Identification and characterization of upstream regulators of Arabidopsis Metacaspase 9

Programmed cell death (PCD) refers to a genetically controlled process causing the death of certain cells or tissues. In plants PCD is critical in normal development of for instance xylem vessels. A group of proteins called metacaspases are believed to play a pivotal role in PCD in plants. As Metacaspase 9 have been shown to be upregulated in Populus during xylem maturation this study attempted to identify genes affecting its expression in Arabidopsis thaliana by forward genetics using a reporter line with GFP fused to the promoter of Metacaspase 9 (AtMC9). Ethyl methanesulfonate seed mutagenesis was used to generate mutants resulting in eleven mutant lineages with a GFP expression pattern deviating from that of the reporter line. These mutants fell into two categories; low/no-signal mutants and ectopic expressors. Several of the low/no-signal mutants had longer roots at five to eight days after germination, a time point shown to be critical for metaxylem differentiation. Further studies of their roots would reveal whether the developing xylem is abnormal or not. Deep sequencing provided evidence for involvement of abscisic acid and polyamines in regulation of AtMC9 expression. Sequencing from a low/no-signal mutant suggests that AtMC9 expression might be affected also by disturbed lignin biosynthesis. Rescuing mutant lineages through transformations with fully functional forms of the candidate genes is the next step to experimentally validate that the candidate genes are involved in the observed changes in AtMC9 expression in each of the isolated mutants