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Plantenwetenschap en meer
Hieronder een overzicht van mijn academische publicaties. Voor elk heb ik de samenvatting en een link naar het volledige artikel toegevoegd. Helaas zijn niet alle publicaties open access.
Membranes are essential for cells and organelles to function. As membranes are impermeable to most polar and charged molecules, they provide electrochemical energy to transport molecules across and create compartmentalized micro-environments for specific enzymatic and cellular processes. Membranes are also responsible for guided transport of cargoes between organelles and during endo- and exocytosis. In addition, membranes play key roles in cell signaling by hosting receptors and signal transducers, and as substrates and products of lipid second messengers. Anionic lipids and their specific interaction with target proteins play an essential role in these processes, which are facilitated by specific lipid binding domains (LBDs). Protein crystallography, lipid-binding studies, subcellular localization analyses, and computer modelling have greatly advanced our knowledge over the years of how these domains achieve precision binding and what their function is in signaling and membrane trafficking, as well as in plant development and stress acclimation.
Satish Kulasekaran, Sergio Cerezo-Medina, Claudia Harflett, Charlotte Lomax, Femke de Jong, Amelie Rendour, Gianluca Ruvo, Steven J Hanley, Michael H Beale, Jane L Ward (2021) A willow UDP-glycosyltransferase involved in salicinoid biosynthesis. Journal of Experimental Botany 72:1634–1648
The salicinoids are phenolic glycosides that are characteristic secondary metabolites of the Salicaceae, particularly willows and poplars. Despite the well-known pharmacology of salicin, that led to the development of aspirin over 100 years ago, the biosynthetic pathways leading to salicinoids have yet to be defined. Here, we describe the identification, cloning and biochemical characterisation of SpUGT71L2 and SpUGT71L3, – isozymic glycosyl transferases from Salix purpurea – that function in the glucosylation of ortho-substituted phenols. The best substrate in vitro was salicyl-7-benzoate. Its product, salicyl-7-benzoate glucoside, was shown to be endogenous in poplar and willow. Together they are inferred to be early intermediates in the biosynthesis of salicortin and related metabolites in planta. The role of this UGT was confirmed via the metabolomic analysis of transgenic plants produced by RNAi knockdown of the poplar orthologue (UGT71L1) in the hybrid clone Populus tremula x P. alba, INRA 717 1-B4.
de Jong F., Hanley S.J., Beale M.H., Karp A. (2015) Characterisation of the willow phenylalanine ammonia-lyase (PAL) gene family reveals expression differences compared with poplar. Phytochemistry 117:90-97
Willow is an important biomass crop for the bioenergy industry, and therefore optimal growth with minimal effects of biotic and abiotic stress is essential. The phenylpropanoid pathway is responsible for the biosynthesis of not only lignin but also of flavonoids, condensed tannins, benzenoids and phenolic glycosides which all have a role in protecting the plant against biotic and abiotic stress. All products of the phenylpropanoid pathway are important for the healthy growth of short rotation cropping species such as willow. However, the phenylpropanoid pathway in willow remains largely uncharacterised. In the current study we identified and characterised five willow phenylalanine ammonia-lyase (PAL) genes, which encode enzymes that catalyse the deamination of l-phenylalanine to form trans-cinnamic acid, the entry point into the phenylpropanoid pathway. Willow PAL1, PAL2, PAL3 and PAL4 genes were orthologous to the poplar genes. However no orthologue of PAL5 appears to be present in willow. Moreover, two tandemly repeated PAL2 orthologues were identified in a single contig. Willow PALs show similar sub-cellular localisation to the poplar genes. However, the enzyme kinetics and gene expression of the willow PAL genes differed slightly, with willow PAL2 being more widely expressed than its poplar orthologues implying a wider role for PALs in the production of flavonoids, condensed tannins, benzenoids, and phenolic glycosides, in willow.
de Jong F., Thodey K., Lejay L.V., Bevan M.W. (2014). Glucose Elevates NITRATE TRANSPORTER2.1 Protein Levels and Nitrate Transport Activity Independently of Its HEXOKINASE1-Mediated Stimulation of NITRATE TRANSPORTER2.1 Expression. Plant Physiology 164(1): 308-320.
Mineral nutrient uptake and assimilation is closely coordinated with the production of photosynthate to supply nutrients for growth. In Arabidopsis, nitrate uptake from the soil is mediated by genes encoding high- and low-affinity transporters that are transcriptionally regulated by both nitrate and photosynthate availability. In this study we have studied the interactions of nitrate and glucose on gene expression, nitrate transport and growth using gin2-1, which is defective in sugar responses. We confirm and extend previous work (Lejay et al., 2003; Lejay et al., 2008) by showing that HXK1-mediated oxidative pentose phosphate pathway (OPPP) metabolism is required for glucose-mediated NRT2.1 expression. Treatment with pyruvate and shikimate, two products derived from intermediates of the oxidative pentose phosphate pathway OPPP destined for amino acid production, restores wild-type levels of NRT2.1 expression, suggesting that metabolites derived from OPPP metabolism can, together with glucose, directly stimulate high levels of NRT2.1 expression. Nitrate-mediated NRT2.1 expression is not influenced by gin2-1, showing that glucose does not influence NRT2.1 expression through nitrate-mediated mechanisms. We also show that glucose stimulates NRT2.1 protein levels and transport activity independently of its HXK1-mediated stimulation of NRT2.1 expression, demonstrating another possible post-transcriptional mechanism influencing nitrate uptake. In gin2-1 plants nitrate responsive biomass growth was strongly reduced, showing that the supply of OPPP metabolites is essential for assimilating nitrate for growth.
Correct interpretation of the coding capacity of RNA polymerase II transcribed eukaryotic genes is determined by the recognition and removal of intronic sequences of pre-mRNAs by the spliceosome. Our current knowledge on dynamic assembly and subunit interactions of the spliceosome mostly derived from the characterization of yeast, Drosophila, and human spliceosomal complexes formed on model pre-mRNA templates in cell extracts. In addition to sequential structural rearrangements catalyzed by ATP-dependent DExH/D-box RNA helicases, catalytic activation of the spliceosome is critically dependent on its association with the NineTeen Complex (NTC) named after its core E3 ubiquitin ligase subunit PRP19. NTC, isolated recently from Arabidopsis, occurs in a complex with the essential RNA helicase and GTPase subunits of the U5 small nuclear RNA particle that are required for both transesterification reactions of splicing. A compilation of mass spectrometry data available on the composition of NTC and spliceosome complexes purified from different organisms indicates that about half of their conserved homologs are encoded by duplicated genes in Arabidopsis. Thus, while mutations of single genes encoding essential spliceosome and NTC components lead to cell death in other organisms, differential regulation of some of their functionally redundant Arabidopsis homologs permits the isolation of partial loss of function mutations. Non-lethal pleiotropic defects of these mutations provide a unique means for studying the roles of NTC in co-transcriptional assembly of the spliceosome and its crosstalk with DNA repair and cell death signalling pathways.
A proteome study of the first five days of Medicago truncatula protoplast cultures was done to investigate molecular changes taking place during protoplast proliferation. A total of 1556 protein spots were analysed, of which 886 protein spots showed significant (p<0.005) changes in abundance at some time during the first five days of protoplast culture. Of the 886 significantly changing protein spots, 89 proteins were identified by MALDI-TOF MS. The majority of the identified proteins were part of four main cellular processes that may be involved in protoplast proliferation: energy metabolism, defence or stress response, secondary metabolism and protein synthesis and folding. The accumulation pattern of these proteins indicates extensive changes in the energy metabolism of the cells, accompanied by the activation of stress response pathways and modifications of the cell wall. In addition, seven PR10-like (pathogenesis related) proteins were identified. The accumulation pattern of these seven PR10-like proteins suggests that they could have a developmental role during protoplast proliferation.
Imin, N., de Jong, F., Mathesius, U., Noorden, G.v., Saeed, N.A., Wang, X.-D., Rose, R.J., and Rolfe, B.G. (2004). Proteome reference maps of Medicago truncatula embryogenic cell cultures generated from single protoplasts. Proteomics 4, 1883-1896.
Using a combination of two-dimensional gel electrophoresis (2-DE) protein mapping and mass spectrometry (MS) analysis, we have established proteome reference maps of Medicago truncatula embryogenic tissue culture cells. The cultures were generated from single protoplasts, which provided a relatively homogeneous cell population. We used these to analyze protein expression at the globular stages of somatic embryogenesis, which is the earliest morphogenetic embryonic stage. Over 3000 proteins could reproducibly be resolved over a pI range of 4-11. Three hundred and twelve protein spots were extracted from colloidal Coomassie Blue-stained 2-DE gels and analyzed by matrix-assisted laser desorption/ionization-time of flight MS analysis and tandem MS sequencing. This enabled the identification of 169 protein spots representing 128 unique gene products using a publicly available expressed sequence tag database and the MASCOT search engine. These reference maps will be valuable for the investigation of the molecular events which occur during somatic embryogenesis in M. truncatula. The proteome reference maps and supplementary materials will be available and updated for public access at http://semele.anu.edu.au/.