Reporter‐based forward genetic screen to identify bundle sheath anatomy mutants in A. thaliana

Abstract

SummaryThe evolution of C4 photosynthesis proceeded stepwise with each small step increasing the fitness of the plant. An important pre‐condition for the introduction of a functional C4 cycle is the photosynthetic activation of the C3 bundle sheath by increasing its volume and organelle number. Therefore, to engineer C4 photosynthesis into existing C3 crops, information about genes that control the bundle sheath cell size and organelle content is needed. However, very little information is known about the genes that could be manipulated to create a more C4–like bundle sheath. To this end, an ethylmethanesulfonate (EMS)‐based forward genetic screen was established in the Brassicaceae C3 species Arabidopsis thaliana. To ensure a high‐throughput primary screen, the bundle sheath cells of A. thaliana were labeled using a luciferase (LUC68) or by a chloroplast‐targeted green fluorescent protein (sGFP) reporter using a bundle sheath specific promoter. The signal strengths of the reporter genes were used as a proxy to search for mutants with altered bundle sheath anatomy. Here, we show that our genetic screen predominantly identified mutants that were primarily affected in the architecture of the vascular bundle, and led to an increase in bundle sheath volume. By using a mapping‐by‐sequencing approach the genomic segments that contained mutated candidate genes were identified.

Photosynthesis in a Changing Global Climate: Scaling Up and Scaling Down in Crops

“…2002; Luo et al, 2018;Döring et al, 2019). Knowledge obtained from mutant screenings can reveal new chloroplast functions, including those necessary for high photosynthetic performance, and accelerate the molecular characterization required for deciphering the genetic basis of plant photosynthesis for future improvements.…”

“…The functional dissection of photosynthesis can be undertaken also by forward genetic screens. Strategies, identification, insights and mutant effects have been reviewed previously ( Somerville, 1986 ; Parinov and Sundaresan, 2000 ; Page and Grossniklaus, 2002 ; Luo et al, 2018 ; Döring et al, 2019 ). Knowledge obtained from mutant screenings can reveal new chloroplast functions, including those necessary for high photosynthetic performance, and accelerate the molecular characterization required for deciphering the genetic basis of plant photosynthesis for future improvements.…”

“…Knowledge obtained from mutant screenings can reveal new chloroplast functions, including those necessary for high photosynthetic performance, and accelerate the molecular characterization required for deciphering the genetic basis of plant photosynthesis for future improvements. For instance, Döring et al (2019) identified genomic segments that contained mutated candidate genes to create a more C4–like bundle sheath by using a mapping-by-sequencing approach. However, a successful forward genetic screen needs an easily identifiable trait followed by a validation of the identified mutated genes by state-of-the-art technologies such as T-DNA knock-out lines, RNAi lines, or by gene-editing tools ( Hahn et al, 2017 ).…”

Enhancer‐mediated reporter gene expression in Arabidopsis thaliana: a forward genetic screen

“…We observed an embryonic‐lethality rate of approximately 40% in the M1 population, which was higher than the basal requirement (at least 25%), but lower than some of the reported EMS mutagenesis experiments (around 50%) (Jander et al ., 2003; Kim et al ., 2006). To achieve a 95% chance of mutation in any given G:C base pair, approximately 45 000 M1 plants would be required (Doring et al ., 2019; Jander et al ., 2003). We harvested seeds from approximately 90 000 M1 plants.…”

Setaria viridis chlorotic and seedling‐lethal mutants define critical functions for chloroplast gene expression

“…This is probably why we were able to recover a mutant allele of psrp7 , for example. An analogous EMS mutant population was recently used to screen for BS abnormalities in the C 3 plant Arabidopsis (Döring et al ., 2019). This much larger collection (45 000 M1 plants) led to a successful screen based on brute force imaging, suggesting that the Setaria approach also holds promise.…”