Periodic Reporting for period 1 - HiPhy (High Phytosterol variants towards improved feedstocks and biofortification of crops)
Período documentado: 2020-09-01 hasta 2022-08-31
Phytosterols are valuable biomolecules used as nutrients and as feedstock material for the pharmaceutical and cosmetic industries. The innovative and fundamental challenge biologists and breeders are faced with is to develop cell technology engineering programs and valuable crops dedicated to the large scale production of phytosterols, which are currently extracted from agricultural waste materials. The goal of the proposal is to identify master regulators of phytosterol biosynthesis and accumulation in genetic models. In fact, phytosterols have essential cellular functions in plant growth and development, acting as key membrane components and membrane reinforcers, as signals in development, and as precursors of brassinosteroids, a class of growth regulators. As such, the concentration of phytosterols and the equilibrium between free sterols and conjugated sterols is highly regulated in order to comply with cellular homeostasis. The goal of the HiPhy project was to identify key players of phytosterol biosynthesis and accumulation in the genetic models thale cress (Arabidopsis thaliana) and tobacco (Nicotiana tabacum).
New forward genetic approaches were designed to screen for high phytosterol phenotypes and to identify novel alleles of genes governing the production of these biomolecules. In a first workpackage, the characterization of a tobacco (Nicotiana tabacum) variant called NtOIL was carried out using genetic and transcriptomic approaches. Molecular markers based on available tobacco genome references and in-house tobacco genome sequencing data were designed and mapped on both the genetic and physical tobacco genome maps. A recombinant population of NtOIL was phenotyped for a further coarse and fine mapping, which is in progress at the host laboratory. Additionally, a transcriptomic analysis of the tobacco variant has revealed specific gene clusters upregulated in the NtOIL mutant, which allowed the identification of genes of interest carrying gained-stop mutations for a further targeted analysis. In a second workpackage, A. thaliana lines altered in the regulation of the lipid isoprenoid pathway including phytosterols were analyzed by next-generation mapping based on intensive use of NGS and bioinformatics, to perform a bulk segregant analysis of a set of unique Arabidopsis lines. The result of this project is a list of candidate mutations, which forms a sound data set for a further targeted analysis of the phytosterol pathway at the host laboratory.
The output of the action is a valuable series of novel plant lines and genetic resource that form an unprecedented data set in the field of plant metabolic biology. Novel targets for pre-breeding and breeding programs and the innovative metabolic engineering of the phytosterol pathway in plants and also other heterologous organisms like yeast or microalgae is considered further.