrated transcriptomic and metabolomic analysis to understand the molecular responses to water deficit in a marker-class common bean accession cultivated under N2 fixation or fertilized with nitrate (NO3-). RNA-seq revealed more transcriptional changes in the plants fertilized with NO3- than in the N2-fixing plants. However, changes in N2-fixing plants were more associated with drought tolerance than in those fertilized with NO3-. N2-fixing plants accumulated more ureides in response to drought, and GC/MS and LC/MS analysis of primary and secondary metabolite profiles revealed that N2-fixing plants also had higher levels of abscisic acid, proline, raffinose, amino acids, sphingolipids, and triacylglycerols than those fertilized with NO3-. Moreover, plants grown under nitrogen fixation recovered from drought better than plants fertilized with NO3-. Altogether we show that common bean plants grown under symbiotic nitrogen fixation were more protected against drought than the plants fertilized with nitrate.

A Comprehensive Study of the WRKY Transcription Factor Family in Strawberry.

WRKY transcription factors play critical roles in plant growth and development or stress responses. Using up-to-date genomic data, a total of 64 and 257 WRKY genes have been identified in the diploid woodland strawberry, Fragaria vesca, and the more complex allo-octoploid commercial strawberry, Fragaria × ananassa cv. Camarosa, respectively. The completeness of the new genomes and annotations has enabled us to perform a more detailed evolutionary and functional study of the strawberry WRKY family members, particularly in the case of the cultivated hybrid, in which homoeologous and paralogous FaWRKY genes have been characterized. Analysis of the available expression profiles has revealed that many strawberry WRKY genes show preferential or tissue-specific expression. Furthermore, significant differential expression of several FaWRKY genes has been clearly detected in fruit receptacles and achenes during the ripening process and pathogen challenged, supporting a precise functional role of these strawberry genes in such processes. Further, an extensive analysis of predicted development, stress and hormone-responsive cis-acting elements in the strawberry WRKY family is shown. Our results provide a deeper and more comprehensive knowledge of the WRKY gene family in strawberry.

Constructing a de novo transcriptome and a reference proteome for the bivalve Scrobicularia plana: Comparative analysis of different assembly strategies and proteomic analysis.

Scrobicularia plana is a coastal and estuarine bivalve widely used in ecotoxicological studies. However, the underlying molecular mechanisms for S. plana pollutant responses are hardly known due to the lack of molecular databases. Thus, in this study we present a holistic approach to assess a robust reference transcriptome and proteome of this clam. A mixture of control and metal-exposed individuals was used for mRNA isolation. Four sets of high quality filtered preprocessed reads were generated (two quality scores and two sequenced lengths) and assembled with Mira, Ray and Trinity algorithms. The sixty-four generated assemblies were refined, filtered and evaluated for their proteomic quality. Eight assemblies presented top Detonate scores but one was selected due to its compactness and biological representation, which was generated: (i) from the highest quality dataset (Q20L100), (ii) using Trinity algorithm with all k-mers (AtKa), (iii) removing redundancy by CD-HIT (RR80), and (iv) filtering out poor contigs (F), that was subsequently named Q20L100AtKaRR80F. S. plana proteomic analysis revealed 10,017 peptide groups that corresponded to 2066 proteins with a wide coverage of molecular functions and biological processes, confirming the strength of the database generated.

Development of New Antiproliferative Compound against Human Tumor Cells from the Marine Microalgae Nannochloropsis gaditana by Applied Proteomics.

Proteomics is a crucial tool for unravelling the molecular dynamics of essential biological processes, becoming a pivotal technique for basic and applied research. Diverse bioinformatic tools are required to manage and explore the huge amount of information obtained from a single proteomics experiment. Thus, functional annotation and protein-protein interactions are evaluated in depth leading to the biological conclusions that best fit the proteomic response in the system under study. To gain insight into potential applications of the identified proteins, a novel approach named "Applied Proteomics" has been developed by comparing the obtained protein information with the existing patents database. The development of massive sequencing technology and mass spectrometry (MS/MS) improvements has allowed the application of proteomics nonmodel microorganisms, which have been deeply described as a novel source of metabolites. Between them, Nannochloropsis gaditana has been pointed out as an alternative source of biomolecules. Recently, our research group has reported the first complete proteome analysis of this microalga, which was analysed using the applied proteomics concept with the identification of 488 proteins with potential industrial applications. To validate our approach, we selected the UCA01 protein from the prohibitin family. The recombinant version of this protein showed antiproliferative activity against two tumor cell lines, Caco2 (colon adenocarcinoma) and HepG-2 (hepatocellular carcinoma), proving that proteome data have been transformed into relevant biotechnological information. From Nannochloropsis gaditana has been developed a new tool against cancer-the protein named UCA01. This protein has selective effects inhibiting the growth of tumor cells, but does not show any effect on control cells. This approach describes the first practical approach to transform proteome information in a potential industrial application, named "applied proteomics". It is based on a novel bioalgorithm, which is able to identify proteins with potential industrial applications. From hundreds of proteins described in the proteome of N. gaditana, the bioalgorithm identified over 400 proteins with potential uses; one of them was selected as UCA01, "in vitro" and its potential was demonstrated against cancer. This approach has great potential, but the applications are potentially numerous and undefined.

An "omic" approach to Pyrocystis lunula: New insights related with this bioluminescent dinoflagellate.

Pyrocystis lunula (Schutt) is a photoautotrophic dinoflagellate without armored form, frequently found in marine environments. Today, there are several biotechnological applications derived from the bioluminescent system of this species. From a post-genomic perspective, in order to have a starting point for studying the proteome of P. lunula, an "omics" approach (transcriptomics-proteomics) was assessed using fresh microalgae samples. A total of 80,874,825 raw reads were generated (11,292,087,505 bp; 55.82% GC) by mRNA sequencing. Very high-quality sequences were assembled into 414,295 contigs (219,203,407 bp; 55.38% GC) using Trinity software, generating a comprehensive reference transcriptome for this species. Then, a P. lunula proteome was inferred and further employed for its analysis on this species. A total of 17,461 peptides were identified, yielding 3182 protein identification hits, including 175 novel proteins. The identified proteins were further categorized according to functional description and gene ontology classification. SIGNIFICANCE: The major contribution of the present work is making available a reference transcriptome and proteome of P. lunula, that is now accessible for the research community, and a functional description of the 3182 proteins inferred from the transcriptome, including 175 novel proteins, which have already been deposited in the ProteomeXchange and NCBI SRA databases, respectively. In addition to this, a series of important factors related to the bioluminescent system and the regulation of gene expression, were identified and described.

Proteomic analysis of goat milk kefir: Profiling the fermentation-time dependent protein digestion and identification of potential peptides with biological activity.

Kefir is a fermented dairy product, associated to health benefits because of being a probiotic and due to the presence of molecules with biological activity. In this work, we have profiled the peptide composition of goat milk kefir at three different fermentation times using a peptidomics approach, in order to study changes in peptide concentrations and patterns of protein digestion throughout the fermentation time. We identified 2328 unique peptides corresponding to 22 protein annotations, with a maximum of peptides found after 24 h fermentation. We established different digestion patterns according to the nature of the proteins, and quantified the changes in the peptides appearing in all the fermentation times. We also identified 11 peptides that matched exactly to sequences with biological activity in databases, almost all of them belonging to caseins. This is the most comprehensive proteomic analysis of goat milk kefir to date.

The Strawberry FaWRKY1 Transcription Factor Negatively Regulates Resistance to Colletotrichum acutatum in Fruit Upon Infection.

Strawberry (Fragaria ×ananassa) is a major food crop worldwide, due to the flavor, aroma and health benefits of the fruit, but its productivity and quality are seriously limited by a large variety of phytopathogens, including Colletotrichum spp. So far, key factors regulating strawberry immune response remain unknown. The FaWRKY1 gene has been previously proposed as an important element mediating defense responses in strawberry to Colletotrichum acutatum. To get further insight into the functional role that FaWRKY1 plays in the defense mechanism, Agrobacterium-mediated transient transformation was used both to silence and overexpress the FaWRKY1 gene in strawberry fruits (Fragaria ×ananassa cv. Primoris), which were later analyzed upon C. acutatum inoculation. Susceptibility tests were performed after pathogen infection comparing the severity of disease between the two agroinfiltrated opposite halves of the same fruit, one half bearing a construct either for FaWRKY1 overexpression or RNAi-mediated silencing and the other half bearing the empty vector, as control. The severity of tissue damage was monitored and found to be visibly reduced at five days after pathogen inoculation in the fruit half where FaWRKY1 was transiently silenced compared to that of the opposite control half and statistical analysis corroborated a significant reduction in disease susceptibility. Contrarily, a similar level of susceptibility was found when FaWRKY1 overexpression and control fruit samples, was compared. These results unravel a negative regulatory role of FaWRKY1 in resistance to the phytopathogenic fungus C. acutatum in strawberry fruit and contrast with the previous role described for this gene in Arabidopsis as positive regulator of resistance against the bacteria Pseudomonas syringae. Based on previous results, a tentative working model for WRKY75 like genes after pathogen infection is proposed and the expression pattern of potential downstream FaWRKY1 target genes was also analyzed in strawberry fruit upon C. acutatum infection. Our results highlight that FaWRKY1 might display different function according to species, plant tissue and/or type of pathogen and underline the intricate FaWRKY1 responsive defense regulatory mechanism taking place in strawberry against this important crop pathogen.

The VQ motif-containing proteins in the diploid and octoploid strawberry.

The plant VQ motif-containing proteins are a recently discovered class of plant regulatory proteins interacting with WRKY transcription factors capable of modulate their activity as transcriptional regulators. The short VQ motif (FxxhVQxhTG) is the main element in the WRKY-VQ interaction, whereas a newly identified variable upstream amino acid motif appears to be determinant for the WRKY specificity. The VQ family has been studied in several species and seems to play important roles in a variety of biological processes, including response to biotic and abiotic stresses. Here, we present a systematic study of the VQ family in both diploid (Fragaria vesca) and octoploid (Fragaria x ananassa) strawberry species. Thus, twenty-five VQ-encoding genes were identified and twenty-three were further confirmed by gene expression analysis in different tissues and fruit ripening stages. Their expression profiles were also studied in F. ananassa fruits affected by anthracnose, caused by the ascomycete fungus Colletotrichum, a major pathogen of strawberry, and in response to the phytohormones salicylic acid and methyl-jasmonate, which are well established as central stress signals to regulate defence responses to pathogens. This comprehensive analysis sheds light for a better understanding of putative implications of members of the VQ family in the defence mechanisms against this major pathogen in strawberry.

Ion Torrent and lllumina, two complementary RNA-seq platforms for constructing the holm oak (Quercus ilex) transcriptome.

Transcriptome analysis is widely used in plant biology research to explore gene expression across a large variety of biological contexts such as those related to environmental stress and plant-pathogen interaction. Currently, next generation sequencing platforms are used to obtain a high amount of raw data to build the transcriptome of any plant. Here, we compare Illumina and Ion Torrent sequencing platforms for the construction and analysis of the holm oak (Quercus ilex) transcriptome. Genomic analysis of this forest tree species is a major challenge considering its recalcitrant character and the absence of previous molecular studies. In this study, Quercus ilex raw sequencing reads were obtained from Illumina and Ion Torrent and assembled by three different algorithms, MIRA, RAY and TRINITY. A hybrid transcriptome combining both sequencing technologies was also obtained in this study. The RAY-hybrid assembly generated the most complete transcriptome (1,116 complete sequences of which 1,085 were single copy) with a E90N50 of 1,122 bp. The MIRA-Illumina and TRINITY-Ion Torrent assemblies annotated the highest number of total transcripts (62,628 and 74,058 respectively). MIRA-Ion Torrent showed the highest number of shared sequences (84.8%) with the oak transcriptome. All the assembled transcripts from the hybrid transcriptome were annotated with gene ontology grouping them in terms of biological processes, molecular functions and cellular components. In addition, an in silico proteomic analysis was carried out using the translated assemblies as databases. Those from Ion Torrent showed more proteins compared to the Illumina and hybrid assemblies. This new generated transcriptome represents a valuable tool to conduct differential gene expression studies in response to biotic and abiotic stresses and to assist and validate the ongoing Q. ilex whole genome sequencing.

Valorisation of the microalgae Nannochloropsis gaditana biomass by proteomic approach in the context of circular economy.

Nannochloropsis gaditana is a non-flagellated microalgae that has been widely used for different purposes, mostly related with the industrial production of biofuels or aquiculture. However, in order to increase the economic viability of the obtained microalgae biomass from a production plant coupled to a coal power plant, a proteomic approach was initiated by using fresh and atomized microalgae samples, as the main used commercial forms. Above 51,000 high quality spectra were obtained per sample in the MS/MS analysis of whole proteome of N. gaditana, yielding above 7,500 peptides, leading the identification of 1,950 proteins, from the N. gaditana protein database, where 655 proteins were presented in all the replicates. The identified proteins were categorized according to gene ontology classification by molecular function and biological process. In this study, it has been described the first proteomic analysis of the microalgae N. gaditana under industrial conditions containing an important number of identified proteins. A significative presence of proteins with a potential role in different agri-food and biomedical applications was detected and studied being the core of future N. gaditana research to expand the current biotechnological applications of this microalga. SIGNIFICANCE OF THE STUDY: Three quarters of the planet earth correspond to seas and oceans, however its potential biotechnological use is still unknown. We described the first proteomic description of the microalgae N. gaditana under industrial conditions. Following the spirit of the EU initiatives of blue growth and the statements of circular economy, CO2 waste from a coal plant power has been transformed in a resource for microalgae biomass production, common product presentations were evaluated by proteomic, and its potential use of identified proteins in Agri-food and Biomedicine has been revealed.

Hotspots in the genomic architecture of field drought responses in wheat as breeding targets.

Wheat can adapt to most agricultural conditions across temperate regions. This success is the result of phenotypic plasticity conferred by a large and complex genome composed of three homoeologous genomes (A, B, and D). Although drought is a major cause of yield and quality loss in wheat, the adaptive mechanisms and gene networks underlying drought responses in the field remain largely unknown. Here, we addressed this by utilizing an interdisciplinary approach involving field water status phenotyping, sampling, and gene expression analyses. Overall, changes at the transcriptional level were reflected in plant spectral traits amenable to field-level physiological measurements, although changes in photosynthesis-related pathways were found likely to be under more complex post-transcriptional control. Examining homoeologous genes with a 1:1:1 relationship across the A, B, and D genomes (triads), we revealed a complex genomic architecture for drought responses under field conditions, involving gene homoeolog specialization, multiple gene clusters, gene families, miRNAs, and transcription factors coordinating these responses. Our results provide a new focus for genomics-assisted breeding of drought-tolerant wheat cultivars.

Variation in functional responses to water stress and differentiation between natural allopolyploid populations in the Brachypodium distachyon species complex.

Background and Aims: Some polyploid species show enhanced physiological tolerance to drought compared with their progenitors. However, very few studies have examined the consistency of physiological drought response between genetically differentiated natural polyploid populations, which is key to evaluation of the importance of adaptive evolution after polyploidization in those systems where drought exerts a selective pressure. Methods: A comparative functional approach was used to investigate differentiation of drought-tolerance-related traits in the Brachypodium species complex, a model system for grass polyploid adaptive speciation and functional genomics that comprises three closely related annual species: the two diploid parents, B. distachyon and B. stacei, and the allotetraploid derived from them, B. hybridum. Differentiation of drought-tolerance-related traits between ten genetically distinct B. hybridum populations and its ecological correlates was further analysed. Key Results: The functional drought response is overall well differentiated between Brachypodium species. Brachypodium hybridum allotetraploids showed a transgressive expression pattern in leaf phytohormone content in response to drought. In contrast, other B. hybridum physiological traits correlated to B. stacei ones. Particularly, proline and water content were the traits that best discriminated these species from B. distachyon under drought. Conclusions: After polyploid formation and/or colonization, B. hybridum populations have adaptively diverged physiologically and genetically in response to variations in aridity.

Partial Activation of SA- and JA-Defensive Pathways in Strawberry upon Colletotrichum acutatum Interaction.

Understanding the nature of pathogen host interaction may help improve strawberry (Fragaria × ananassa) cultivars. Plant resistance to pathogenic agents usually operates through a complex network of defense mechanisms mediated by a diverse array of signaling molecules. In strawberry, resistance to a variety of pathogens has been reported to be mostly polygenic and quantitatively inherited, making it difficult to associate molecular markers with disease resistance genes. Colletotrichum acutatum spp. is a major strawberry pathogen, and completely resistant cultivars have not been reported. Moreover, strawberry defense network components and mechanisms remain largely unknown and poorly understood. Assessment of the strawberry response to C. acutatum included a global transcript analysis, and acidic hormones SA and JA measurements were analyzed after challenge with the pathogen. Induction of transcripts corresponding to the SA and JA signaling pathways and key genes controlling major steps within these defense pathways was detected. Accordingly, SA and JA accumulated in strawberry after infection. Contrastingly, induction of several important SA, JA, and oxidative stress-responsive defense genes, including FaPR1-1, FaLOX2, FaJAR1, FaPDF1, and FaGST1, was not detected, which suggests that specific branches in these defense pathways (those leading to FaPR1-2, FaPR2-1, FaPR2-2, FaAOS, FaPR5, and FaPR10) were activated. Our results reveal that specific aspects in SA and JA dependent signaling pathways are activated in strawberry upon interaction with C. acutatum. Certain described defense-associated transcripts related to these two known signaling pathways do not increase in abundance following infection. This finding suggests new insight into a specific putative molecular strategy for defense against this pathogen.

MYB10 plays a major role in the regulation of flavonoid/phenylpropanoid metabolism during ripening of Fragaria x ananassa fruits.

This work characterized the role of the R2R3-MYB10 transcription factor (TF) in strawberry fruit ripening. The expression of this TF takes place mainly in the fruit receptacle and is repressed by auxins and activated by abscisic acid (ABA), in parallel to the ripening process. Anthocyanin was not produced when FaMYB10 expression was transiently silenced in fruit receptacles. An increase in FaMYB10 expression was observed in water-stressed fruits, which was accompanied by an increase in both ABA and anthocyanin content. High-throughput transcriptomic analyses performed in fruits with downregulated FaMYB10 expression indicated that this TF regulates the expression of most of the Early-regulated Biosynthesis Genes (EBGs) and the Late-regulated Biosynthesis Genes (LBGs) genes involved in anthocyanin production in ripened fruit receptacles. Besides, the expression of FaMYB10 was not regulated by FaMYB1 and vice versa. Taken together, all these data clearly indicate that the Fragaria × ananassa MYB10 TF plays a general regulatory role in the flavonoid/phenylpropanoid pathway during the ripening of strawberry.

Identification and validation of reference genes for transcript normalization in strawberry (Fragaria × ananassa) defense responses.

Strawberry (Fragaria spp) is an emerging model for the development of basic genomics and recombinant DNA studies among rosaceous crops. Functional genomic and molecular studies involve relative quantification of gene expression under experimental conditions of interest. Accuracy and reliability are dependent upon the choice of an optimal reference control transcript. There is no information available on validated endogenous reference genes for use in studies testing strawberry-pathogen interactions. Thirteen potential pre-selected strawberry reference genes were tested against different tissues, strawberry cultivars, biotic stresses, ripening and senescent conditions, and SA/JA treatments. Evaluation of reference candidate's suitability was analyzed by five different methodologies, and information was merged to identify best reference transcripts. A combination of all five methods was used for selective classification of reference genes. The resulting superior reference genes, FaRIB413, FaACTIN, FaEF1α and FaGAPDH2 are strongly recommended as control genes for relative quantification of gene expression in strawberry. This report constitutes the first systematic study to identify and validate optimal reference genes for accurate normalization of gene expression in strawberry plant defense response studies.

FaGAST2, a strawberry ripening-related gene, acts together with FaGAST1 to determine cell size of the fruit receptacle.

Numerous GAST-like genes have been reported in higher plants, but only one GAST-like gene (FaGAST1) has been described in strawberry so far. Herein, we have identified a novel strawberry FaGAST gene (FaGAST2) whose expression showed an increase throughout fruit receptacle development and ripening, coinciding with those stages where a decrease in fruit expansion processes (G3-W and R-OR stages) occurs. FaGAST2 only shares 31% and 15.7% amino acid and nucleotide sequence homology, respectively, with the previously reported FaGAST1 gene, but both genes contain a signal peptide and a highly conserved GASA domain (cysteine-rich domain) in the C-terminal region. FaGAST2 expression is mainly confined to the fruit receptacle and is not regulated by auxins, GA(3) or ABA, but is regulated by ethephon, an intracellular generator of ethylene. In addition, the expression of the FaGAST2 gene also increased under oxidative stress conditions (H(2)O(2) or Colletotrichum acutatum infection), suggesting a direct role for FaGAST2 protein in reactive oxygen species scavenging during fruit growth and ripening and during fungal infection. On the other hand, the overexpression of the FaGAST2 gene in different transgenic lines analyzed caused a delay in the growth of strawberry plants and a reduction in the size of the transgenic fruits. The histological studies performed in these fruits showed that their parenchymal cells were smaller than those of the controls, supporting a relationship between FaGAST2 gene expression, strawberry fruit cell elongation and fruit size. However, transitory silencing of FaGAST2 gene expression through RNA interference approaches revealed an increase in FaGAST1 expression, but no changes in fruit cell size were observed. These results support the hypothesis that both genes must act synergistically to determine fruit cell size during fruit development and ripening.

The strawberry plant defense mechanism: a molecular review.

Strawberry, a small fruit crop of great importance throughout the world, has been considered a model plant system for Rosaceae, and is susceptible to a large variety of phytopathogenic organisms. Most components and mechanisms of the strawberry defense network remain poorly understood. However, from current knowledge, it seems clear that the ability of a strawberry plant to respond efficiently to pathogens relies first on the physiological status of injured tissue (pre-formed mechanisms of defense) and secondly on the general ability to recognize and identify the invaders by surface plant receptors, followed by a broad range of induced mechanisms, which include cell wall reinforcement, production of reactive oxygen species, phytoalexin generation and pathogenesis-related protein accumulation. Dissection of these physiological responses at a molecular level will provide valuable information to improve future breeding strategies for new strawberry varieties and to engineer strawberry plants for durable and broad-spectrum disease resistance. In turn, this will lead to a reduction in use of chemicals and in environmental risks. Advances in the understanding of the molecular interplay between plant (mainly those considered model systems) and various classes of microbial pathogens have been made in the last two decades. However, major progress in the genetics and molecular biology of strawberry is still needed to uncover fully the way in which this elaborate plant innate immune system works. These fundamental insights will provide a conceptual framework for rational human intervention through new strawberry research approaches. In this review, we will provide a comprehensive overview and discuss recent advances in molecular research on strawberry defense mechanisms against pathogens.

Evidence for a positive regulatory role of strawberry (Fragaria x ananassa) Fa WRKY1 and Arabidopsis At WRKY75 proteins in resistance.

Knowledge of the molecular basis of plant resistance to pathogens in species other than Arabidopsis is limited. The function of Fa WRKY1, the first WRKY gene isolated from strawberry (Fragaria x ananassa), an important agronomical fruit crop, has been investigated here. Fa WRKY1 encodes a IIc WRKY transcription factor and is up-regulated in strawberry following Colletotrichum acutatum infection, treatments with elicitors, and wounding. Its Arabidopsis sequence homologue, At WRKY75, has been described as playing a role in regulating phosphate starvation responses. However, using T-DNA insertion mutants, a role for the At WRKY75 and Fa WRKY1 in the activation of basal and R-mediated resistance in Arabidopsis is demonstrated. At wrky75 mutants are more susceptible to virulent and avirulent isolates of Pseudomonas syringae. Overexpression of Fa WRKY1 in At wrky75 mutant and wild type reverts the enhanced susceptible phenotype of the mutant, and even increases resistance to avirulent strains of P. syringae. The resistance phenotype is uncoupled to PATHOGENESIS-RELATED (PR) gene expression, but it is associated with a strong oxidative burst and glutathione-S-transferase (GST) induction. Taken together, these results indicate that At WRKY75 and Fa WRKY1 act as positive regulators of defence during compatible and incompatible interactions in Arabidopsis and, very likely, Fa WRKY1 is an important element mediating defence responses to C. acutatum in strawberry. Moreover, these results provide evidence that Arabidopsis can be a useful model for functional studies in Rosacea species like strawberry.