Abstract
BackgroundHigh light tolerance of microalgae is a desired phenotype for efficient cultivation in large scale production systems under fluctuating outdoor conditions. Outdoor cultivation requires the use of either wild-type or non-GMO derived mutant strains due to safety concerns. The identification and molecular characterization of such mutants derived from untagged forward genetics approaches was limited previously by the tedious and time-consuming methods involving techniques such as classical meiotic mapping. The combination of mapping with next generation sequencing technologies offers alternative strategies to identify genes involved in high light adaptation in untagged mutants.ResultsWe used the model alga Chlamydomonas reinhardtii in a non-GMO mutation strategy without any preceding crossing step or pooled progeny to identify genes involved in the regulatory processes of high light adaptation. To generate high light tolerant mutants, wildtype cells were mutagenized only to a low extent, followed by a stringent selection. We performed whole-genome sequencing of two independent mutants hit1 and hit2 and the parental wildtype. The availability of a reference genome sequence and the removal of shared bakground variants between the wildtype strain and each mutant, enabled us to identify two single nucleotide polymorphisms within the same gene Cre02.g085050, hereafter called LRS1 (putative Light Response Signaling protein 1). These two independent single amino acid exchanges are both located in the putative WD40 propeller domain of the corresponding protein LRS1. Both mutants exhibited an increased rate of non-photochemical-quenching (NPQ) and an improved resistance against chemically induced reactive oxygen species. In silico analyses revealed homology of LRS1 to the photoregulatory protein COP1 in plants.ConclusionsIn this work we identified the nuclear encoded gene LRS1 as an essential factor for high light adaptation in C. reinhardtii. The causative random mutation within this gene was identified by a rapid and efficient method, avoiding any preceding crossing step, meiotic mapping, or pooled progeny. Our results open up new insights into mechanisms of high light adaptation in microalgae and at the same time provide a simplified strategy for non-GMO forward genetics, a crucial precondition that could result in the identification of key factors for economically relevant biological processes within algae.
Highlights
High light tolerance of microalgae is a desired phenotype for efficient cultivation in large scale production systems under fluctuating outdoor conditions
Generation and phenotypical characterization of high light tolerant C. reinhardtii mutants Tolerance to fluctuating light conditions, in particular to high light stress, is a desired phenotype for microalgae that are intended to be used for outdoor cultivation in biotechnological approaches [8]
In order to obtain non-genetically modified organisms (GMO) high light tolerant Chlamydomonas strains, we applied a selection method under light intensities of 1500–2000 μmol m-2 s-1 that are known to be lethal to WT cells
Summary
High light tolerance of microalgae is a desired phenotype for efficient cultivation in large scale production systems under fluctuating outdoor conditions. Over recent years several efforts have been reported to identify and construct algae strains with improved light conversion efficiency rates (PCE rates) [9,10,11,12,13,14,15,16] as well as more robust variants surviving elevated and fluctuating light conditions, which is crucial for establishing efficient outdoor cultivation [8,17,18,19] Such fluctuating or high light regimes cause an imbalance in the absorbtion and utilization of light energy that can lead to photooxydative damage due to the production of reactive oxygen species (ROS) resulting in severe cell damage or even cell death [18,20,21,22,23] The rather complex regulation of light adaptation mechanisms is not yet fully understood and key factors still need to be identified
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