Transcriptional regulation os phenylalaline biosynthesis and utilization

Conifer trees divert large quantities of carbon into the biosynthesis of phenylpropanoids, particularly to generate lignin, an important constituent of wood. Since phenylalanine is the precursor for phenylpropanoid biosynthesis, the precise regulation of phenylalanine synthesis and utilization should occur simultaneously. This crucial pathway is finely regulated primarily at the transcriptional level. Transcriptome analyses indicate that the transcription factors (TFs) preferentially expressed during wood formation in plants belong to the MYB and NAC families. Craven-Bartle et al. (2013) have shown in conifers that Myb8 is a candidate regulator of key genes in phenylalanine biosynthesis involved in the supply of the phenylpropane carbon skeleton necessary for lignin biosynthesis. This TF is able to bind AC elements present in the promoter regions of these genes to activate transcription. Constitutive overexpression of Myb8 in white spruce increased secondary-wall thickening and led to ectopic lignin deposition (Bomal et al. 2008). In Arabidopsis, the transcriptional network controlling secondary cell wall involves NAC-domain regulators operating upstream Myb transcription factors. Functional orthologues of members of this network described have been identified in poplar and eucalyptus, but in conifers functional evidence had only been obtained for MYBs. We have identified in the P. pinaster genome 37 genes encoding NAC proteins, which 3 NAC proteins could be potential candidates to be involved in vascular development (Pascual et al. 2015). The understanding of the transcriptional regulatory network associated to phenylpropanoids and lignin biosynthesis in conifers is crucial for future applications in tree improvement and sustainable forest management. This work is supported by the projects BIO2012-33797, BIO2015-69285-R and BIO-474 References: Bomal C, et al. (2008) Involvement of Pinus taeda MYB1 and MYB8 in phenylpropanoid metabolism and secondary cell wall biogenesis: a comparative in planta analysis. J Exp Bot. 59: 3925-3939. Craven-Bartle B, et al. (2013) A Myb transcription factor regulates genes of the phenylalanine pathway in maritime pine. Plant J, 74: 755-766. Pascual MB, et al. (2015) The NAC transcription factor family in maritime pine (Pinus pinaster): molecular regulation of two genes involved in stress responses. BMC Plant Biol, 15: 254.

Mineral stress response of Vitis cell wall transcriptome: identification, characterization and expression of genes from key families involved in wall biosynthesis and modification

Doutoramento em Engenharia Agronómica - Instituto Superior de Agronomia - UL

NAC-MYB basedtranscriptional networkinvolved in the regulation of phenylalanine biosynthesis in P. pinaster

P2-2 NAC-MYB-BASED TRANSCRIPCIONAL NETWORK INVOLVED IN THE REGULATION OF PHENYLALANINE BIOSYNTHESIS IN P. PINASTER Mª Belén Pascual, Rafael A. Cañas, Blanca Craven-Bartle, Francisco M. Cánovas and Concepción Ávila Departamento de Biología Molecular y Bioquímica. Facultad de Ciencias. Universidad de Málaga. Campus de teatinos s/n, Málaga, Spain Email: cavila@uma.es Conifer trees divert large quantities of carbon into the biosynthesis of phenylpropanoids, particularly to generate lignin, an important constituent of wood. Since phenylalanine is the precursor for phenylpropanoid biosynthesis, the precise regulation of phenylalanine synthesis and use should occur simultaneously. This crucial pathway is finely regulated primarily at the transcriptional level. Transcriptome analyses indicate that the transcription factors (TFs) preferentially expressed during wood formation in plants belong to the MYB and NAC families. Craven-Bartle et al. (2013) have shown that Myb8 is a candidate regulator of key genes in phenylalanine biosynthesis involved in the supply of the phenylpropane carbon skeleton necessary for lignin biosynthesis. This TF is able to bind AC elements present in the promoter regions of these genes to activate transcription. In Arabidopsis, the transcriptional network controlling secondary cell wall involves NAC-domain regulators operating upstream Myb transcription factors. We have identified in the P. pinaster genome three NAC proteins as potential candidates to be involved in vascular development. One of them, PpNAC1 is expressed both in xylem and compression wood from adult trees and has been thoroughly characterized. Its role upstream the transcriptional network involving Myb8 will be discussed. The understanding of the transcriptional regulatory network associated to phenylpropanoids and lignin biosynthesis in conifers is crucial for future applications in tree improvement and sustainable forest management.

Metabolic channelingof phe for lignin biosynthesis in maritime pine

METABOLIC CHANNELING OF PHE FOR LIGNIN BIOSYNTHESIS IN MARITIME PINE Jorge El-Azaz, Fernando de la Torre, Belén Pascual, Concepción Ávila and Francisco M. Cánovas Departamento de Biología Molecular y Bioquímica, Universidad de Málaga. Málaga, Spain Email: jelazaz@alu.uma.es The amino acid phenylalanine (Phe) is the main precursor of phenylpropanoids biosynthesis in plants. This vast family of Phederived compounds can represent up to 30% of captured photosynthetic carbon, playing essential roles in plants such as cell wall components, defense molecules, pigments and flavors. In addition to its physiological importance, phenylpropanoids and particularly lignin, a component of wood, are targets in plant biotechnology. The arogenate pathway has been proposed as the main pathway for Phe biosynthesis in plants (Maeda et al., 2010). The final step in Phe biosynthesis, catalyzed by the enzyme arogenate dehydratase (ADT), has been considered as a key regulatory point in Phe biosynthesis, due to its key branch position in the pathway, the multiple isoenzymes identified in plants and the existence of a feedback inhibition mechanism by Phe. So far, the regulatory mechanisms underlying ADT genes expression have been poorly characterized, although a strong regulation of the Phe metabolic flux should be expected depending on its alternative use for protein biosynthesis versus phenylpropanoid biosynthesis. This second fate involves a massive carbon flux compared to the first one. In this study we report our current research activities in the transcriptional regulation of ADT genes by MYB transcription factors in the conifer Pinus pinaster (maritime pine). The conifers channels massive amounts of photosynthetic carbon for phenylpropanoid biosynthesis during wood formation. We have identified the complete ADT gene family in maritime pine (El-Azaz et al., 2016) and a set of ADT isoforms specifically related with the lignification process. The potential control of transcription factors previously reported as key regulators in pine wood formation (Craven-Bartle et al., 2013) will be presented. Maeda et al. (2010) Plant Cell 22: 832-849. El-Azaz et al. (2016) The Plant Jounal. Accepted article, doi: 10.1111/tpj.13195 Craven-Bartle et al. (2013). The Plant Journal 74(5):755-766

Intra- and extracellular osmotic regulation in the hololimnetic Caridea and Anomura: a phylogenetic perspective on the conquest of fresh water by the decapod Crustacea

We investigate extra- and intracellular osmoregulatory capability in two species of hololimnetic Caridea and Anomura: Macrobrachium brasiliense, a palaemonid shrimp, and Aegla franca, an aeglid anomuran, both restricted to continental waters. We also appraise the sharing of physiological characteristics by the hololimnetic Decapoda, and their origins and role in the conquest of fresh water. Both species survive salinity exposure well. While overall hyperosmoregulatory capability is weak in A. franca and moderate in M. brasiliense, both species strongly hyporegulate hemolymph [Cl-] but not osmolality. Muscle total free amino acids (FAA) increase slowly but markedly in response to the rapid rise in hemolymph osmolality consequent to hyperosmotic challenge: 3.5-fold in A. franca and 1.9-fold in M. brasiliense. Glycine, taurine, arginine, alanine and proline constitute a parts per thousand 85% of muscle FAA pools in fresh water; taurine, arginine, alanine each contribute a parts per thousand 22% in A. franca, while glycine predominates (70%) in M. brasiliense. These FAA also show the greatest increases on salinity challenge. Muscle FAA titers correlate strongly (R = 0.82) with hemolymph osmolalities across the main decapod sub/infraorders, revealing that marine species with high hemolymph osmolalities achieve isosmoticity of the intra- and extracellular fluids partly through elevated intracellular FAA concentrations; freshwater species show low hemolymph osmolalities and exhibit reduced intracellular FAA titers, consistent with isosmoticity at a far lower external osmolality. Given the decapod phylogeny adopted here and their multiple, independent invasions of fresh water, particularly by the Caridea and Anomura, our findings suggest that homoplastic strategies underlie osmotic and ionic homeostasis in the extant freshwater Decapoda.

Candida albicans Modifies the Protein Composition and Size Distribution of THP1 macrophages-derived Extracellular Vesicles.

The effectiveness of macrophages in the response to systemic candidiasis is crucial to an effective clearance of the pathogen. The secretion of proteins, mRNAs, non-coding RNAs and lipids through extracellular vesicles (EVs) is one of the mechanisms of communication between immune cells. EVs change their cargo to mediate different responses, and may play a role in the response against infections. Thus, we have undertaken the first quantitative proteomic analysis on the protein composition of THP1 macrophages-derived EVs during the interaction with Candida albicans. This study revealed changes in EVs sizes and in protein composition, and allowed the identification and quantification of 717 proteins. Of them, 133 proteins changed their abundance due to the interaction. The differentially abundant proteins were involved in functions relating to immune response, signaling, or cytoskeletal reorganization. THP1-derived EVs, both from control and from Candida-infected macrophages, had similar effector functions on other THP1-differenciated macrophages, activating ERK and p38 kinases, and increasing both the secretion of proinflammatory cytokines and the candidacidal activity; while in THP1 non-differenciated monocytes, only EVs from infected macrophages increased significantly the TNF-α secretion. Our findings provide new information on the role of macrophage-derived EVs in response to C. albicans infection and in macrophages communication.

Plant Genes Related to Gibberellin Biosynthesis and Signaling Are Differentially Regulated during the Early Stages of AM Fungal Interactions

Dear Editor, Phytohormones are essential regulators of plant development, but their role in the signaling processes between plants and fungi during arbuscular mycorrhizal (AM) establishment is far from being understood (Ludwig-Müller, 2010). AM colonization leads to extensive effects on host metabolism, as revealed by transcriptome studies of AM plants (Hogekamp et al., 2011). Some genes have been specified as an AM core set, since they are mycorrhizal-responsive, irrespective of the identity of the plant, of the fungus, and of the investigated organ. These data support the idea that, on colonization, plants activate a wide reprogramming of their major regulatory networks and argue that mobile factors of fungal or plant origin are involved in such generalized metabolic changes. In this context, hormones may be good candidates (Bonfante and Genre, 2010). However, the emerging picture of the interaction between phytohormones and AMs is very patchy, and information on gibberellin (GA) involvement is still more limited (García-Garrido et al., 2010). The role of GA during nodulation is instead known to control the nodulation signaling pathway (Ferguson et al., 2011).

Natural variation in expression of genes associated with carotenoid biosynthesis and accumlation in cassava (Manihot esculenta Crantz) storage root.

2016

Natural variation in expression of genes associated with carotenoid biosynthesis and accumulation in cassava (Manihot esculenta Crantz) storage root.

ABSTRACT: BACKGROUND: Cassava (Manihot esculenta Crantz) storage root provides a staple food source for millions of people worldwide. Increasing the carotenoid content in storage root of cassava could provide improved nutritional and health benefits. Because carotenoid accumulation has been associated with storage root color, this study characterized carotenoid profiles, and abundance of key transcripts associated with carotenoid biosynthesis, from 23 landraces of cassava storage root ranging in color from white-to-yellow-to-pink. This study provides important information to plant breeding programs aimed at improving cassava storage root nutritional quality. RESULTS: Among the 23 landraces, five carotenoid types were detected in storage root with white color, while carotenoid types ranged from 1 to 21 in storage root with pink and yellow color. The majority of storage root in these landraces ranged in color from pale-to-intense yellow. In this color group, total ß-carotene, containing all-E-, 9-Z-, and 13-Z-ß-carotene isomers, was the major carotenoid type detected, varying from 26.13 to 76.72 %. Although no ?-carotene was observed, variable amounts of a ?-ring derived xanthophyll, lutein, was detected; with greater accumulation of ?-ring xanthophylls than of ß-ring xanthophyll. Lycopene was detected in a landrace (Cas51) with pink color storage root, but it was not detected in storage root with yellow color. Based on microarray and qRT-PCR analyses, abundance of transcripts coding for enzymes involved in carotenoid biosynthesis were consistent with carotenoid composition determined by contrasting HPLC-Diode Array profiles from storage root of landraces IAC12, Cas64, and Cas51. Abundance of transcripts encoding for proteins regulating plastid division were also consistent with the observed differences in total ß-carotene accumulation. CONCLUSIONS: Among the 23 cassava landraces with varying storage root color and diverse carotenoid types and profiles, landrace Cas51 (pink color storage root) had low LYCb transcript abundance, whereas landrace Cas64 (intense yellow storage root) had decreased HYb transcript abundance. These results may explain the increased amounts of lycopene and total ß-carotene observed in landraces Cas51 and Cas64, respectively. Overall, total carotenoid content in cassava storage root of color class representatives were associated with spatial patterns of secondary growth, color, and abundance of transcripts linked to plastid division. Finally, a partial carotenoid biosynthesis pathway is proposed.

ABC transporters of the A subfamily: potential prognostic markers and candidates for antibody selection from phage-display libraries for therapeutic use in Ewing sarcoma

The prognostic value of ABC transporters in Ewing sarcoma is still poorly explored and controversial. We described for the first time the impact of various ABCs on Ewing sarcoma prognosis by assessment of their gene expression in two independent cohorts of patients. Unexpected associations with favourable outcomes were observed for two ABCs of the A-subfamily, ABCA6 and ABCA7, whereas no associations with the canonical multidrug ABC transporters were identified. The ABCs of the A-subfamily are involved in cholesterol/phospholipids transportation and efflux from cells. Our clinical data support the drug-efflux independent contribution to cancer progression of the ABCAs, which has been confirmed in PDX-derived cell lines. The impact of these ABCA transporters on tumor progression seems to be mediated by lowering intracellular cholesterol, supporting the role of these proteins in lipid transport. In addition, the gene expression of ABCA6 and ABCA7 is regulated by transcription factors which control lipid metabolism: ABCA6 was induced by the binding of FoxO1/FoxO3a to its promoter and repressed by IGF1R/Akt signaling, whereas the expression of ABCA7 was regulated by p53. The data point to ABCA6 and ABCA7 as potential prognostic markers in Ewing sarcoma and suggest the IGF1/ABCA/lipid axis as an intriguing therapeutic target. Agonist monoclonal antibodies towards ABCA6/7 or inhibitors of cholesterol biosynthesis, such as statins or aminobiphoshonates, may be investigated as therapeutic options in combination with chemotherapy. Considering that no monoclonal antibodies selectively targeting extracellular domains of ABCA6/7 are available, the second part of the project has been dedicated to the generation of human antibody phage-display libraries as tools for selecting monoclonal antibodies. A novel synthetic human antibody phage-display library has been designed, cloned and characterized. The library takes advantages of the high variability of a designed naïve repertoire to be a useful tool for isolating antibodies towards all potential antigens, including the ABCAs.

Analysis of extracellular vesicles from patients with advanced pancreatic cancer identifies miRNAs with predictive value for treatment with gemcitabine + nab-paclitaxel

Pancreatic cancer (PC) is the seventh leading cause of cancer death. Despite recent therapy advancements, 5-year survival is 11%. Resistance to therapy is common, and no predictive factors, except for BRCA1/2 and PALB2 mutations, can drive treatment selection. Based on the easy isolation of extracellular vesicles (EVs) from blood and the role of EV-borne miRNAs in chemoresistance, we analyzed EVs and their miRNA content in order to identify predictive factors. First, we analyzed samples from 28 PC patients and 7 healthy subjects, in order to establish methods for isolation and analysis of EVs and their miRNA content. We observed a significantly different expression of 28 miRNAs, including oncogenic or tumor suppressor miRNAs, showing the ability of our approach to detect candidate biomarkers. Then, we analyzed samples of 21 advanced PC patients, collected before first-line treatment with gemcitabine + nab-paclitaxel, and compared findings in responders and non-responders. EVs have been analyzed with Nanoparticle tracking analysis, flow cytometry and RNA-Seq; then, laboratory results have been matched with clinical data. Nanoparticle tracking analysis did not show any significant difference. Flow cytometry showed a lower expression of SSE4 and CD81 in responders. Finally, miRNA analysis showed 25 upregulated and 19 downregulated miRNAs in responders. In particular, in responders we observed upregulation of miR-141-3p, miR-141-5p, miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-375-3p, miR-429, miR-545-5p. These miRNAs have targets with a previously reported role in PC. In conclusion, we show the feasibility of the proposed approach to identify EV-derived biomarkers with predictive value for therapy with gemcitabine + nab-paclitaxel in PC. Our findings highlight the possibility to exploit liquid biopsy for personalized treatment in PC, in order to maximize chances of response and patients’ outcome. These findings are worthy of further investigation: in the same setting, with different chemotherapy schedules, and in different disease settings such as preoperative therapy.