High-throughput chlorophyll fluorescence screening of Setaria viridis for mutants with altered CO2 compensation points

To assist with efforts to engineer a C-4 photosynthetic pathway into rice, forward-genetic approaches are being used to identify the genes modulating key C-4 traits. Currently, a major challenge is how to screen for a variety of different traits in a high-throughput manner. Here we describe a method for identifying C-4 mutant plants with increased CO2 compensation points. This is used as a signature for decreased photosynthetic efficiency associated with a loss of C-4 function. By exposing plants to a CO2 concentration close to the CO2 compensation point of a wild-type plant, individuals can be identified from measurements of chlorophyll a fluorescence. We use this method to screen a mutant population of the C-4 monocot Setaria viridis (L.) P.Beauv. generated using N-nitroso-N-methylurea (NMU). Mutants were identified at a frequency of 1 per 157 lines screened. Forty-six candidate lines were identified and one line with a heritable homozygous phenotype selected for further characterisation. The CO2 compensation point of this mutant was increased to a value similar to that of C-3 rice. Photosynthesis and growth was significantly reduced under ambient conditions. These data indicate that the screen was capable of identifying mutants with decreased photosynthetic efficiency. Characterisation and next-generation sequencing of all the mutants identified in this screen may lead to the discovery of novel genes underpinning C-4 photosynthesis. These can be used to engineer a C-4 photosynthetic pathway into rice.