Publications - Cell and Metabolic Biology

Genetic resources for the model plant Arabidopsis comprise mutant lines defective in almost any single gene in reference accession Columbia. However, gene redundancy and/or close linkage often render it extremely laborious or even impossible to isolate a desired line lacking a specific function or set of genes from segregating populations. Therefore, we here evaluated strategies and efficiencies for the inactivation of multiple genes by Cas9-based nucleases and multiplexing. In first attempts, we succeeded in isolating a mutant line carrying a 70 kb deletion, which occurred at a frequency of ~ 1.6% in the T2 generation, through PCR-based screening of numerous individuals. However, we failed to isolate a line lacking Lhcb1 genes, which are present in five copies organized at two loci in the Arabidopsis genome. To improve efficiency of our Cas9-based nuclease system, regulatory sequences controlling Cas9 expression levels and timing were systematically compared. Indeed, use of DD45 and RPS5a promoters improved efficiency of our genome editing system by approximately 25–30-fold in comparison to the previous ubiquitin promoter. Using an optimized genome editing system with RPS5a promoter-driven Cas9, putatively quintuple mutant lines lacking detectable amounts of Lhcb1 protein represented approximately 30% of T1 transformants. These results show how improved genome editing systems facilitate the isolation of complex mutant alleles, previously considered impossible to generate, at high frequency even in a single (T1) generation.

BackgroundCharacterization of plant terpene synthases is typically done by production of recombinant enzymes in Escherichia coli. This is often difficult due to solubility and codon usage issues. Furthermore, plant terpene synthases which are targeted to the plastids, such as diterpene synthases, have to be shortened in a more or less empirical approach to improve expression. We report here an optimized Agrobacterium-mediated transient expression assay in Nicotiana benthamiana for plant diterpene synthase expression and product analysis.ResultsAgrobacterium-mediated transient expression of plant diterpene synthases in N. benthamiana led to the accumulation of diterpenes within 3 days of infiltration and with a maximum at 5 days. Over 50% of the products were exported onto the leaf surface, thus considerably facilitating the analysis by reducing the complexity of the extracts. The robustness of the method was tested by expressing three different plant enzymes, cembratrien-ol synthase from Nicotiana sylvestris, casbene synthase from Ricinus communis and levopimaradiene synthase from Gingko biloba. Furthermore, co-expression of a 1-deoxy-D-xylulose-5-phosphate synthase from tomato and a geranylgeranyl diphosphate synthase from tobacco led to a 3.5-fold increase in the amount of cembratrien-ol produced, with maximum yields reaching 2500 ng/cm2.ConclusionWith this optimized method for diterpene synthase expression and product analysis, a single infiltrated leaf of N. benthamiana would be sufficient to produce quantities required for the structure elucidation of unknown diterpenes. The method will also be of general use for gene function discovery, pathway reconstitution and metabolic engineering of diterpenoid biosynthesis in plants.

BackgroundPhytohormones are the key metabolites participating in the regulation of multiple functions of plant organism. Among them, jasmonates, as well as abscisic and salicylic acids are responsible for triggering and modulating plant reactions targeted against pathogens and herbivores, as well as resistance to abiotic stress (drought, UV-irradiation and mechanical wounding). These factors induce dramatic changes in phytohormone biosynthesis and transport leading to rapid local and systemic stress responses. Understanding of underlying mechanisms is of principle interest for scientists working in various areas of plant biology. However, highly sensitive, precise and high-throughput methods for quantification of these phytohormones in small samples of plant tissues are still missing.ResultsHere we present an LC-MS/MS method for fast and highly sensitive determination of jasmonates, abscisic and salicylic acids. A single-step sample preparation procedure based on mixed-mode solid phase extraction was efficiently combined with essential improvements in mobile phase composition yielding higher efficiency of chromatographic separation and MS-sensitivity. This strategy resulted in dramatic increase in overall sensitivity, allowing successful determination of phytohormones in small (less than 50 mg of fresh weight) tissue samples. The method was completely validated in terms of analyte recovery, sensitivity, linearity and precision. Additionally, it was cross-validated with a well-established GC-MS-based procedure and its applicability to a variety of plant species and organs was verified.ConclusionThe method can be applied for the analyses of target phytohormones in small tissue samples obtained from any plant species and/or plant part relying on any commercially available (even less sensitive) tandem mass spectrometry instrumentation.