A K homology (KH) domain protein identified by a forward genetic screen affects bundle sheath anatomy in Arabidopsis thaliana

Because of their photosynthetic capacity, leaves function as solar panels providing the basis for the growth of the entire plant. Although the molecular mechanisms of leaf development have been well studied in model dicot and monocot species, a lot of information is still needed about the interplay of the genes that regulate cell division and differentiation and thereby affect the photosynthetic performance of the leaf. We were specifically interested in understanding the differentiation of mesophyll and bundle sheath cells in Arabidopsis thaliana and aimed to identify genes that are involved in determining bundle sheath anatomy. To this end, we established a forward genetic screen by using ethyl methanesulfonate (EMS) for mutagenizing a reporter line expressing a chloroplast‐targeted green fluorescent protein (sGFP) under the control of a bundle sheath‐specific promoter. Based on the GFP fluorescence phenotype, numerous mutants were produced, and by pursuing a mapping‐by‐sequencing approach, the genomic segments containing mutated candidate genes were identified. One of the lines with an enhanced GFP fluorescence phenotype (named ELEVATED BUNDLE SHEATH CELLS SIGNAL 1 [ebss1]) was selected for further study, and the responsible gene was verified by CRISPR/Cas9‐based mutagenesis of candidate genes located in the mapped genomic segment. The verified gene, At2g25970, encodes a K homology (KH) domain‐containing protein.