And Only Four Remain: Functionally Characterizing the Peptidoglycan-Related Genes in Flowering Plants

An ancient endosymbiosis involving early eukaryotic cells and cyanobacteriaresulted in the modern chloroplast organelle of the Archaeplastida clade, including all land plants. Throughout chloroplast evolution, many genes from the chloroplast genome were transferred to the nuclear genome, including the genetic pathway for synthesis of the bacterial polymer peptidoglycan (PG). In the ~1 billion years since the acquisition of the chloroplast, seed plants can now be grouped into species that retain ten PG genes (“full-PG”) or only four of the ten (“4-PG”). In Arabidopsis thaliana, a 4-PG flowering plant, knockout mutants for the PG gene murE show a proplastid phenotype, where chloroplast development is completely halted. In this report, A. thaliana mutants for the remaining three PG genes (mraY, murG, and ddl) were phenotyped to test for their functions. Two T-DNA insertion lines for each gene were selected from the Arabidopsis Biological Resource Center, and either confirmed as homozygotes or selfed to produce homozygous lines. These lines were then grown for at least two generations to confirm homozygous status and the location of the T-DNA insertion was identified using Sanger sequencing. All mutant lines had an insertion in either an exon or 5’ UTR of target genes. All lines were then grown on soil in conjunction with wild type plants for comparison of germination, morphology, and transcriptomics. No differences were observed in time to germination or morphology compared to the wild type control. Transcriptomics showed that target PG genes were differentially expressed in each line, but usually at a non-significant value. Genes related to flowering and reproduction were significantly differentially expressed in the mraY lines. Protoplasts were produced to image chloroplasts and identify any morphological or developmental effects. Chloroplast morphology was comparable to wild type; additionally, measurements of photosynthetic efficiency were not found to be significantly different than wild type. Together these data show that A. thaliana mutants for three PG genes do not display a phenotype like those for mutants of the fourth gene, murE. The results suggest that PG genes in A. thaliana are no longer linked to PG production and have diverged from the ancestral bacterial pathway.