Final published version
Licence: CC BY: Creative Commons Attribution 4.0 International License
Research output: Contribution to Journal/Magazine › Journal article › peer-review
Research output: Contribution to Journal/Magazine › Journal article › peer-review
}
TY - JOUR
T1 - Reporter-based forward genetic screen to identify bundle sheath anatomy mutants in A. thaliana
AU - Döring, Florian
AU - Billakurthi, Kumari
AU - Gowik, Udo
AU - Sultmanis, Stefanie
AU - Khoshravesh, Roxana
AU - Das Gupta, Shipan
AU - Sage, Tammy L
AU - Westhoff, Peter
PY - 2019/3/31
Y1 - 2019/3/31
N2 - The evolution of C(4) photosynthesis proceeded stepwise with each small step increasing the fitness of the plant. An important pre-condition for the introduction of a functional C(4) cycle is the photosynthetic activation of the C(3) bundle sheath by increasing its volume and organelle number. Therefore, to engineer C(4) photosynthesis into existing C(3) 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 C(4) -like bundle sheath. To this end, an ethylmethanesulfonate (EMS)-based forward genetic screen was established in the Brassicaceae C(3 ) 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.
AB - The evolution of C(4) photosynthesis proceeded stepwise with each small step increasing the fitness of the plant. An important pre-condition for the introduction of a functional C(4) cycle is the photosynthetic activation of the C(3) bundle sheath by increasing its volume and organelle number. Therefore, to engineer C(4) photosynthesis into existing C(3) 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 C(4) -like bundle sheath. To this end, an ethylmethanesulfonate (EMS)-based forward genetic screen was established in the Brassicaceae C(3 ) 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.
KW - GFP
KW - LUC
KW - Arabidopsis thaliana
KW - C4 photosynthesis
KW - EMS mutagenesis
KW - bundle sheath cell
KW - technical advance
KW - Arabidopsis/anatomy & histology/genetics/physiology
KW - Chloroplasts/metabolism
KW - Chromosome Mapping
KW - Ethyl Methanesulfonate
KW - Genes, Reporter
KW - Genome, Plant/genetics
KW - Green Fluorescent Proteins
KW - Luciferases
KW - Mutagenesis
KW - Photosynthesis
KW - Plant Leaves/anatomy & histology/genetics/physiology
U2 - 10.1111/tpj.14165
DO - 10.1111/tpj.14165
M3 - Journal article
VL - 97
SP - 984
EP - 995
JO - The Plant Journal
JF - The Plant Journal
SN - 1365-313X
IS - 5
ER -