Final Report Summary - NEW ROOT STEM CELLS (A MOLECULAR, GENOMICS AND GENETIC ANALYSIS OF NEW STEM CELL FORMATION DURING ROOT MORPHOGENESIS)
Stem cells are indispensable for development and growth in all multicellular organisms. Plants can specify new stem cells after embryogenesis as part of their normal development while this is severally restricted in animals. Plants can then be used to unravel reprogramming of differentiated cells into stem cells in the context of multicellular organisms. My previous research has identified a novel regulatory mechanism, the Root Clock (Moreno-Risueno et al. (2010) Science 329: 1306) that regulates the mechanism by which new branches (lateral roots) are formed in a spatial-temporal pattern in the model plant Arabidopsis thaliana. Formation of lateral roots requires the specification of pluripotent stem cells. My research focuses on understanding the molecular mechanisms and cues that underlie the basis of new stem cell formation in time and in space.
During the first 24 month period of the CIG we have addressed how the Root Clock selects populations of cells to specify new stem cells (objective 1). We dissected the role of the different oscillations through the analysis of oscillating transcription factors mutants. We focused on two transcription factors, one oscillating in phase and the other one oscillating in antiphase. Using a combined Luciferase and confocal imaging followed by lineage analyses of cells exposed to the oscillations we determined if abnormal oscillations in different oscillatory phases caused impairment in reprogramming of pericycle cells to form pluripotent lateral root founder cells and their progeny (stem cells). Our findings provide a novel insight into the specification of pluripotent and stem cells showing how oscillating transcription factors select cells to become lateral root founder cells. We also addressed how new stem cells are specified (objective 2). Our research, during this first 24 month period, focused on the identification of regulators specifying lateral root founder cells or required for their asymmetric divisions that occur up to stage II of lateral root formation. We screened for EMS mutants in a triply-marked line. As a result of this screen we identified two heritable mutations. Through macroscopic and microscopic analysis we determined that one of these mutants showed altered pluripotency, involving increased cell reprogramming, and impairment in development of adult plant organs because asymmetric divisions required for stem cell specification were blocked. The second mutant was also required for specification of stem cells. We have mapped these mutations and identified the changes in the DNA responsible of the observed phenotypes.
During the second period of the Career Integration Grant –CIG- (months 25-48), we have addressed (objective 1) the molecular role of a number of transcriptional regulators in establishing the periodic gene expression oscillations of the Root Clock. We have identified a transcriptional module that appears to integrate the activity of the LR clock and auxin signaling to generate robust in-phase gene expression oscillations thus positioning new branching sites and/or lateral root founder cells in a spatial-temporal pattern. We have also addressed how new stem cells are specified (objective 2). We have followed up the two mutants identified during this first 24 month period of the CIG. Our research efforts have characterized the molecular role of these genes in regulation of the first asymmetric division of lateral root founder cells to initiate organogenesis. In a different approach we have identified 3 transcriptional factors that are involved in specifying cell fate in the new stem cell niche during lateral root morphogenesis.
The results derived from my research will help to further understanding the complex regulation that is required to make up multicellularity, plant architecture and to establish paradigms of how stem cell specification and activity is controlled. The knowledge gained from this research may derive in future applications or be the basis of new biotechnological tools aimed to increase biomass, improve stress adaptation through root architecture modification, molecular farming, and propagation of recalcitrant species.
Finally, I have developed of a strategic research line and gained two grants (with associated PhD contracts) as principal investigator. This has allowed me to start my own research group while I collaborated with my initial host group (Laboratory of Dr. Del Pozo, who became my mentor) at my host institution: Center for Plant Biotechnology and Genomics (CBGP) at the Universidad Politecnica de Madrid. As result of my participation in his group and in the course of collaboration we have developed a system to grow roots in vitro in light conditions closer to the natural ones. This system was called D-Root. This system allowed us to assess the effect of light on roots that was published in a journal in the first quartile of its area. Roots have negative phototropism and bend to avoid light growing in the opposite direction. We modified D-Root to expose roots to a spotlight that allowed us to elucidate most of the regulatory molecular mechanism using a system biology approach. I co-supervised this work together with Dr. del Pozo, and we are both co-corresponding authors in a paper published in the journal with highest impact factor of primary research journals in plant biology. Finally, we screened for factors involved in root system architecture using an inducible activation tagging approach. This allowed us to identify a transcription factor required for proliferation during cell reprogramming and/or transdiferentiation. This paper was published a journal in the first quartile of its area.
In conclusion, the CIG has been highly beneficial for my re-integration in Spain allowing me to develop my career and to start my own research group while integrated at the host institution, the CBGP. Based on my achievements during this period (which had not been possible without the CIG), I am currently being evaluated for a permanent position at the CBGP. The CIG has, therefore, enormously benefited my scientific career facilitating my lasting professional integration in the European Research Area.
During the first 24 month period of the CIG we have addressed how the Root Clock selects populations of cells to specify new stem cells (objective 1). We dissected the role of the different oscillations through the analysis of oscillating transcription factors mutants. We focused on two transcription factors, one oscillating in phase and the other one oscillating in antiphase. Using a combined Luciferase and confocal imaging followed by lineage analyses of cells exposed to the oscillations we determined if abnormal oscillations in different oscillatory phases caused impairment in reprogramming of pericycle cells to form pluripotent lateral root founder cells and their progeny (stem cells). Our findings provide a novel insight into the specification of pluripotent and stem cells showing how oscillating transcription factors select cells to become lateral root founder cells. We also addressed how new stem cells are specified (objective 2). Our research, during this first 24 month period, focused on the identification of regulators specifying lateral root founder cells or required for their asymmetric divisions that occur up to stage II of lateral root formation. We screened for EMS mutants in a triply-marked line. As a result of this screen we identified two heritable mutations. Through macroscopic and microscopic analysis we determined that one of these mutants showed altered pluripotency, involving increased cell reprogramming, and impairment in development of adult plant organs because asymmetric divisions required for stem cell specification were blocked. The second mutant was also required for specification of stem cells. We have mapped these mutations and identified the changes in the DNA responsible of the observed phenotypes.
During the second period of the Career Integration Grant –CIG- (months 25-48), we have addressed (objective 1) the molecular role of a number of transcriptional regulators in establishing the periodic gene expression oscillations of the Root Clock. We have identified a transcriptional module that appears to integrate the activity of the LR clock and auxin signaling to generate robust in-phase gene expression oscillations thus positioning new branching sites and/or lateral root founder cells in a spatial-temporal pattern. We have also addressed how new stem cells are specified (objective 2). We have followed up the two mutants identified during this first 24 month period of the CIG. Our research efforts have characterized the molecular role of these genes in regulation of the first asymmetric division of lateral root founder cells to initiate organogenesis. In a different approach we have identified 3 transcriptional factors that are involved in specifying cell fate in the new stem cell niche during lateral root morphogenesis.
The results derived from my research will help to further understanding the complex regulation that is required to make up multicellularity, plant architecture and to establish paradigms of how stem cell specification and activity is controlled. The knowledge gained from this research may derive in future applications or be the basis of new biotechnological tools aimed to increase biomass, improve stress adaptation through root architecture modification, molecular farming, and propagation of recalcitrant species.
Finally, I have developed of a strategic research line and gained two grants (with associated PhD contracts) as principal investigator. This has allowed me to start my own research group while I collaborated with my initial host group (Laboratory of Dr. Del Pozo, who became my mentor) at my host institution: Center for Plant Biotechnology and Genomics (CBGP) at the Universidad Politecnica de Madrid. As result of my participation in his group and in the course of collaboration we have developed a system to grow roots in vitro in light conditions closer to the natural ones. This system was called D-Root. This system allowed us to assess the effect of light on roots that was published in a journal in the first quartile of its area. Roots have negative phototropism and bend to avoid light growing in the opposite direction. We modified D-Root to expose roots to a spotlight that allowed us to elucidate most of the regulatory molecular mechanism using a system biology approach. I co-supervised this work together with Dr. del Pozo, and we are both co-corresponding authors in a paper published in the journal with highest impact factor of primary research journals in plant biology. Finally, we screened for factors involved in root system architecture using an inducible activation tagging approach. This allowed us to identify a transcription factor required for proliferation during cell reprogramming and/or transdiferentiation. This paper was published a journal in the first quartile of its area.
In conclusion, the CIG has been highly beneficial for my re-integration in Spain allowing me to develop my career and to start my own research group while integrated at the host institution, the CBGP. Based on my achievements during this period (which had not been possible without the CIG), I am currently being evaluated for a permanent position at the CBGP. The CIG has, therefore, enormously benefited my scientific career facilitating my lasting professional integration in the European Research Area.