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

Potential Dissociative Glucocorticoid Receptor Activity for Protopanaxadiol and Protopanaxatriol

Glucocorticoids are steroid hormones that regulate inflammation, growth, metabolism, and apoptosis via their cognate receptor, the glucocorticoid receptor (GR). GR, acting mainly as a transcription factor, activates or represses the expression of a large number of target genes, among them, many genes of anti-inflammatory and pro-inflammatory molecules, respectively. Transrepression activity of glucocorticoids also accounts for their anti-inflammatory activity, rendering them the most widely prescribed drug in medicine. However, chronic and high-dose use of glucocorticoids is accompanied with many undesirable side effects, attributed predominantly to GR transactivation activity. Thus, there is a high need for selective GR agonist, capable of dissociating transrepression from transactivation activity. Protopanaxadiol and protopanaxatriol are triterpenoids that share structural and functional similarities with glucocorticoids. The molecular mechanism of their actions is unclear. In this study applying induced-fit docking analysis, luciferase assay, immunofluorescence, and Western blot analysis, we showed that protopanaxadiol and more effectively protopanaxatriol are capable of binding to GR to activate its nuclear translocation, and to suppress the nuclear factor-kappa beta activity in GR-positive HeLa and HEK293 cells, but not in GR-low level COS-7 cells. Interestingly, no transactivation activity was observed, whereas suppression of the dexamethasone-induced transactivation of GR and induction of apoptosis in HeLa and HepG2 cells were observed. Thus, our results indicate that protopanaxadiol and protopanaxatriol could be considered as potent and selective GR agonist

Microcompartmentation of aldolase in Arabidopsis

Understanding the internal organization of cells from the molecular up to organelle level is a current challenge for biology if we are to better comprehend the mechanisms by which cellular processes occur. A prevailing view of the cell interior is that biochemical reactions and molecular movements are dominated by random diffusion. However, in addition to being compartmented into organelles, cells may well be organized at a finer level. Proteins may localise to specific areas within sub-cellular compartments, by associating with each other, the cytoskeleton and organelle membranes. Thus giving rise to distinct microcompartments. Considerable in vitro evidence exists for such interactions between enzymes and larger cellular components. This strengthens the idea that cells may be microcompartmented. However, little in vivo evidence supports this hypothesis, especially in plants. As a test-case for the concept of microcompartmentation this project investigated the sub-cellular distribution of the glycolytic enzyme fructose-bisphosphate aldolase in Arabidopsis thaliana; the ultimate aim being to establish whether it is microcompartmented in vivo and, further, to test the potential function(s) of such microcompartmentation.</p

Microcompartmentation of aldolase in Arabidopsis

Understanding the internal organization of cells from the molecular up to organelle level is a current challenge for biology if we are to better comprehend the mechanisms by which cellular processes occur. A prevailing view of the cell interior is that biochemical reactions and molecular movements are dominated by random diffusion. However, in addition to being compartmented into organelles, cells may well be organized at a finer level. Proteins may localise to specific areas within sub-cellular compartments, by associating with each other, the cytoskeleton and organelle membranes. Thus giving rise to distinct microcompartments. Considerable in vitro evidence exists for such interactions between enzymes and larger cellular components. This strengthens the idea that cells may be microcompartmented. However, little in vivo evidence supports this hypothesis, especially in plants. As a test-case for the concept of microcompartmentation this project investigated the sub-cellular distribution of the glycolytic enzyme fructose-bisphosphate aldolase in Arabidopsis thaliana; the ultimate aim being to establish whether it is microcompartmented in vivo and, further, to test the potential function(s) of such microcompartmentation.This thesis is not currently available on ORA

Microcompartmentation of cytosolic aldolase by interaction with the actin cytoskeleton in Arabidopsis

Evidence is accumulating for molecular microcompartments formed when proteins interact in localized domains with the cytoskeleton, organelle surfaces, and intracellular membranes. To understand the potential functional significance of protein microcompartmentation in plants, we studied the interaction of the glycolytic enzyme fructose bisphosphate aldolase with actin in Arabidopsis thaliana. Homology modelling of a major cytosolic isozyme of aldolase, FBA8, suggested that the tetrameric holoenzyme has two actin binding sites and could therefore act as an actin-bundling protein, as was reported for animal aldolases. This was confirmed by in vitro measurements of an increase in viscosity of F-actin polymerized in the presence of recombinant FBA8. Simultaneously, interaction with F-actin caused non-competitive inhibition of aldolase activity. We did not detect co-localization of an FBA8–RFP fusion protein, expressed in an fba8-knockout background, with the actin cytoskeleton using confocal laser-scanning microscopy. However, we did find evidence for a low level of interaction using FRET-FLIM analysis of FBA8–RFP co-expressed with the actin-binding protein GFP–Lifeact. Furthermore, knockout of FBA8 caused minor alterations of guard cell actin cytoskeleton morphology and resulted in a reduced rate of stomatal closure in response to decreased humidity. We conclude that cytosolic aldolase can be microcompartmented in vivo by interaction with the actin cytoskeleton and may subtly modulate guard cell behaviour as a result

A metabolic gene cluster in Lotus japonicus discloses novel enzyme functions and products in triterpene biosynthesis

Genes for triterpene biosynthetic pathways exist as metabolic gene clusters in oat and Arabidopsis thaliana plants. We characterized the presence of an analogous gene cluster in the model legume Lotus japonicus. In the genomic regions flanking the oxidosqualene cyclase AMY2 gene, genes for two different classes of cytochrome P450 and a gene predicted to encode a reductase were identified. Functional characterization of the cluster genes was pursued by heterologous expression in Nicotiana benthamiana. The gene expression pattern was studied under different developmental and environmental conditions. The physiological role of the gene cluster in nodulation and plant development was studied in knockdown experiments. A novel triterpene structure, dihydrolupeol, was produced by AMY2. A new plant cytochrome P450, CYP71D353, which catalyses the formation of 20-hydroxybetulinic acid in a sequential three-step oxidation of 20-hydroxylupeol was characterized. The genes within the cluster are highly co-expressed during root and nodule development, in hormone-treated plants and under various environmental stresses. A transcriptional gene silencing mechanism that appears to be involved in the regulation of the cluster genes was also revealed. A tightly co-regulated cluster of functionally related genes is involved in legume triterpene biosynthesis, with a possible role in plant development