Final Report Summary - STEMRB (Role of siRNA-mediated DNA methylation in the root stem cell niche)
The main objective of my proposal was to identify the role of Retinoblastoma-related (RBR) and its interactors in the root stem cell niche. When I first applied to this fellowship, in summer 2008, I thought the best interactor-candidates were proteins in the small-RNA pathway and I had already started a preliminary characterisation of the phenotypes involved.
By the end of 2010, we had uncovered a new and potentially more interesting interactor of RBR to affect the root stem cell niche. This interactor is scarecrow (SCR), that is involved in the stem niche specification, together with its binding partner, Shortroot. So, we decided to refocus my project in this direction, as a part of a team of both researchers and computer modelers.
As a result, we have discovered that RBR inhibits SCR activity in the mature endodermal cells, thus preventing extra assymetric cell divisions. A genetic network, in which SCR activates the transcription of an inhibitor of RBR, CycD6, assures that the circuit has a bistable behaviour. Hormonal masimum in the meristem enhances CycD6 expression, thus biasing the system to the ON state in the stem cell area. We have also observed that protein degradation during cell cycle allows for the ON state to be turned OFF again after the asymmetic cell division. Thus, we have unveiled a new network motif, that we call flip-flop bistable switch. This is a very robust system to specify one asymmetric cell division within the root meristem, allowing a very tight control of where the asymmetric cell division will occur. This work has been accepted in the prestigious journal 'Cell'.
As part of this work, I also have more specific data on RBR role in the root stem cell niche. Silencing of RBR in cell type specific manner (artificial microRNA for gene-silencing overcome (amiGO) RBR) shows that RBR cell autonomously regulates asymmetric cell division in the quiescent center (QC) (root organising centre), and two kinds of stem cells (cortex endodermis initial (CEI) and columella stem cell (CSC)). In the QC and CEI, the action of RBR is dependent on the LxCxE motif, as we demonstrated by mutating RBR in aminoacid N849. When introgressed into the amiGO RBR background, N849F mutant was not able to complement the phenotype, pointing to a role of LxCxE containing proteins in the asymmetric cell division of the QC and CSC. SCR protein does contain an LxCxE motif, and we were able to demonstrate that RBR action in the QC is SCR dependent. The fact that QC is dividing faster in the amiGO RBR background does not affect root growth or architecture, but it does impair the response to genotoxic damage. This suggest that RBR-SCR control of QC division is mantaining the organising center as a quiescent stem cell, that will only divide upon genotoxic damage to re-establish the stem cell. This work is ready for submission into Plant Cell.
By the end of 2010, we had uncovered a new and potentially more interesting interactor of RBR to affect the root stem cell niche. This interactor is scarecrow (SCR), that is involved in the stem niche specification, together with its binding partner, Shortroot. So, we decided to refocus my project in this direction, as a part of a team of both researchers and computer modelers.
As a result, we have discovered that RBR inhibits SCR activity in the mature endodermal cells, thus preventing extra assymetric cell divisions. A genetic network, in which SCR activates the transcription of an inhibitor of RBR, CycD6, assures that the circuit has a bistable behaviour. Hormonal masimum in the meristem enhances CycD6 expression, thus biasing the system to the ON state in the stem cell area. We have also observed that protein degradation during cell cycle allows for the ON state to be turned OFF again after the asymmetic cell division. Thus, we have unveiled a new network motif, that we call flip-flop bistable switch. This is a very robust system to specify one asymmetric cell division within the root meristem, allowing a very tight control of where the asymmetric cell division will occur. This work has been accepted in the prestigious journal 'Cell'.
As part of this work, I also have more specific data on RBR role in the root stem cell niche. Silencing of RBR in cell type specific manner (artificial microRNA for gene-silencing overcome (amiGO) RBR) shows that RBR cell autonomously regulates asymmetric cell division in the quiescent center (QC) (root organising centre), and two kinds of stem cells (cortex endodermis initial (CEI) and columella stem cell (CSC)). In the QC and CEI, the action of RBR is dependent on the LxCxE motif, as we demonstrated by mutating RBR in aminoacid N849. When introgressed into the amiGO RBR background, N849F mutant was not able to complement the phenotype, pointing to a role of LxCxE containing proteins in the asymmetric cell division of the QC and CSC. SCR protein does contain an LxCxE motif, and we were able to demonstrate that RBR action in the QC is SCR dependent. The fact that QC is dividing faster in the amiGO RBR background does not affect root growth or architecture, but it does impair the response to genotoxic damage. This suggest that RBR-SCR control of QC division is mantaining the organising center as a quiescent stem cell, that will only divide upon genotoxic damage to re-establish the stem cell. This work is ready for submission into Plant Cell.