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Flax is one of the oldest crop being widely grown for its oil, fiber content and food nutrients such as high quality omega-3 fatty acids (?-linolenic acid), proteins, lignin and phenolic acids (lignans). Besides industrial and nutritional importance, flaxseeds exhibit numerous pharmacological activities due to the presence of biologically active components (lignans) that show antioxidant, cytotoxic, antifungal, antiviral and phytoestrogenic properties. Furthermore, numerous studies have demonstrated the role of these compounds in reducing the risk of certain types of cancers especially the breast, colon and prostate cancers. Therefore, due to the growing need of these medically and nutritionally important metabolites, it is crucial to find sustainable and eco-friendly alternative system for their production on large scale. In vitro plant cell cultures represents a promising source for rapid and enhanced production of desired plant metabolites. Indeed, the production of these metabolites can be enhanced by the use of specific elicitors that in small quantities promote the biosynthesis of secondary metabolites. Flax comprises over 180 species taxonomically divided into 2 clades. The species belonging to the first clade are characterized by the accumulation of podophyllotoxin type lignans, which show cytotoxic activity and are successfully used as anticancer compounds. The species belonging to the second clade accumulate the justicidin B and secoisolaricilresinol (SECO) as major lignans, which show an anti-inflammatory, anti-viral and cytotoxic activity. The aim of this research project was to exploit different flax species for improving lignans production in vitro cell cultures. To this purpose, we analyzed the total phenol content, the antioxidant capacity and the metabolite profile of the cell extracts in order to identify flax species that produce the highest level of metabolites. We have also evaluated the effect of the elicitor methyl-jasmonate on metabolites production, and we are now planning to apply a metabolic engineering approach to improve the flax bioactive compound production.

The importance of perilla frutescens as a species rich in medicinal properties and with a high antioxidant content is increasingly know. The use of this asian plant is spreading worldwide. Our previous studies regarding visits by honey bees and other insect pollinators on Perilla aimed to explore the possible effects on the pollinator action on the content of active ingredients of seeds from this species . The positive effects of pollinating insects and in particular the honey bee, are well known in crop plants such as fruit, oilseed and fodder, but have not been studied in medicinal plants. Pollination tests were conducted in september (2013) by isolating plants of Perilla with mesh cages before blooming and considering another plants left open to insect pollination. At maturity, seeds were collected counted and weighed. In (2015) additional trials were carried out to assess possible differences in antioxidant content of open pollination plants. The difference in seeds number and weight between open pollinated flowers recorded using a 600 MHz spectrometer showed the presence of rosmarinic acid, luteolin and apigenin, in line with previous studies, while their content was increased in plants with open insect pollination. In particular seeds from open (2013) compared to caged plants indicated the presence of glucosylated flavonoid, most likely due the proteolitic activity of glucosidic enzymes. Seeds from open-pollinated (2015) plants showed a content of luteolin and epigenin that was increased by 3-fold compared to self-polinated (2013) plants. As he honey bee is the main pollinator of Perilla, its important role in increasing the presence of antioxidants is becoming more evident.

The aim of this work was to study the ability of flax and hemp Liquid Cell Cultures (LCCs) to produce and accumulate secondary metabolites of interest for industrial/pharmaceutical purposes. At first, seven cultivars of hemp and flax were tested with a combined approach of biochemistry, molecular biology and 1H-NMR profiling to select the most promising cultivar of both species. qRT-PCR of two key genes of phenylpropanoid pathway (PAL and 4CL) and their relative specific enzymatic activity have been determined, as well as the metabolic profile of leaves extracts. On the basis of these results two cultivars, 'Valoal' for flax and 'Futura' for hemp, have been chosen to set up the cell cultures. The obtained LCCs have been characterized for total phenolics and flavonoids content, free radical scavenging activity (DPPH assay) and metabolic profiles- (1H-NMR). According to the class of metabolites identified the relative biosynthetic pathway has been examined by mean of key enzymatic activities and relative mRNA expression (qRT-PCR).

One of the major objectives of Italian rice (Oryza sativa L.) breeding programs is the development of less water-demanding varieties, for the adaptation of rice to suboptimal climate and soil conditions. Polyethylene glycol (PEG)-mediated osmotic stress is an experimental systems which decreases the water potential of the nutrient solution and therefore it has been often used to study drought response in plants. Previously, we performed a physiological characterization of 17 Italian rice cultivars for their response to PEG-mediated osmotic treatment. Differential responses among the genotypes were highlighted and two cultivars showing opposite phenotypes were selected for transcriptome analysis: the high sensitive cultivar Loto and the high tolerant Eurosis. A RNA-Seq analysis (Illumina technology) was performed on leaf and root samples of these cultivars, after 3h and 24h of osmotic stress treatment. The analysis of transcriptomic data revealed that a 3h-treatment led to a substantial modulation of genes in the roots of Eurosis, whereas fewer DEGs were called in the roots of the sensitive cultivar Loto. After 24h of treatment, a similar number of DEGs was found in Eurosis and Loto. In leaves, the highest number of DEGs was observed in Loto after 24h of stress treatment. GO enrichment analysis of DEGs in the various contrasts highlighted some differences between the cultivars, with several genotype-specific GO terms (i.e. enriched GO terms from one genotype/tissue/treatment missing in the alternative genotype in the same tissue/treatment). Some enriched GO terms specific to the tolerant genotype Eurosis were related to chromatin and cytoskeleton. Many genes associated with these GO terms were down-regulated in 3h-treated Eurosis roots, whereas in Loto they were down-regulated only after 24h, suggesting a delay in the Loto response. These data indicated a different modulation of gene sets between the tolerant and the sensitive cultivars. In particular, Eurosis showed an earlier regulation of gene expression in response to osmotic stress which is not paralleled, at least at early stages, in Loto. The analysis of the expression data is in progress to reveal genes putatively responsible for the contrasting phenotypes of the analyzed genotypes. Many genes showed a contrasting regulation between the two cultivars, especially in roots at 3h of stress treatment; among them, several transcription factors and two helicases encoding genes. Helicases are involved in RNA metabolism and may be important for increasing transcriptional and translational activity under stress conditions. Interestingly, some myb and WRKY genes seemed to be involved in the different response to the osmotic stress, since they showed a different regulation in the two genotypes. For instance in roots, at 3h of treatment, OsMYB55, known to be involved in the heat stress response, and the myb-related NIGT1 gene, known to be involved in salt stress and nitrate response, were up-regulated only in Eurosis; WRKY30 was up-regulated in Loto and down-regulated in Eurosis, whereas WRKY106 and WRKY117 were down-regulated only in Eurosis. This work was supported by Progetto AGER, grant n° 2010-2369 (RISINNOVA - Integrated genetic and genomic approaches for new Italian rice breeding strategies).

Abiotic stresses are the primary cause of crop loss worldwide, reducing average yields for most major crop plants by more than 50%. Rice is particularly susceptible to dehydration, and cultivars may considerably differ in their vegetative response to water deficit. The identification of key genes regulated by water shortage is a major objective for the development of drought-tolerant varieties by molecular breeding. Here, we provide a physiological analysis of the response to osmotic stress in 17 Italian rice cultivars (Oryza sativa L. ssp japonica). Rice plants were grown in hydroponic culture and then subjected to PEG treatment (20% PEG 6000). Based on the measurement of two physiological parameters (leaf Relative Water Content, RWC, and Electrolyte Leakage, EL), the cultivars exhibited differential responses to osmotic stress. After 48h of treatment, Carnaroli, Gigante Vercelli, Loto, Maratelli and Vialone Nano exhibited the lowest RWC values (80%. Similarly, the first group of cultivars exhibited the highest EL values (>94%), whereas the second group maintained EL values lower than 40%. The other varieties exhibited an intermediate sensitivity. Therefore, the cultivars Eurosis (tolerant) and Loto (sensitive) were selected for transcriptome analysis, to identify differentially regulated genes (DEGs) and pathways. Total RNAs were separately extracted from leaves and roots, after 3h and 24h of osmotic stress treatment, and used for RNA sequencing through Illumina (HiSeq2000) technology. The analysis of transcriptomic data revealed that a 3h osmotic treatment led to a substantial modulation of genes in the roots of the tolerant genotype Eurosis (6007 genes), whereas fewer DEGs were called in the roots of the sensitive cv Loto (3962 genes). At 24h of treatment, a similar number of DEGs was found in Eurosis and Loto (3065 and 3102 genes, respectively). In leaves, the highest number of DEGs was observed in Loto at 24h of stress treatment. The responses of the two genotypes were different, both in roots and leaves, especially at 3h of treatment. In roots, at 3h of treatment 2552 DEGs were common to Eurosis and Loto, whereas 3455 were specific to Eurosis and 1410 to Loto. At 24h, 1789 DEGs were common to the two cultivars, whereas 1276 and 1313 DEGs were specific to Eurosis and Loto, respectively. In leaves, at 3h of treatment 2062 DEGs were common to the two cultivars, whereas 915 were specific to Eurosis and 2751 to Loto. At 24h, 2004 DEGs were common to the two cultivars, whereas 2219 and 1084 were specific to Eurosis and Loto, respectively. GO enrichment analysis of DEGs in the various contrasts is in progress to reveal biological trends differentiating tolerant and sensitive genotypes.

The constitutive expression of the rice Osmyb4 gene in Arabidopsis plants gives rise to enhanced abiotic and biotic stress tolerance, probably by activating several stress-inducible pathways. However, the effect of Osmyb4 on stress tolerance likely depends on the genetic background of the transformed species. In this study, we explored the potential of Osmyb4 to enhance the cold and freezing tolerance of Osteospermum ecklonis, an ornamental and perennial plant native to South Africa, because of an increasing interest in growing this species in Europe where winter temperatures are low. Transgenic O. ecklonis plants were obtained through transformation with the Osmyb4 rice gene under the control of the CaMV35S promoter. We examined the phenotypic adaptation of transgenic plants to cold and freezing stress. We also analysed the ability of wild-type and transgenic Osteospermum to accumulate several solutes, such as proline, amino acids and sugars. Using nuclear magnetic resonance, we outlined the metabolic profile of this species under normal growth conditions and under stress for the first time. Indeed, we found that overexpression of Osmyb4 improved the cold and freezing tolerance and produced changes in metabolite accumulation, especially of sugars and proline. Based on our data, it could be of agronomic and economic interest to use this gene to produce Osteospermum plants capable of growing in open field, even during the winter season in climatic zone Z9. (C) 2010 Elsevier Masson SAS. All rights reserved.

Constitutive expression of the rice cold-inducible Osmyb4 gene in transgenic Arabidopsis (Arabidopsis thaliana) plants improves adaptive responses to cold and drought stress, most likely due to the constitutive activation of several stress-inducible pathways and to the accumulation of several compatible solutes (e.g., glucose, fructose, sucrose, proline, glycine betaine and some aromatic compounds). Although the Osmyb4 gene seems able to activate stress responsive pathways in different species, we previously reported that its specific effect on stress tolerance depends on the transformed species. In the present work, we report the effects of the Osmyb4 expression for improving the stress response in apple (Malus pumila Mill.) plants. Namely, we found that the ectopic expression of the Myb4 transcription factor improved physiological and biochemical adaptation to cold and drought stress and modified metabolite accumulation. Based on these results it may be of interest to use Osmyb4 as a tool for improving the productivity of woody perennials under environmental stress conditions.