Clonal analysis of gene function during Arabidopsis root development

Abstract

Mutant genotypes are essential to uncover the role of any gene of interest (GOI), however, the absence of a wt (normal) allele in the entire organism might be obstructive for taking such approaches. If a GOI is essential in early developmental stages (e.g., embryogenesis), homozygous knockout individuals do not reach adulthood and mutant alleles cannot be studied in later stages. Together with advances in molecular biological techniques that are designed to address more specific and fine-tuned questions, cell/tissue specific knockout (or knockdown) methods are continuously developed. Several methods make use of two-component systems such as the Cre/lox mechanism to excise a genomic sequence, which can subsequently lead to elimination and/or activation of a GOI. One of our focuses was the cell specific role of the RETINOBLASTOMA-RELATED (RBR) gene in the Arabidopsis root stem cell (SC) niche. In animals, the homologous gene, RETINOBLASTOMA (Rb), was the first identified tumor suppressor, leading to 2.5 decades of research that disclosed Rb as one of the chief regulators of cell cycle, cell differentiation and cell death. Previous studies in plants suggested that RBR functions in a variety of developmental processes, amongst which, the maintenance of shoot and root meristem. We sought to create cell specific RBR loss of function using the Cre/lox system to study its distinct (and/or overlapping) activity in several types of root meristem cells. This kind of analysis, also known as clonal deletion, relies on loss of heterozygosity, where null and wt sectors are compared to study the local effect of a knockout genotype. However, these methods are not applicable for RBR since the protein is essential in the parental gametophytes. Therefore, hemizygous genotypes are rare and practically not available for research. To circumvent this obstacle, we design a new recombination system, Brother Of Brainbow (BOB) and also made use of tissue specific, inducible Cre recombinase to create and visually identify mutant clones. We found that RBR is required cell-autonomously in the QC and columella SCs to suppress unscheduled proliferation. Using the BOB system as well as artificial microRNA based silencing method (amiGO), we showed that the QC serves as a reservoir for columella SCs and that RBR interaction with SCR is necessary to keep its quiescent state as well as to protect it from genotoxic stress. Further examination of hypomorphic RBR genotypes revealed that reduced RBR levels lead to cell death. This phenotype partially requires the ATM/ATR machinery, whereas several mechanisms that induce differentiation including RBR over-expression partially reduced the cell death. Last, we investigated transcriptional regulation by the root specific transcription factor, PLETHORA2. We identified two targets, HAN and ARF10 that could either mimic PLT2 over-expression phenotype or complement plt1;plt2 mutant, respectively. Over-expression of either PLT2 or HAN was especially intriguing since it was sufficient to revert differentiating cells back to proliferation state. These findings together with the BOB and amiGO tools can set the basis for further studies on the mechanisms that underlie proliferation and differentiation in the root SC niche and the interaction between the two pathways