Transportomics for the characterization of plant apocarotenoid transmembrane transporters

Apocarotenoids are carotenoid derivatives produced by the nonenzymatic or enzymatic cleavage of carotenoids, followed by different enzymatic modifications. In plants, apocarotenoids play different roles, such as attraction of pollinators and seeds dispersal, defense against pathogens and herbivores, protection against photo-oxidative stresses, stimulation and inhibition of plant growth and regulation of biological processes in the case of phytohormones abscisic acid and strigolactones. While carotenoids are in general plastid-localized metabolites, apocarotenoids can reach different final destinations inside or outside the cell. The mechanisms of apocarotenoid transport through biological membranes have been poorly studied. This chapter describes a method to characterize transmembrane transporters involved in the transport of polar and amphipathic apocarotenoids. This protocol was successfully used to in vitro characterize the transport activity of ATP-binding cassette (ABC) and multidrug and toxic extrusion (MATE) in microsomes isolated from Saccharomyces cerevisiae expressing these plant transporters

Agrobacterium-mediated and electroporation-mediated transformation of Chlamydomonas reinhardtii: a comparative study

Abstract Background Chlamydomonas reinhardtii is an unicellular green alga used for functional genomics studies and heterologous protein expression. A major hindrance in these studies is the low level and instability of expression of nuclear transgenes, due to their rearrangement and/or silencing over time. Results We constructed dedicated vectors for Agrobacterium-mediated transformation carrying, within the T-DNA borders, the Paromomycin (Paro) selectable marker and an expression cassette containing the Luciferase (Luc) reporter gene. These vectors and newly developed co-cultivation methods were used to compare the efficiency, stability and insertion sites of Agrobacterium- versus electroporation-mediated transformation. The influence of different transformation methods, of the cell wall, of the virulence of different Agrobacterium strains, and of transgene orientation with respect to T-DNA borders were assessed. False positive transformants were more frequent in Agrobacterium-mediated transformation compared to electroporation, compensating for the slightly lower proportion of silenced transformants observed in Agrobacterium-mediated transformation than in electroporation. The proportion of silenced transformants remained stable after 20 cycles of subculture in selective medium. Next generation sequencing confirmed the nuclear insertion points, which occurred in exons or untraslated regions (UTRs) for 10 out of 10 Agrobacterium-mediated and 9 out of 13 of electroporation-mediated insertions. Electroporation also resulted in higher numbers of insertions at multiple loci. Conclusions Due to its labor-intensive nature, Agrobacterium transformation of Chlamydomonas does not present significant advantages over electroporation, with the possible exception of its use in insertional mutagenesis, due to the higher proportion of within-gene, single-locus insertions. Our data indirectly support the hypothesis that rearrangement of transforming DNA occurs in the Chlamydomonas cell, rather than in the extracellular space as previously proposed

A plant protein signal sequence improved humoral immune response to HPV prophylactic and therapeutic DNA vaccines

Signal sequences (ss) play a critical role in the sorting of nascent secretory and membrane proteins. This function has been conserved from bacteria through eukaryotes, although ss appear diverse in length and amino acid composition. Sorting of proteins is also critical to instruct antigens for a proper immunological response. Thus, a plant ss was used to drive Human Papillomavirus (HPV) model antigens into the human secretory pathway: the HPV16 E7 oncoprotein, its chimera with the coat protein (CP) of the Potato Virus X (PVX), the first 200 amino acids of the HPV16 minor capsid protein L2 (known to harbour cross-reacting epitopes) and its chimera with E7 gene. These genes were used to transfect HEK-293 cells and to immunize C57BL/6 mice. The ss-provided genes were expressed, and proteins detected by immunofluorescence and immunoblotting. Mouse immunization with DNA constructs carrying the ss elicited a strong humoral response against both E7 and L2 and a weak cell-mediated immunity. To our knowledge this is the first demonstration that a signal sequence derived from a plant can modulate the sorting of a heterologous protein in mammalian cells. This activity in mammalian cells may be responsible for the observed increased humoral response to DNA-based vaccines that are generally weak inducers of IgG response. This might open new perspectives in the design of DNA vaccines, especially to counteract infections where a strong humoral response is needed

Production of Saffron Apocarotenoids in Nicotiana benthamiana Plants Genome-Edited to Accumulate Zeaxanthin Precursor

Crocins are glycosylated apocarotenoids with strong coloring power and anti-oxidant, anticancer, and neuro-protective properties. We previously dissected the saffron crocin biosynthesis pathway, and demonstrated that the CsCCD2 enzyme, catalyzing the carotenoid cleavage step, shows a strong preference for the xanthophyll zeaxanthin in vitro and in bacterio. In order to investigate substrate specificity in planta and to establish a plant-based bio-factory system for crocin production, we compared wild-type Nicotiana benthamiana plants, accumulating various xanthophylls together with α- and β-carotene, with genome-edited lines, in which all the xanthophylls normally accumulated in leaves were replaced by a single xanthophyll, zeaxanthin. These plants were used as chassis for the production in leaves of saffron apocarotenoids (crocins, picrocrocin) using two transient expression methods to overexpress CsCCD2: agroinfiltration and inoculation with a viral vector derived from tobacco etch virus (TEV). The results indicated the superior performance of the zeaxanthin-accumulating line and of the use of the viral vector to express CsCCD2. The results also suggested a relaxed substrate specificity of CsCCD2 in planta, cleaving additional carotenoid substrates

Anti-cancer activity of grape seed semi-polar extracts in human mesothelioma cell lines

Malignant mesothelioma is a tumor that affects pleural surface and has very poor prognosis. The standard therapeutic modalities for this cancer have yielded unsatisfactory outcomes, therefore the development of alternative and effective therapies is currently an urgent requirement. Grapevine is a plant rich of bioactive compounds, known for its therapeutic effects. Here, we describe the anti-cancer activity of grape seeds semi-polar extracts of two Italian grape varieties (Aglianico and Falanghina) in mesothelioma in vitro. Seed extracts from both varieties induced intrinsic apoptosis in a dose and time-dependent manner in three different human mesothelioma cell lines. Global metabolic analysis of this fraction revealed a higher accumulation of phenylpropanoid precursors and proanthocyanidins and expression of genes involved in the aforementioned pathways. These findings suggest that new phenolic molecules from grape seeds could be viewed as new drugs to be used alone or in combinations with standard chemotherapeutics in mesothelioma treatment

Integrative analysis of metabolome and transcriptome profiles to highlight aroma determinants in Aglianico and Falanghina grape berries

Abstract Background The biochemical makeup of grape berries at harvest is essential for wine quality and depends on a fine transcriptional regulation occurring during berry development. In this study, we conducted a comprehensive survey of transcriptomic and metabolomic changes occurring in different berry tissues and developmental stages of the ancient grapes Aglianico and Falanghina to establish the patterns of the secondary metabolites contributing to their wine aroma and investigate the underlying transcriptional regulation. Results Over two hundred genes related to aroma were found, of which 107 were differentially expressed in Aglianico and 99 in Falanghina. Similarly, 68 volatiles and 34 precursors were profiled in the same samples. Our results showed a large extent of transcriptomic and metabolomic changes at the level of isoprenoids (terpenes, norisoprenoids), green leaf volatiles (GLVs), and amino acid pathways, although the terpenoid metabolism was the most distinctive for Aglianico, and GLVs for Falanghina. Co-expression analysis that integrated metabolome and transcriptome data pinpointed 25 hub genes as points of biological interest in defining the metabolic patterns observed. Among them, three hub genes encoding for terpenes synthases (VvTPS26, VvTPS54, VvTPS68) in Aglianico and one for a GDP-L-galactose phosphorylase (VvGFP) in Falanghina were selected as potential active player underlying the aroma typicity of the two grapes. Conclusion Our data improve the understanding of the regulation of aroma-related biosynthetic pathways of Aglianico and Falanghina and provide valuable metabolomic and transcriptomic resources for future studies in these varieties

Aglianico Grape Seed Semi-Polar Extract Exerts Anticancer Effects by Modulating MDM2 Expression and Metabolic Pathways

Grapevine (Vitis vinifera L.) seeds are rich in polyphenols including proanthocyanidins, molecules with a variety of biological effects including anticancer action. We have previously reported that the grape seed semi-polar extract of Aglianico cultivar (AGS) was able to induce apoptosis and decrease cancer properties in different mesothelioma cell lines. Concomitantly, this extract resulted in enriched oligomeric proanthocyanidins which might be involved in determining the anticancer activity. Through transcriptomic and metabolomic analyses, we investigated in detail the anticancer pathway induced by AGS. Transcriptomics analysis and functional annotation allowed the identification of the relevant causative genes involved in the apoptotic induction following AGS treatment. Subsequent biological validation strengthened the hypothesis that MDM2 could be the molecular target of AGS and that it could act in both a p53-dependent and independent manner. Finally, AGS significantly inhibited tumor progression in a xenograft mouse model of mesothelioma, confirming also in vivo that MDM2 could act as molecular player responsible for the AGS antitumor effect. Our findings indicated that AGS, exerting a pro-apoptotic effect by hindering MDM2 pathway, could represent a novel source of anticancer molecules