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Lignans are the main secondary metabolites synthetized by Linum species as plant defense compounds but they are also valuable for human health, in particular, for novel therapeutics. In this work, Linum austriacum in vitro cultures, cells (Cc), adventitious roots (ARc) and hairy roots (HRc) were developed for the production of justicidin B through elicitation with methyl jasmonate (MeJA) and coronatine (COR). The performances of the cultures were evaluated for their stability, total phenols content and antioxidant ability. NMR was used to identify justicidin B and isojusticidin B and HPLC to quantify the production, highlighting ARc and HRc as the highest productive tissues. MeJA and COR treatments induced the synthesis of justicidin B more than three times and the synthesis of other compounds. RNA-sequencing and a de novo assembly of L. austriacum ARc transcriptome was generated to identify the genes activated by MeJA. Furthermore, for the first time, the intracellular localization of justicidin B in ARc was investigated through microscopic analysis. Then, HRc was chosen for small-scale production in a bioreactor. Altogether, our results improve knowledge on justicidin B pathway and cellular localization in L. austriacum for future scale-up processes.

Globulins are the predominant class of seed storage proteins in a wide variety of plants. In many plant species globulins are present in several isoforms encoded by gene families. The major seed storage protein of Cannabis sativa L. is the globulin edestin, widely known for its nutritional potential. In this work, we report the isolation of seven cDNAs encoding for edestin from the C. sativa variety Carmagnola. Southern blot hybridization is in agreement with the number of identified edestin genes. All seven sequences showed the characteristic globulin features, but they result to be divergent members/forms of two edestin types. According to their sequence similarity four forms named CsEde1A, CsEde1B, CsEde1C, CsEde1D have been assigned to the edestin type 1 and the three forms CsEde2A, CsEde2B, CsEde2C to the edestin type 2. Analysis of the coding sequences revealed a high percentage of similarity (98-99%) among the different forms belonging to the same type, which decreased significantly to approximately 64% between the forms belonging to different types. Quantitative RT-PCR analysis revealed that both edestin types are expressed in developing hemp seeds and the amount of CsEde1 was 4.44 ± 0.10 higher than CsEde2. Both edestin types exhibited a high percentage of arginine (11-12%), but CsEde2 resulted particularly rich in methionine residues (2.36%) respect to CsEde1 (0.82%). The amino acid composition determined in CsEde1 and CsEde2 types suggests that these seed proteins can be used to improve the nutritional quality of plant food-stuffs., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Phenylalanine ammonia-lyase (PAL), Cinnamic acid 4-hydroxylase (C4H) and 4-Coumarate: CoA ligase (4CL) catalyze the first three steps of the general phenylpropanoid pathway whereas chalcone synthase (CHS) catalyzes the first specific step towards flavonoids production. This class of specialized metabolites has a wide range of biological functions in plant development and defence and a broad spectrum of therapeutic activities for human health. In this study, we report the isolation of hemp PAL and 4CL cDNA and genomic clones. Through in silico analysis of their deduced amino acid sequences, more than an 80% identity with homologues genes of other plants was shown and phylogenetic relationships were highlighted. Quantitative expression analysis of the four above mentioned genes, PAL and 4CL enzymatic activities, lignin content and NMR metabolite fingerprinting in different Cannabis sativa tissues were evaluated. Furthermore, the use of different substrates to assay PAL and 4CL enzymatic activities indicated that different isoforms were active in different tissues. The diversity in secondary metabolites content observed in leaves (mainly flavonoids) and roots (mainly lignin) was discussed in relation to gene expression and enzymatic activities data.

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., (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.

Mybleu is a natural incomplete transcription factor of rice (Oryza sativa), consisting of a partial Myb repeat followed by a short leucine zipper. We previously showed its localization to the apical region of rice roots and coleoptiles. Specifically, in coleoptiles, Mybleu is expressed under both aerobic and anaerobic conditions, whereas in roots, it is expressed only under aerobic conditions. Mybleu is able to dimerize with canonical leucine zippers and to activate transcription selectively. To investigate Mybleu function in vivo, we transformed Arabidopsis thaliana and evaluated several morphological, physiological and biochemical parameters. In agreement with a hypothesized role of Mybleu in cell elongation in the differentiation zone, we found that the constitutive expression of this transcription factor in Arabidopsis induced elongation in the primary roots and in the internodal region of the floral stem; we also observed a modification of the root apex morphology in transformed lines. Based on the high expression of Mybleu in anaerobic rice coleoptiles, we studied the role of this transcription factor in transgenic plants grown under low-oxygen conditions. We found that overexpression of this transcription factor increased tolerance to oxygen deficit. In transgenic plants, this effect may depend both on the maintenance of a higher metabolism during stress and on the higher expression levels of certain genes involved in the anaerobic response.

The expression of the gene Osmyb4, detected at low level in rice (Oryza sativa) coleoptiles grown for 3 days at 29 degrees C, is strongly induced by treatments at 4 degrees C. At sublethal temperatures of 10 and 15 degrees C, its expression in rice seedlings is already evident, but this effect cannot be vicariated by other stresses or ABA treatment. We demonstrate by transient expression that Myb4 transactivates the PAL2, ScD9 SAD and COR15a cold-inducible promoters. The Osmyb4 function in vivo is demonstrated overexpressing its cDNA in Arabidopsis thaliana plants (ecotype Wassilewskija) under the control of the constitutive CaMV 35S promoter. Myb4 overexpressing plants show a significant increased cold and freezing tolerance, measured as membrane or Photosystem II (PSII) stability and as whole plant tolerance. Finally, in Osmyb4 transgenic plants, the expression of genes participating in different cold-induced pathways is affected, suggesting that Myb4 represents a master switch in cold tolerance.