A hairy-root transformation protocol for Trigonella foenum-graecum L. as a tool for metabolic engineering and specialised metabolite pathway elucidation

The development of genetic transformation methods is critical for enabling the thorough characterization of an organism and is a key step in exploiting any species as a platform for synthetic biology and metabolic engineering approaches. In this work we describe the development of an Agrobacterium rhizogenes-mediated hairy root transformation protocol for the crop and medicinal legume fenugreek (Trigonella foenum-graecum). Fenugreek has a rich and diverse content in bioactive specialised metabolites, notably diosgenin, which is a common precursor for synthetic human hormone production. This makes fenugreek a prime target for identification and engineering of specific biosynthetic pathways for the production of triterpene and steroidal saponins, phenolics, and galactomanans. Through this transformation protocol, we identified a suitable promoter for robust transgene expression in fenugreek. Finally, we establish the proof of principle for the utility of the fenugreek system for metabolic engineering programs, by heterologous expression of known triterpene saponin biosynthesis regulators from the related legume Medicago truncatula in fenugreek hairy roots

Breeding, molecular markers and molecular biology of the olive tree

Olive (Olea europaea L.) is a typical crop species of the Mediterranean Basin. A number of cultivars were selected and propagated mainly vegetatively over the centuries for their qualitative and quantitative traits. Due to the long juvenile phase of the tree, few breeding programs have been performed. Therefore the most appropriate process is a selection scheme from heterogeneous populations or cultivars varying in oil quantity and quality, harvest regimes, and biotic and abiotic resistance. Molecular marker techniques have been applied recently on olive to relate, identify, distinguish and characterize different cultivars or genotypes and in order to provide information on olive origin and dispersal and to evaluate olive germplasm for traits with agronomical importance. To understand the regulation of biosynthetic pathways of oil and antioxidants on the molecular level, we have isolated a number of genes encoding for key enzymes in fatty acid and antioxidant biosynthesis, modification and triacylglycerol storage. The gene expression during fruit growth and seed development as well as their transient and temporal expression in different tissues is discussed in relation to storage of fatty acids and to provision of signaling molecules important in plant defense mechanisms and reproduction

The Role of PME2 and PME3 in Arabidopsis Stomatal Development and Morphology †

Pectin methylesterases (PMEs) are enzymes, encoded by multigene families, that catalyze the demethylesterification of cell wall homogalacturonans. The removal of methyl groups, if performed block-wise in large series of methylesters, leads to the production of homogalacturonans that can be cross-linked with calcium bridges. This fine modulation of the methylesterification status of the pectin network alters the mechanical properties of the cell wall and has been proven crucial for stomatal complex ontogenesis and function. Considering this significant role of PMEs, as well as their involvement in numerous plant development processes, we investigated the phenotypic implications of two Arabidopsis thaliana PME compromised mutants (pme2 and pme3) and the corresponding double mutant (pme2 pme3) in stomatal development and morphology. The cotyledons of the double mutant were larger and wider, while the ratio of length/width was smaller compared to that of WT plants. The stomatal patterning was also affected since the pme2 pme3 mutant displayed a higher number of mature stomata as well as a higher percentage of stomatal clustering. Furthermore, the guard cells of the double mutant displayed a lower ratio of cell length to width, indicating alterations in the morphology of mature stomata. As far as the cell wall matrix composition is concerned, callose and pectins’ epitope distribution displayed significant differences in pme single and double mutants compared to WT plants. Taken together, our results underline the indispensable role of PME2 and PME3 in stomatal development, since their functional disruption affects not only stomatal patterning but also the morphology and function of the guard cells

The SAH7 Homologue of the Allergen Ole e 1 Interacts with the Putative Stress Sensor SBP1 (Selenium-Binding Protein 1) in Arabidopsis thaliana

In this study, we focused on a member of the Ole e 1 domain-containing family, AtSAH7, in Arabidopsis thaliana. Our lab reports for the first time on this protein, AtSAH7, that was found to interact with Selenium-binding protein 1 (AtSBP1). We studied by GUS assisted promoter deletion analysis the expression pattern of AtSAH7 and determined that the sequence 1420 bp upstream of the transcription start can act as a minimal promoter inducing expression in vasculature tissues. Moreover, mRNA levels of AtSAH7 were acutely increased under selenite treatment in response to oxidative stress. We confirmed the aforementioned interaction in vivo, in silico and in planta. Following a bimolecular fluorescent complementation approach, we determined that the subcellular localization of the AtSAH7 and the AtSAH7/AtSBP1 interaction occur in the ER. Our results indicate the participation of AtSAH7 in a biochemical network regulated by selenite, possibly associated with responses to ROS production

TRH1 Encodes a Potassium Transporter Required for Tip Growth in Arabidopsis Root Hairs

Root hair initiation involves the formation of a bulge at the basal end of the trichoblast by localized diffuse growth. Tip growth occurs subsequently at this initiation site and is accompanied by the establishment of a polarized cytoplasmic organization. Arabidopsis plants homozygous for a complete loss-of-function tiny root hair 1 (trh1) mutation were generated by means of the T-DNA–tagging method. Trichoblasts of trh1 plants form initiation sites but fail to undergo tip growth. A predicted primary structure of TRH1 indicates that it belongs to the AtKT/AtKUP/HAK K(+) transporter family. The proposed function of TRH1 as a K(+) transporter was confirmed in (86)Rb uptake experiments, which demonstrated that trh1 plants are partially impaired in K(+) transport. In line with these results, TRH1 was able to complement the trk1 potassium transporter mutant of Saccharomyces, which is defective in high-affinity K(+) uptake. Surprisingly, the trh1 phenotype was not restored when mutant seedlings were grown at high external potassium concentrations. These data demonstrate that TRH1 mediates K(+) transport in Arabidopsis roots and is responsible for specific K(+) translocation, which is essential for root hair elongation

BRI1 and BAK1 Canonical Distribution in Plasma Membrane Is HSP90 Dependent

The activation of BRASSINOSTEROID INSENSITIVE1 (BRI1) and its association with the BRI1 ASSOCIATED RECEPTOR KINASE1 (BAK1) are key steps for the initiation of the BR signaling cascade mediating hypocotyl elongation. Heat shock protein 90 (HSP90) is crucial in the regulation of signaling processes and the activation of hormonal receptors. We report that HSP90 is required for the maintenance of the BRI1 receptor at the plasma membrane (PM) and its association with the BAK1 co-receptor during BL-ligand stimulation. HSP90 mediates BR perception and signal transduction through physical interactions with BRI1 and BAK1, while chaperone depletion resulted in lower levels of BRI1 and BAK1 receptors at the PM and affected the spatial partitioning and organization of BRI1/BAK1 heterocomplexes at the PM. The BRI1/BAK1 interaction relies on the HSP90-dependent activation of the kinase domain of BRI1 which leads to the confinement of the spatial dynamics of the membrane resident BRI1 and the attenuation of the downstream signaling. This is evident by the impaired activation and transcriptional activity of BRI1 EMS SUPPRESSOR 1 (BES1) upon HSP90 depletion. Our findings provide conclusive evidence that further expands the commitment of HSP90 in BR signaling through the HSP90-mediated activation of BRI1 in the control of the BR signaling cascade in plants