Molecular and morphological analysis by transmission electron microscopy (TEM) and inducible RNAi of two genes that encode mitochondrial recombination proteins found in Arabidopsis

The completion of the genomic sequence of Arabidopsis thaliana has accelerated analysis of gene expression for many basic cellular processes, including DNA recombination. Our focus is to examine both the global and mitochondrial morphological effect, as well as the mitochondrial DNA repair efficiency of Arabidopsis plants that contain mutations in two genes (single stranded DNA binding protein (ssb), At4g11060 , and RecA, At3g10140 ) that encode mitochondrial targeted DNA recombination proteins. DNA mutants were created in these two genes by transfer DNA (T‐DNA) inserts and single base mutations created by site directed mutagenesis (TILLING plants). We have also created an RNAi construct that can be induced at different plant developmental stages. Under standard and DNA damaging growth conditions we have observed hindered to lethal plant development when T‐DNA is inserted into the mitochondrial targeted ssb gene. Depending on the location of the mutation, TILLING RecA plants appear to have similar results to T‐DNA ssb plants. We have used RNAi and TILLING lines with RecA because all RecA plants failed to germinate that contain a T‐DNA insertion near the 5′ end. TEM analysis has been conducted on all RecA and ssb gene silenced and mutant plants grown under standard and DNA damaging chemical conditions. Real time PCR has been conducted to quantify the abundance of mitochondrial DNA under the same conditions as listed above. The results from these studies indicate that these two proteins are needed for plant development and strongly suggest they are essential for DNA recombination and repair in mitochondria. This work was funded in part by a grant from the NIH.