Novel Mutations That Affect Stomata Development in Arabidopsis thaliana

Located on the epidermal surface of plants, stomata are small, pore-like structures that act as channels to exchange gas and water vapor between plant cells and the environment. Concentrations of gases and water within the plant cell are regulated through opening and closing of the stomata by turgor-driven movements. In Arabidopsis thaliana, development and differentiation of cells is controlled by the ERECTA (ER) family of genes (ERECTA, ERL1, and ERL2) which encode leucine-rich repeat-receptor-like kinases (LRR-RLKs). Acting synergistically, they direct cell division in different tissues and formation of stomata in epidermis. To better understand how ERECTA family genes regulate stomata development we conducted a forward genetic screen. Approximately 10,000 seeds of erl1erl2 were mutagenized using ethyl methanesulfonate (EMS). The M1 plants were grown and the M2 seeds were collected. Then, M2 seedlings were microscopically screened for stomata clustering. Two mutants, JMC19 and MC12 were chosen to pursue further because a high percentage of stomata in clusters was observed in their cotyledons. Both mutant lines were crossed with erl1erl2 in order to rid their genomes of other EMS induced mutations and to determine the nature of obtained mutations (recessive versus dominant; single or double). The phenotype of novel mutants (stomata index and stomata clustering) was compared to the erl1erl2. The two lines were also crossed with Col to see if the phenotype of novel mutations depended on erl1and/or erl2 mutations. After characterization of mutations, determining the location of the mutated JMC19 and MC12 genes through positional cloning is the next step. JMC19 and MC12 were crossed with Landsberg erecta (Ler) to analyze recombination frequency between mutant phenotype and a set of genetic markers. The frequency at which the mutant gene(s) recombined with markers on Ler chromosomes determined the location of the MC12. This method will also be used for JMC19 in the future. The overall goal of the study is to understand, through the use of forward genetics, the mechanism by which stomata are spaced and to identify the gene(s) that control this developmental process.